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WO2025113648A1 - Mrna molecule and use thereof in preparation of beauty and skin care products - Google Patents

Mrna molecule and use thereof in preparation of beauty and skin care products Download PDF

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Publication number
WO2025113648A1
WO2025113648A1 PCT/CN2024/135712 CN2024135712W WO2025113648A1 WO 2025113648 A1 WO2025113648 A1 WO 2025113648A1 CN 2024135712 W CN2024135712 W CN 2024135712W WO 2025113648 A1 WO2025113648 A1 WO 2025113648A1
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WO
WIPO (PCT)
Prior art keywords
mrna
collagen
skin
introduction device
present
Prior art date
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Pending
Application number
PCT/CN2024/135712
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French (fr)
Chinese (zh)
Inventor
胡勇
韩金雨
谢暮晴
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Rhegen Biotechnology Co Ltd
Wuhan Rhegen Biotechnology Co Ltd
Original Assignee
Shenzhen Rhegen Biotechnology Co Ltd
Wuhan Rhegen Biotechnology Co Ltd
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Publication of WO2025113648A1 publication Critical patent/WO2025113648A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A61K8/606—Nucleosides; Nucleotides; Nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/18—Applying electric currents by contact electrodes
    • A61N1/20—Applying electric currents by contact electrodes continuous direct currents
    • A61N1/30—Apparatus for iontophoresis, i.e. transfer of media in ionic state by an electromotoric force into the body, or cataphoresis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/18—Applying electric currents by contact electrodes
    • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/18—Applying electric currents by contact electrodes
    • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
    • A61N1/327—Applying electric currents by contact electrodes alternating or intermittent currents for enhancing the absorption properties of tissue, e.g. by electroporation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/16—Emollients or protectives, e.g. against radiation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/18—Antioxidants, e.g. antiradicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
    • A61M2037/0007—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin having means for enhancing the permeation of substances through the epidermis, e.g. using suction or depression, electric or magnetic fields, sound waves or chemical agents

Definitions

  • the invention belongs to the field of medical cosmetology and cosmetics, and relates to an mRNA molecule and application thereof in the preparation of beauty and skin care products.
  • Skin is the largest organ in the human body, accounting for about 16% of body weight. Skin covers the entire body surface and is the only organ that has the functions of protection, temperature regulation, secretion and excretion of sweat and oil, absorption, sensation, participation in the body's metabolism and immunity to external invasion.
  • the skin is mainly divided into three layers, from the outside to the inside, divided into the epidermis, dermis, and subcutaneous tissue.
  • Skin aging is a process in which human functions develop naturally at a faster or slower pace due to the combined influence of internal and external factors. It is mainly caused by endogenous aging and exogenous aging. The main manifestations are the gradual slowing down of cell metabolism, the gradual loss of collagen, the destruction of collagen, the thinning of skin thickness, the rupture of elastic fibers, and the appearance of dryness, roughness, wrinkles, sagging and other skin aging problems. As the skin ages, protein synthesis decreases, proteolysis increases, humidity generally decreases, and the skin barrier, connective tissue and cohesion are generally damaged.
  • Collagen plays a very important supporting role in the skin and can maintain the firmness and elasticity of the skin, especially type I collagen, which is the most abundant collagen in the skin, accounting for about 90% of the total collagen content.
  • Collagen is the main structural protein in the extracellular matrix (ECM).
  • ECM extracellular matrix
  • a large amount of water, extracellular matrix and functional cells are distributed between the fibers, which is an important biochemical reaction site for the skin.
  • the ECM interacts with the dermal fibroblasts, and the collagen fiber scaffold is constantly reshaped.
  • mRNA messenger RNA
  • genomic DNA a nucleic acid molecule that plays a role in information transmission in cells. It is transcribed from genomic DNA and participates in the process of protein synthesis in cells. Compared with DNA, mRNA can mediate better transfection efficiency and longer protein expression time. mRNA can guide skin cells to produce specific proteins, such as collagen, thereby enhancing the elasticity and moisturizing ability of the skin and reducing the appearance of fine lines and wrinkles.
  • the purpose of the present invention is to provide a method for delivering mRNA into the skin to promote collagen growth, so that mRNA can penetrate the skin and be absorbed by skin cells to promote the efficient expression of collagen, reverse the loss of collagen in damaged areas of aging skin, improve the cellular microenvironment of damaged skin, and regulate the skin condition to achieve skin repair.
  • the present invention adopts the following technical solutions:
  • the present invention provides an mRNA molecule for introduction into the bottom layer of the skin for beauty treatment, which comprises a nucleic acid sequence encoding collagen.
  • the mRNA molecule contains a nucleic acid sequence encoding collagen, and the nucleic acid sequence encoding collagen contains a sequence shown in any one of SEQ ID NOs: 1 to 18.
  • SEQ ID NO: 1 to 18 are only characteristic display sequences. According to the characteristics of mRNA technology, theoretically, the nucleotide sequence of the collagen protein can also be the sequence of other types of collagen proteins. When other conditions remain unchanged, other types of collagen proteins are expressed, and the related technical mechanism is the same, and the purpose of the present invention can also be achieved.
  • the second aspect of the present invention provides an mRNA molecule, wherein the mRNA molecule comprises a nucleic acid sequence encoding collagen, and the nucleic acid sequence encoding collagen contains a sequence shown in any one of SEQ ID NOs: 1 to 8 and SEQ ID NOs: 10 to 17.
  • the mRNA molecule comprises at least one mRNA sequence encoding type I collagen ⁇ 1 chain and at least one mRNA sequence encoding type I collagen ⁇ 2 chain, the mRNA sequence encoding type I collagen ⁇ 1 chain has a sequence shown in any one of SEQ ID NOs: 1 to 9, and the mRNA sequence encoding type I collagen ⁇ 2 chain has a sequence shown in any one of SEQ ID NOs: 10 to 18.
  • the mRNA sequence encoding type I collagen ⁇ 1 chain has the sequence shown in SEQ ID NO: 3
  • the mRNA sequence encoding type I collagen ⁇ 2 chain has the sequence shown in SEQ ID NO: 12.
  • the mRNA sequence encoding type I collagen ⁇ 1 chain has the sequence shown in SEQ ID NO: 5
  • the mRNA sequence encoding type I collagen ⁇ 2 chain has the sequence shown in SEQ ID NO: 14.
  • the mRNA sequence encoding type I collagen ⁇ 1 chain has the sequence shown in SEQ ID NO: 3
  • the mRNA sequence encoding type I collagen ⁇ 2 chain has the sequence shown in SEQ ID NO: 16.
  • part or all of the nucleosides in the mRNA molecule of the present invention may be chemically modified nucleosides.
  • the chemically modified nucleoside is selected from one or more of 2-fluoro-2-deoxyadenosine, 2-fluoro-2-deoxyuridine, 2-fluoro-2-deoxycytidine, 2-fluoro-2-deoxyguanosine, 2-fluoro-2-deoxy-5-methylcytidine, 2-fluoro-2-deoxy-pseudouridine, 2-fluoro-2-deoxy-N1-methyl-pseudouridine, 2-fluoro-2-deoxy-N7-methyl-guanosine, 2-fluoro-2-deoxy-5-methoxyuridine, 2-fluoro-2-deoxy-N4-acetylcytidine, 2-fluoro-2-deoxy-N6-methyladenosine, 5-methylcytidine, pseudouridine, N1-methyl-pseudouridine, N7-methyl-guanosine, 5-methoxyuridine, N4-acetylcytidine and N
  • the mRNA molecule further has a 5' cap structure.
  • the 5' cap structure is selected from one of m7G, Cap0, Cap1, Cap2, modified Cap0 and modified Cap1 structures, specifically, it can be Cap0, Cap1, Cap2, ARCA, inosine, N1-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine, or one of the 5' cap structures mentioned in patent application CN202310091020.3 or CN202310391954.9.
  • the mRNA molecule further has PolyA.
  • the PolyA structure is selected from one of the PolyAs mentioned in CN202211032978.7 or CN202311309226.5.
  • the third aspect of the present invention provides a cosmetic or medicinal composition, wherein the cosmetic or medicinal composition comprises the mRNA molecule described in the first aspect or the second aspect of the present invention.
  • the cosmetic or pharmaceutical composition further comprises a base material acceptable for medical, cosmetic and cosmetic use.
  • the matrix material includes currently common matrix materials with good compatibility with RNA, which will not cause the denaturation or degradation of RNA components, and can be one or more natural polymer materials, one or more artificially synthesized polymer materials, or a mixed system of natural polymer materials and artificially synthesized polymer materials.
  • Common matrix materials include but are not limited to starch, plant gum, animal gelatin, sodium hyaluronate, hyaluronic acid, polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, ethyl cellulose, sodium carboxymethyl cellulose, hydroxyethyl cellulose, guar gum and its derivatives, polyvinyl alcohol, polyvinyl pyrrolidone, acrylic polymers.
  • the cosmetic or pharmaceutical composition further comprises a cosmetically, food or pharmaceutically acceptable carrier.
  • the carrier is selected from one or more of an excipient, a disintegrant, a binder, and a lubricant.
  • excipients include, but are not limited to, microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof.
  • the disintegrant includes, but is not limited to, sodium starch glycolate, anhydrous calcium hydrogen phosphate or a combination thereof.
  • the binder includes, but is not limited to, polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose, hydroxypropyl cellulose or a combination thereof.
  • the lubricant includes, but is not limited to, magnesium stearate, silicon dioxide, talc, or a combination thereof.
  • the cosmetic or pharmaceutical composition further comprises water, glycerin, propylene glycol, sodium hyaluronate, astaxanthin, citric acid, arbutin, PEG-40, mineral oil, 1.4-butylene glycol, isopropyl palmitate, caprylic/capric triglyceride, dimethicone, PEG-60 sorbitan stearate, cetearyl alcohol, polysorbate-60, glyceryl stearate, phenoxyethanol, and methylparaben.
  • the cosmetic or pharmaceutical composition further comprises a preservative, a stabilizer, a surfactant, a solvent, a moisturizer, an emollient, an ultraviolet absorber, a preservative, a bactericide, an antioxidant, a pH adjuster, an organic and inorganic pigment, a fragrance, a cooling agent or an antiperspirant.
  • a preservative e.g., a preservative, a stabilizer, a surfactant, a solvent, a moisturizer, an emollient, an ultraviolet absorber, a preservative, a bactericide, an antioxidant, a pH adjuster, an organic and inorganic pigment, a fragrance, a cooling agent or an antiperspirant.
  • the cosmetic or pharmaceutical composition is sterile.
  • the cosmetic or pharmaceutical composition is suitable for topical, transdermal, subcutaneous, intradermal, oral, intranasal, intratracheal, sublingual, buccal, rectal, vaginal, transurethral, inhalation, intravenous, intraarterial, intramuscular, intracardiac, intraosseous, intraperitoneal, transmucosal, intravitreal, subretinal, intraarticular, periarticular, local or epidermal administration.
  • the cosmetic or pharmaceutical composition is suitable for intradermal administration and superficial injection.
  • the cosmetic or pharmaceutical composition can be prepared into a non-oral dosage form.
  • the non-oral dosage form can be an injection or a skin topical agent.
  • the skin topical agent can be a cream, a gel, an ointment, a skin emulsifier, a skin suspension, a transdermal patch, a medicated bandage, an emulsion or a combination thereof.
  • the skin external preparation can be appropriately mixed with ingredients commonly used in skin external preparations such as cosmetics or medicines, such as aqueous ingredients, oily ingredients, powder ingredients, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, and various skin nutrients, as needed.
  • ingredients commonly used in skin external preparations such as cosmetics or medicines, such as aqueous ingredients, oily ingredients, powder ingredients, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, and various skin nutrients, as needed.
  • the skin external preparation can be appropriately mixed with metal chelating agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconic acid, etc., caffeine, tannin, verapamil, glycyrrhizic acid, tranexamic acid and its derivatives or salts thereof, vitamin C, magnesium ascorbyl phosphate, ascorbyl glucoside, arbutin, kojic acid, glucose, fructose, trehalose, etc.
  • metal chelating agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconic acid, etc., caffeine, tannin, verapamil, glycyrrhizic acid, tranexamic acid and its derivatives or salts thereof, vitamin C, magnesium ascorbyl phosphate, ascorbyl glucoside, arbutin, kojic acid, glucose
  • the cosmetic or medicinal composition is a skin care product.
  • the cosmetic or pharmaceutical composition also includes the following dosage forms: lotion, skin softener, toner, astringent, emulsion, milk lotion, moisturizing lotion, nutrient solution, massage cream, nutrient cream, moisturizing cream, hand cream, foundation, essence, nutrient essence, film, soap, cleansing foam, cleansing milk, cleansing cream, body lotion, body cleansing liquid, suspension, gel, powder, paste, facial mask or sheet mask or spray composition.
  • the cosmetic or medicinal composition of the present invention is a preparation for introduction into the underlying layer of the skin via a radio frequency introduction device, a negative pressure introduction device, a microcrystal introduction device, a microcurrent introduction device, a microneedle introduction device, or a syringe.
  • the fourth aspect of the present invention provides a DNA molecule which can be used to transcribe the mRNA molecule described in the first aspect or the second aspect of the present invention.
  • the DNA molecule further comprises a promoter, a UTR sequence and a PolyA fragment connected to its 3' end, and a UTR sequence and a Cap connected to its 5' end.
  • the fifth aspect of the present invention provides a recombinant plasmid or a recombinant cell, which comprises the DNA molecule described in the fourth aspect of the present invention.
  • the vector of the recombinant plasmid is an expression vector.
  • the recombinant plasmid or recombinant cell can be used to transcribe the mRNA described in the first aspect or the second aspect of the present invention.
  • the sixth aspect of the present invention provides a kit comprising the mRNA molecule described in the first or second aspect of the present invention, the cosmetic or pharmaceutical composition described in the third aspect of the present invention, the DNA molecule described in the fourth aspect of the present invention, or the recombinant plasmid or recombinant cell described in the fifth aspect of the present invention.
  • the kit is a kit for preparing the mRNA molecule described in the first aspect or the second aspect of the present invention.
  • the kit for preparing the mRNA molecule described in the first aspect or the second aspect of the present invention comprises the DNA molecule described in the third aspect of the present invention, or the recombinant plasmid or recombinant cell described in the fourth aspect of the present invention.
  • the kit is a kit for skin care or skin condition improvement.
  • the kit for skin care or skin condition improvement comprises the mRNA molecule described in the first aspect or the second aspect of the present invention or the cosmetic or pharmaceutical composition described in the third aspect of the present invention.
  • the kit for skin care or skin condition improvement of the present invention further comprises an auxiliary introduction device.
  • the auxiliary introduction device promotes the mRNA molecules to pass through the skin into the underlying skin layer, thereby achieving the purpose of effective delivery; wherein the auxiliary introduction device is mainly divided into a radio frequency introduction device, a negative pressure introduction device, a microcrystal introduction device, a microcurrent introduction device, a microneedle introduction device, and a syringe according to its different mechanisms of action.
  • the auxiliary introduction device includes a negative pressure introduction device, a microcurrent introduction device, and a microneedle introduction device. More preferably, the auxiliary introduction device is a microneedle introduction device.
  • the auxiliary introduction device is a syringe.
  • the seventh aspect of the present invention provides a method for preparing the mRNA molecule according to the first aspect or the second aspect of the present invention, comprising:
  • RNA in vitro synthesis system including the DNA template to perform in vitro synthesis of mRNA to obtain the mRNA molecule.
  • the mRNA molecule when the mRNA molecule is a mixture of an mRNA molecule containing an mRNA sequence encoding type I collagen ⁇ 1 chain and an mRNA molecule containing an mRNA sequence encoding type I collagen ⁇ 2 chain, the mixture can be obtained by mixing the mRNA molecules obtained above in proportion.
  • a DNA fragment of the mRNA is synthesized and transcribed, and the DNA fragment is cloned into an expression plasmid to obtain a recombinant plasmid.
  • the present invention does not specifically limit the method for synthesizing the DNA fragment corresponding to the mRNA, and a conventional DNA synthesis method in the art can be used. In the specific implementation of the present invention, it is preferably commissioned to a biotechnology company for synthesis.
  • the DNA fragment is preferably cloned into the expression plasmid by enzyme digestion and ligation; in the present invention, the DNA fragment is preferably double digested by BamHI and NheI enzymes to obtain a digested DNA fragment; the expression plasmid is preferably double digested by BamHI and NheI enzymes to obtain a digested plasmid; and then the digested DNA fragment and the digested plasmid are ligated to obtain a recombinant plasmid.
  • the present invention transfers the recombinant plasmid into a host cell to obtain a recombinant cell, extracts the plasmid from the expanded recombinant cell, and uses the extracted plasmid as a template to perform PCR amplification to obtain a DNA template for in vitro expression of mRNA.
  • the host cell is preferably an Escherichia coli competent cell;
  • the present invention preferably performs screening of positive recombinant cells and colony sequencing.
  • the screening of the positive recombinant cells is preferably performed on an amp-resistant solid culture medium.
  • a single colony on the amp-resistant solid culture medium is selected for colony PCR, and a colony containing a target band in the colony PCR result is selected for sequencing.
  • the present invention does not specifically limit the specific steps of the colony PCR, and the conventional colony PCR steps in the art can be used.
  • the plasmid of the correctly sequenced recombinant cell is extracted; the present invention does not specifically limit the method for extracting the plasmid, and preferably a plasmid extraction kit is used.
  • the extracted plasmid is used as a template for PCR amplification to obtain a DNA template for in vitro expression of mRNA.
  • the concentration of the DNA template is preferably 1 ng/ ⁇ l.
  • the PCR amplification procedure is preferably as follows: pre-denaturation at 98°C for 3 minutes; denaturation at 98°C for 10 seconds, annealing at 60°C for 5 seconds, extension at 72°C for 2 minutes, 34 cycles; final extension at 72°C for 10 minutes.
  • the amplification product is preferably subjected to agarose gel electrophoresis to determine whether the reaction is successful; the parameters of the agarose gel electrophoresis detection are preferably as follows: 1.5% agarose, 5V/min, 40 minutes. In the present invention, the appearance of a band of the target size in agarose gel electrophoresis is considered to be a successful reaction.
  • the amplification product is preferably concentrated and purified.
  • the concentration is preferably performed using a Millipore 30Kd ultrafiltration tube; the purification is preferably performed using FPLC; after the purification, the present invention preferably uses Nano Drop to detect the concentration of the purified template, as well as the ratios of 260/280 and 260/230.
  • the range of 260/280 is 1.8-2.1, and the range of 260/230 is greater than 2.0.
  • the present invention constructs an RNA in vitro synthesis system including the DNA template to perform in vitro synthesis of mRNA to obtain the active ingredient mRNA.
  • the RNA in vitro synthesis system calculated in 1600 ⁇ l, includes the following components:
  • the conditions for the in vitro synthesis of RNA are preferably 36-38°C, 8-12 hours, more preferably 37°C, 10 hours.
  • the in vitro synthesis of RNA is preferably carried out in a constant temperature reactor; the in vitro synthesis system of RNA is preferably placed in a 2ml RNase-free Tube tube, and multiple tubes are reacted at the same time; the reaction reagents in the in vitro synthesis system of RNA are added in the above order.
  • the present invention preferably also includes the steps of removing the DNA template, recovering mRNA and purifying mRNA.
  • the removal of the DNA template is preferably achieved by DNase I digestion; the digestion preferably includes mixing DNase I with the solution after the RNA in vitro synthesis reaction; the volume ratio of the DNase I to the solution after the RNA in vitro synthesis reaction is preferably 3:40; the mixing is preferably achieved by turning the RNase-free Tube upside down, and the number of upside downs is preferably 8 to 12 times, more preferably 10 times; after the mixing, the present invention preferably performs centrifugation to collect the solution to the bottom of the RNase-free Tube.
  • the speed of the centrifugation is preferably 800 to 1200 rpm, more preferably 1000 rpm; the time of the centrifugation is preferably 8 to 12 seconds, more preferably 10 seconds.
  • the temperature of the digestion is preferably 37°C; the time of the digestion is preferably 1 hour.
  • the present invention preferably performs residual DNA fragment detection after the digestion is completed.
  • the recovery of mRNA is preferably achieved by precipitation with the ammonium acetate solution; the specific implementation method is described in the examples; after the mRNA is recovered, the present invention performs a quality test on the mRNA; the quality test includes the concentration of the mRNA, the ratios of 260/280 and 260/230 of the mRNA, and the A260/A280 value of the pure mRNA is 2.0 to 2.1, and the A260/A230 range is 1.8 to 2.2.
  • the purification of mRNA is achieved by HPLC purification. After the mRNA is purified according to the present invention, the purified mRNA is preferably packaged.
  • the eighth aspect of the present invention provides a cosmetic method, which comprises applying the mRNA molecule described in the first aspect or the second aspect of the present invention, or the cosmetic or pharmaceutical composition described in the third aspect of the present invention to the skin.
  • the method further includes promoting the mRNA molecules to pass through the skin into the underlying skin by means of an auxiliary introduction device to achieve effective delivery; wherein the auxiliary introduction device is mainly divided into a radio frequency introduction device, a negative pressure introduction device, a microcrystal introduction device, a microneedle introduction device, and a syringe according to its different mechanisms of action.
  • the auxiliary introduction device includes a negative pressure introduction device, a microcurrent introduction device, and a microneedle introduction device. More preferably, the auxiliary introduction device is a microneedle introduction device.
  • the auxiliary introduction device is a syringe.
  • the working concentration of the mRNA molecule is 0.001 ng/ml-10 mg/ml.
  • the working concentration of the mRNA molecule is 0.01 ⁇ g/ml-1.0 mg/ml.
  • the working concentration of the mRNA molecule is 0.01-1.0 ⁇ g/ml.
  • the working concentration of the mRNA molecule is 1.0 mg/ml.
  • the working concentration of the mRNA molecule is 0.01 ⁇ g/ml.
  • the cosmetic method helps to improve skin wrinkles
  • the targeted skin wrinkles refer to nasolabial folds, crow's feet, frown lines, worry lines, scars, glabellar lines, drooping eyebrows, tear troughs, nasal cheek lines, bunny lines, cheek/mid-face sagging, marionette lines, poppy pits, smile lines, laugh lines, chin wrinkles, neck lines, platysma bands and any combination thereof formed by superficial depressions.
  • the ninth aspect of the present invention provides the use of the mRNA molecule described in the first or second aspect of the present invention, the cosmetic or pharmaceutical composition described in the third aspect of the present invention, the DNA molecule described in the fourth aspect of the present invention, the recombinant plasmid or recombinant cell described in the fifth aspect of the present invention, or the kit described in the sixth aspect of the present invention in the preparation of a product for skin care or improving skin condition.
  • the product is a medicine, a cosmetic or a kit.
  • the improvement of skin condition is to promote the expression of collagen.
  • the improvement of skin condition is to stimulate the proliferation of collagen fibers.
  • the present invention provides an mRNA molecule and its use, wherein the mRNA molecule comprises a nucleic acid sequence encoding collagen.
  • the mRNA sequence constructed by the present invention largely avoids sequence-independent apoptosis in mammalian cells, and shows better serum stability and improved in vivo activity.
  • the present invention also relates to the application of medical, beauty and cosmetic compositions, using liposomes or other gene introduction technologies, the composition is introduced into skin cells or absorbed by skin cells, and then the biological mechanism of mRNA translation of protein is used to make skin cells produce collagen, and the collagen content in skin tissue is increased to normalize aging skin.
  • the preparation method of the cosmetic composition provided by the present invention is simple, fast, and has a high expression of active ingredients. Combined with the drug introduction method in the present invention, the efficiency of mRNA preparation introduction into the skin can be enhanced, and the utilization rate of effective preparations can be improved, thereby achieving the purpose of more effective skin transdermal delivery and achieving the effect of skin repair.
  • the mRNA dosage form cosmetic preparation provided by the present invention includes mRNA that stimulates collagen regeneration. After the preparation is introduced into skin cells, it can promote the expression of collagen in fibroblasts, making up for skin wrinkles and the like caused by aging, light exposure, water loss, etc.
  • the data in the specific embodiments show the effectiveness data; according to the records of the embodiments, the mRNA that stimulates collagen growth factor provided by the present invention can be expressed at a high level specifically in cells; during use, the present invention is combined with auxiliary equipment such as a radio frequency introduction instrument, a negative pressure introduction instrument, a microcrystal introduction instrument, a microcurrent introduction instrument, etc.
  • FIG1 is a schematic diagram of the structure of mRNA encoding collagen growth factor
  • Figure 2 is a comparison of the collagen content of cells transfected with different sequences of SEQ ID No. 1-18 alone and in combination;
  • FIG3 is the quantitative result of collagen content at the cellular level after cells were transfected with mRNA
  • Figure 4 is a comparison of the up-regulation rates of collagen content in cells after transfection of mRNA
  • FIG5 is a graph showing changes in the content of type I collagen in the skin after 3D skin test mRNA enters the skin using the ELISA detection method
  • FIG6 is a comparison of the up-regulation rate of type I collagen in the skin after the 3D skin test mRNA enters the skin using the ELISA detection method
  • FIG7 is the immunofluorescence result of type I collagen in skin sections after 3D skin test mRNA enters the skin using immunofluorescence method
  • FIG8 is a comparison of the results of the upregulation of the immunofluorescence intensity of type I collagen in skin sections after the 3D skin test mRNA entered the skin using the immunofluorescence method;
  • Figure 9 shows the results of observing the expression of fluorescent protein GFP mRNA in the skin of mice in each experimental group using the auxiliary introduction instrument
  • Figure 10 shows rat skin sections and Masson staining results, observing the expression of type I collagen
  • FIG11 is an image of the dermis layer thickness of skin sections of rats in each group, which reflects the promoting effect of the mRNA preparation on the production of type I collagen.
  • This embodiment provides an mRNA molecule, which is formed by connecting an mRNA molecule to a 3' end Poly A.
  • the sequence of the mRNA molecule comprises a 5' cap structure, a 5' UTR, a target gene sequence, a 3' UTR and Poly A.
  • the mRNA molecule encoding collagen is prepared by the following steps:
  • Step S1 design and synthesize a plasmid vector with a promoter sequence and a target gene sequence.
  • the plasmid vector is a company vector, and the connection order of each part is 5'UTR-collagen gene nucleotide sequence-3'UTR;
  • Step S2 using the plasmid vector of step S1 as a template for in vitro transcription to obtain an mRNA molecule, wherein the sequence of the mRNA molecule comprises a 5' cap structure, a 5' UTR, a target gene sequence, a 3' UTR and plolyA;
  • the mRNA molecule is synthesized in a one-step process. Except for the cap structure, all other elements are integrated into the DNA template during plasmid construction.
  • nucleotide sequence of the collagen gene is shown in SEQ ID No.1 ⁇ 18.
  • the 5’ cap structure is m7G(5’)ppp(5’)(2’OMeA)pGp.
  • the 5’UTR sequence is shown as SEQ ID No.19.
  • the 3’UTR sequence is shown as SEQ ID No.20.
  • the polyA sequence is shown as SEQ ID No.21.
  • step S2 the specific method for synthesizing the mRNA molecule in step S2 is as follows:
  • Reaction volume 50 ⁇ l (reaction volume of a single tube, multiple tubes reacted at the same time); the PCR amplification system (50 ⁇ l): Prime STAR Max Premix (2 ⁇ ) 25 ⁇ l, primer F 10 ⁇ mol/L 1.2 ⁇ l, primer R 10 ⁇ mol/L 1.2 ⁇ l, DNA template (1ng/ ⁇ l) 1 ⁇ l and water 21.6 ⁇ l.
  • the PCR amplification program is as follows: pre-denaturation 98°C 3min; denaturation 98°C 10s, annealing 60°C 5s, extension 72°C 2min, 34 cycles; final extension 72°C, 10min.
  • the reaction solution is combined in a 1.5ml Tube tube. Take 10 ⁇ l for DNA agarose gel electrophoresis (1.5% agarose, 5V/min, 40min). Confirm the success of the reaction based on the size of the electrophoresis target band.
  • the DNA template obtained above was concentrated using Millipore 30Kd ultrafiltration tube.
  • the DNA template purified by FPLC was concentrated by Millipore 30Kd ultrafiltration tube and eluted with RNase-free water. The concentration of the template after ultrafiltration and the ratios of 260/280 and 260/230 were detected by NanoDrop. Finally, the template was diluted to 150ng/ ⁇ l with RNase-free water.
  • RNA-free water 440 ⁇ l, 7.5mM ATP 160 ⁇ l, 7.5mM UTP 160 ⁇ l, 7.5mM CTP 160 ⁇ l, 7.5mM GTP 160 ⁇ l, 7.5mM M7G (2'OMeA) pG 160 ⁇ l, 150ng/ ⁇ l DNA template 40 ⁇ l, 10 ⁇ Buffer 160 ⁇ l and Enzyme Mix 160 ⁇ l.
  • the RNA in vitro synthesis procedure is 37°C, 10h.
  • the mRNA SEQ ID No.1-No.18 obtained by the above steps expresses COL1A1, i.e., type I collagen ⁇ 1 chain
  • SEQ ID No.10-18 expresses COL1A2, i.e., type I collagen ⁇ 2 chain.
  • COL1A1 protein alone can increase the expression of collagen in the skin
  • mRNA SEQ ID No.1-No.18 has the ability to increase collagen expression whether used alone or in combination.
  • the cell transfection results of each mRNA group are shown in Figure 2. In the embodiments described below, all mRNA test groups have two mRNA sequences used in combination.
  • S1 cell culture and inoculation cell digestion and inoculation of fibroblasts. Observe under an inverted microscope. When most of the cells become round and suspended, add about 2 to 3 times the volume of trypsin and serum-containing DMEM medium to terminate digestion, collect them into a centrifuge tube, and centrifuge at 1200r/min for 5 minutes. After centrifugation, discard the supernatant, add a certain volume of cell culture medium to the centrifuge tube, mix the cells with an elbow pipette, and count them with a cell counter. After diluting the cells to the inoculation density, inoculate them into a 96-well plate with 200 ⁇ L of liquid per well. After inoculation, place them in a CO2 incubator and culture for 24h ⁇ 2h.
  • S2 Dosing Discard the culture medium in the 96-well plate and carry out the dosing operation. Add 1.0 ⁇ g/mL, 0.1 ⁇ g/mL, and 0.01 ⁇ g/mL collagen mRNA culture medium to the sample wells. Because the 8 preferred groups of sequences are all effective relative to the wild type during the AI algorithm screening process, the preferred group in this embodiment randomly selects a combination of SEQ ID No.3 and SEQ ID No.12, and the mixing ratio is 1:1 mixed with the same volume; add 250ng/mL TGF culture medium to the positive control well, and add normal cell culture medium to the blank control well, 200 ⁇ L per well. After the dosing is completed, place the 96-well plate in a CO2 incubator and culture for 24h ⁇ 2h.
  • the culture fluid is collected by centrifugation and the type I collagen (COL-1) content is determined using an ELISA kit.
  • the test data of each group were subjected to one-way analysis of variance using SPSS, and the statistical method used was a two-tailed test with a test level of 0.05; the COL-1 upregulation rate (%) of each group was calculated according to the following formula:
  • COL-1 up-regulation rate % (test group content - control group content) / control group content ⁇ 100%
  • the efficiency of the mRNA test group in promoting the expression of collagen in cells is much higher than that of the blank control group.
  • the positive control group has the function of supporting the correctness of the cell experiment and reflecting the effectiveness of the positive substance. Compared with the positive control group, the upregulation rate of the mRNA group is significantly higher. In the cell experiment, the mRNA group has a higher upregulation efficiency than the positive control group. Compared with the blank control group, the content of collagen expressed by cells is increased by 2 times.
  • Test system T-Skin in vitro artificial full-thickness skin model test kit, purchased from Shanghai Sianfunuo Biotechnology Co., Ltd.; culture environment: 37.0 ⁇ 1°C, 5 ⁇ 1% CO2 (v/v), saturated humidity; cell culture medium: T-Skin in vitro artificial full-thickness skin model special culture medium;
  • Main equipment CO2 incubator, multifunctional microplate reader, biological safety cabinet, TS-8 transfer and decolorization shaker, confocal microscope;
  • test substances Preparation of test substances: During the AI algorithm screening process, the 8 preferred groups of sequences were all effective compared to the wild type. Therefore, in this embodiment, the preferred group randomly selected a combination of SEQ ID No. 5 and SEQ ID No. 14, and the mixing ratio was 1:1 with the same volume as the test substance.
  • the T-Skin model was brought into the laboratory on the 18th day of culture and maintained in fresh maintenance medium at 37°C and 5% CO2 for another 48 hours.
  • test substance final concentration of 1 mg/mL
  • positive control 200 ⁇ M vitamin C
  • blank control PBS
  • DAPI nuclear staining solution (1:1000), incubate at room temperature for 2 minutes, and wash 4 times with PBS for 5 minutes each.
  • type I collagen (COL-1) content using ELISA kit After 5 days of treatment, the culture medium below the model was collected, stored at -80°C, and measured using an ELISA kit.
  • COL-1 up-regulation rate (%) (content in the experimental group - content in the control group) / content in the control group ⁇ 100%
  • Example 4 Animal experiment to verify the effect of auxiliary instruments
  • Equipment preparation before the experiment Purchase daily skin care devices in the shopping mall.
  • the skin care devices claim to have the function of increasing the skin permeability of cosmetics. For the convenience of understanding, they are distinguished from the mechanism as microcurrent introduction devices, microneedle introduction devices, and negative pressure introduction devices.
  • the above three introduction devices can be purchased directly from the mall and are daily-use instruments. This patent does not restrict or protect the brands and types of introduction devices.
  • Green fluorescent protein is a protein composed of about 238 amino acids. It can be excited by light from blue to ultraviolet light and emit green fluorescence. It is a commonly used indicator protein in biomolecular experiments. It can quickly locate the position of the expressing cells and facilitate the detection of the distribution of mRNA in the skin after it is introduced into the skin.
  • eGFP mRNA group 25 healthy Balb/C female mice aged 10 weeks that passed the quarantine were selected and randomly divided into blank control group, eGFP mRNA group, eGFP mRNA + microcurrent introduction instrument test group (hereinafter referred to as mRNA microcurrent group), eGFP mRNA + microneedle introduction instrument test group (hereinafter referred to as mRNA microneedle group), eGFP mRNA + negative pressure introduction instrument test group (hereinafter referred to as mRNA negative pressure group), with 5 mice in each group.
  • mRNA microcurrent group eGFP mRNA + microcurrent introduction instrument test group
  • mRNA microneedle group eGFP mRNA + microneedle introduction instrument test group
  • mRNA negative pressure group eGFP mRNA + negative pressure introduction instrument test group
  • mice After depilating the back of the mice, the mice were anesthetized with 1.5% isoflurane and wiped with disinfectant alcohol. After the alcohol evaporated, the eGFP mRNA solution was evenly applied on the back of the mice at a dose of 5 ⁇ g/mouse. The application range was controlled within a 1cm x 1cm square. Then, according to the instruction manual, the auxiliary introduction instrument was used to perform liquid introduction treatment on the back of the mice for 15 minutes. In the blank control group, normal saline was used as a control reagent to apply on the back of the mice.
  • the mRNA group showed varying degrees of green fluorescent groups under a fluorescence microscope (as shown in Figure 9), which proved that the mRNA passed through the skin and was taken up by skin cells to express fluorescent protein; compared with the group that applied mRNA alone, the expression level of fluorescent protein in the experimental group using the auxiliary introduction device increased to varying degrees, among which more obvious green fluorescence could be observed in the epidermis in the mRNA microcurrent group and the mRNA negative pressure group.
  • Equipment preparation before the experiment Purchase daily skin care devices in the shopping mall.
  • the skin care devices claim to have the function of increasing the skin permeability of cosmetics. For the convenience of understanding, they are distinguished from the mechanism as microcurrent introduction devices, microneedle introduction devices, and negative pressure introduction devices.
  • the above three introduction devices can be purchased directly from the mall and are daily-use instruments. This patent does not restrict or protect the brands and types of introduction devices.
  • mRNA encoding collagen was selected as the marker.
  • the eight groups of sequences selected were all effective relative to the wild type. Therefore, the preferred group in this embodiment randomly selected a combination of SEQ ID No. 7 and SEQ ID No. 16, and the mixing ratio was 1:1 with the same volume.
  • Preparation of experimental animals 25 healthy 4-week-old SD female rats that passed the quarantine were selected and randomly divided into a blank control group, a collagen mRNA + microcurrent introduction instrument test group (hereinafter referred to as the mRNA microcurrent group), a collagen mRNA + microneedle introduction instrument test group (hereinafter referred to as the mRNA microneedle group), and a collagen mRNA + negative pressure introduction instrument test group (hereinafter referred to as the mRNA negative pressure group).
  • Collagen mRNA intradermal injection was used as the positive control group, with 5 rats in each group.
  • the auxiliary introduction instrument uses physiological saline as a control reagent, and applies it to the rat's back in the same way.
  • wipe off the excess liquid on the rat's back remove the anesthesia, and observe for 48 hours. Take the skin of the experimental site and fix it overnight with 4% paraformaldehyde to make paraffin sections, and the sections are masson stained and photographed for analysis.
  • Figure 10 shows a direct comparison of the changes in collagen fibers in the dermis after treatment in each group.
  • the dermis is mainly composed of collagen fibers and elastic fibers, which directly determine the elasticity of the skin.
  • the data results in Figure 11 show that the positive control group has a promoting effect on the upregulation of collagen through intradermal injection, and the microneedle introduction method can also increase the expression of collagen in the skin, which is significantly different from the control group.

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Abstract

Provided are an mRNA molecule and a use thereof in preparation of beauty and skin care products. Specifically, the mRNA molecule comprises a nucleic acid sequence encoding collagen. After the mRNA is applied to skin, skin cells can generate collagen, and thus aged skin is normalized by up-regulating the content of the collagen in skin tissue, achieving the repairing effect of skin.

Description

一种mRNA分子及其在制备美容护肤产品中的应用An mRNA molecule and its application in preparing beauty and skin care products 技术领域Technical Field

本发明属于医学美容和化妆品领域，涉及一种mRNA分子及其在制备美容护肤产品中的应用。The invention belongs to the field of medical cosmetology and cosmetics, and relates to an mRNA molecule and application thereof in the preparation of beauty and skin care products.

背景技术Background Art

皮肤是人体最大的器官，约占体重的16％。皮肤覆盖于全身表面，是唯一有保护、体温调节、分泌和排泄汗液及油脂、吸收、感觉、参与机体的代谢和免疫外界侵袭的功能的器官，皮肤主要分为三大层，从外到里，分为表皮层、真皮层、皮下组织。Skin is the largest organ in the human body, accounting for about 16% of body weight. Skin covers the entire body surface and is the only organ that has the functions of protection, temperature regulation, secretion and excretion of sweat and oil, absorption, sensation, participation in the body's metabolism and immunity to external invasion. The skin is mainly divided into three layers, from the outside to the inside, divided into the epidermis, dermis, and subcutaneous tissue.

皮肤衰老是其受内部与外部因素共同影响而使人体机能或快或慢自然发展的一个过程，其主要是由内源性老化和外源性老化造成。主要表现为细胞代谢逐渐减缓，胶原蛋白逐渐流失，胶原质受到破坏，皮肤厚度转薄，弹性纤维断裂，从而出现干燥粗糙、皱纹、松弛等皮肤衰老问题。随着皮肤老化，产生蛋白质合成降低，蛋白酶解增加，湿度普遍性降低并且皮肤屏障、结缔组织以及内聚性的普遍损坏。Skin aging is a process in which human functions develop naturally at a faster or slower pace due to the combined influence of internal and external factors. It is mainly caused by endogenous aging and exogenous aging. The main manifestations are the gradual slowing down of cell metabolism, the gradual loss of collagen, the destruction of collagen, the thinning of skin thickness, the rupture of elastic fibers, and the appearance of dryness, roughness, wrinkles, sagging and other skin aging problems. As the skin ages, protein synthesis decreases, proteolysis increases, humidity generally decreases, and the skin barrier, connective tissue and cohesion are generally damaged.

胶原蛋白在皮肤中发挥着非常重要的支撑作用，能够维持皮肤的紧致度和弹性，尤其是Ⅰ型胶原蛋白，它是皮肤中含量最高的胶原蛋白，占总胶原含量的约90％。胶原蛋白是细胞外基质(ECM)中的主要结构蛋白，胶原纤维(主要由胶原蛋白构成)和弹性纤维(主要由弹力蛋白构成)构成的网状结构有助于增强组织的结构支架和抗拉强度。在纤维之间则分布着大量的水分、细胞外基质和功能性细胞，是皮肤重要的生化反应场所。在皮肤的ECM中，ECM与真皮成纤维细胞相互作用，胶原纤维支架被不断重塑。Collagen plays a very important supporting role in the skin and can maintain the firmness and elasticity of the skin, especially type I collagen, which is the most abundant collagen in the skin, accounting for about 90% of the total collagen content. Collagen is the main structural protein in the extracellular matrix (ECM). The mesh structure composed of collagen fibers (mainly composed of collagen) and elastic fibers (mainly composed of elastin) helps to enhance the structural support and tensile strength of the tissue. A large amount of water, extracellular matrix and functional cells are distributed between the fibers, which is an important biochemical reaction site for the skin. In the ECM of the skin, the ECM interacts with the dermal fibroblasts, and the collagen fiber scaffold is constantly reshaped.

mRNA(messenger RNA)是一种在细胞中起着信息传递的作用的核酸分子。它是由基因组DNA转录而来的，在细胞中参与蛋白质合成的过程。相比DNA，mRNA能介导更优的转染效率和更长的蛋白表达时间。mRNA可以指导皮肤细胞产生特定的蛋白质，如胶原蛋白，从而增强皮肤的弹性和保湿能力，减少细纹和皱纹的出现。mRNA (messenger RNA) is a nucleic acid molecule that plays a role in information transmission in cells. It is transcribed from genomic DNA and participates in the process of protein synthesis in cells. Compared with DNA, mRNA can mediate better transfection efficiency and longer protein expression time. mRNA can guide skin cells to produce specific proteins, such as collagen, thereby enhancing the elasticity and moisturizing ability of the skin and reducing the appearance of fine lines and wrinkles.

经查目前，向皮肤内的递送mRNA促进胶原蛋白增长的相关技术尚无深入研究。Upon investigation, it was found that there is no in-depth research on the technology of delivering mRNA into the skin to promote collagen growth.

发明内容Summary of the invention

为解决现有技术中存在的不足，本发明的目的是提供一种向皮肤内递送mRNA促进胶原蛋白增长的方法，实现mRNA经皮肤渗透后通过与皮肤细胞吸收后促进胶原蛋白的高效表达，逆转衰老皮肤受损处的胶原蛋白流失，重新改善受损肌肤的细胞微环境，调节皮肤状态使其达到肌肤修护。In order to solve the deficiencies in the prior art, the purpose of the present invention is to provide a method for delivering mRNA into the skin to promote collagen growth, so that mRNA can penetrate the skin and be absorbed by skin cells to promote the efficient expression of collagen, reverse the loss of collagen in damaged areas of aging skin, improve the cellular microenvironment of damaged skin, and regulate the skin condition to achieve skin repair.

为实现该目的，本发明采用了如下技术方案：To achieve this purpose, the present invention adopts the following technical solutions:

本发明第一方面提供了一种用于导入皮肤底层以美容的mRNA分子，其包含编码胶原蛋白的核酸序列。In a first aspect, the present invention provides an mRNA molecule for introduction into the bottom layer of the skin for beauty treatment, which comprises a nucleic acid sequence encoding collagen.

根据本发明的具体实施方式，所述的mRNA分子包含编码胶原蛋白的核酸序列，所述的编码胶原蛋白的核酸序列含有SEQ ID NO：1～18中任一项所示的序列。According to a specific embodiment of the present invention, the mRNA molecule contains a nucleic acid sequence encoding collagen, and the nucleic acid sequence encoding collagen contains a sequence shown in any one of SEQ ID NOs: 1 to 18.

本发明中，SEQ ID NO：1～18仅为特征展示序列，根据mRNA技术特点，理论上所述的胶原蛋白的核苷酸序列也可以为其他种类胶原蛋白的序列。在保持其他条件不变的情况下，表达其他种类的胶原蛋白，此相关技术机制相同，也能实现本发明的目的。In the present invention, SEQ ID NO: 1 to 18 are only characteristic display sequences. According to the characteristics of mRNA technology, theoretically, the nucleotide sequence of the collagen protein can also be the sequence of other types of collagen proteins. When other conditions remain unchanged, other types of collagen proteins are expressed, and the related technical mechanism is the same, and the purpose of the present invention can also be achieved.

本发明第二方面提供了一种mRNA分子，其中，所述的mRNA分子包含编码胶原蛋白的核酸序列，所述的编码胶原蛋白的核酸序列含有SEQ ID NO：1～8、SEQ ID NO：10～17中任一项所示的序列。The second aspect of the present invention provides an mRNA molecule, wherein the mRNA molecule comprises a nucleic acid sequence encoding collagen, and the nucleic acid sequence encoding collagen contains a sequence shown in any one of SEQ ID NOs: 1 to 8 and SEQ ID NOs: 10 to 17.

根据本发明的具体实施方案，所述的mRNA分子包含至少一条编码I型胶原蛋白α1链的mRNA序列以及至少一条编码I型胶原蛋白α2链的mRNA序列，所述编码I型胶原蛋白α1链的mRNA序列具有SEQ ID NO：1～9中任一所示序列，所述编码I型胶原蛋白α2链的mRNA序列具有SEQ ID NO：10～18中任一所示序列。According to a specific embodiment of the present invention, the mRNA molecule comprises at least one mRNA sequence encoding type I collagen α1 chain and at least one mRNA sequence encoding type I collagen α2 chain, the mRNA sequence encoding type I collagen α1 chain has a sequence shown in any one of SEQ ID NOs: 1 to 9, and the mRNA sequence encoding type I collagen α2 chain has a sequence shown in any one of SEQ ID NOs: 10 to 18.

作为一种优选的实施方式，所述编码I型胶原蛋白α1链的mRNA序列具有SEQ ID NO：3所示序列，所述的编码I型胶原蛋白α2链的mRNA序列具有SEQ ID NO：12所示序列。As a preferred embodiment, the mRNA sequence encoding type I collagen α1 chain has the sequence shown in SEQ ID NO: 3, and the mRNA sequence encoding type I collagen α2 chain has the sequence shown in SEQ ID NO: 12.

作为一种优选的实施方式，所述编码I型胶原蛋白α1链的mRNA序列具有SEQ ID NO：5所示序列，所述的编码I型胶原蛋白α2链的mRNA序列具有SEQ ID NO：14所示序列。As a preferred embodiment, the mRNA sequence encoding type I collagen α1 chain has the sequence shown in SEQ ID NO: 5, and the mRNA sequence encoding type I collagen α2 chain has the sequence shown in SEQ ID NO: 14.

作为一种优选的实施方式，所述编码I型胶原蛋白α1链的mRNA序列具有SEQ ID NO：3所示序列，所述的编码I型胶原蛋白α2链的mRNA序列具有SEQ ID NO：16所示序列。As a preferred embodiment, the mRNA sequence encoding type I collagen α1 chain has the sequence shown in SEQ ID NO: 3, and the mRNA sequence encoding type I collagen α2 chain has the sequence shown in SEQ ID NO: 16.

根据本发明的具体实施方案，本发明的mRNA分子的部分或全部核苷可以是化学修饰的核苷。According to a specific embodiment of the present invention, part or all of the nucleosides in the mRNA molecule of the present invention may be chemically modified nucleosides.

在本发明中，所述化学修饰核苷选自2-氟-2-脱氧腺苷、2-氟-2-脱氧尿苷、2-氟-2-脱氧胞苷、2-氟-2-脱氧鸟苷、2-氟-2-脱氧-5-甲基胞苷、2-氟-2-脱氧-假尿苷、2-氟-2-脱氧-N1-甲基-假尿苷、2-氟-2-脱氧-N7-甲基-鸟苷、2-氟-2-脱氧-5-甲氧基尿苷、2-氟-2-脱氧-N4-乙酰基胞苷、2-氟-2-脱氧-N6-甲基腺苷、5-甲基胞苷、假尿苷、N1-甲基-假尿苷、N7-甲基-鸟苷、5-甲氧基尿苷、N4-乙酰基胞苷和N6-甲基腺苷中的一种或几种。In the present invention, the chemically modified nucleoside is selected from one or more of 2-fluoro-2-deoxyadenosine, 2-fluoro-2-deoxyuridine, 2-fluoro-2-deoxycytidine, 2-fluoro-2-deoxyguanosine, 2-fluoro-2-deoxy-5-methylcytidine, 2-fluoro-2-deoxy-pseudouridine, 2-fluoro-2-deoxy-N1-methyl-pseudouridine, 2-fluoro-2-deoxy-N7-methyl-guanosine, 2-fluoro-2-deoxy-5-methoxyuridine, 2-fluoro-2-deoxy-N4-acetylcytidine, 2-fluoro-2-deoxy-N6-methyladenosine, 5-methylcytidine, pseudouridine, N1-methyl-pseudouridine, N7-methyl-guanosine, 5-methoxyuridine, N4-acetylcytidine and N6-methyladenosine.

根据本发明的具体实施方案，所述的mRNA分子还具有5'帽结构。According to a specific embodiment of the present invention, the mRNA molecule further has a 5' cap structure.

在本发明中，所述的5'帽结构选自m7G、Cap0、Cap1、Cap2、修饰的Cap0和修饰的Cap1结构中的一种，具体地，可以为Cap0、Cap1、Cap2、ARCA、肌苷、N1-甲基-鸟苷、2'氟-鸟苷、7-脱氮-鸟苷、8-氧代-鸟苷、2-氨基-鸟苷、LNA-鸟苷、2-叠氮基-鸟苷、或专利申请CN202310091020.3或CN202310391954.9所提到的5’帽结构中的一种。In the present invention, the 5' cap structure is selected from one of m7G, Cap0, Cap1, Cap2, modified Cap0 and modified Cap1 structures, specifically, it can be Cap0, Cap1, Cap2, ARCA, inosine, N1-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine, or one of the 5' cap structures mentioned in patent application CN202310091020.3 or CN202310391954.9.

根据本发明的具体实施方式，所述的mRNA分子还具有PolyA。According to a specific embodiment of the present invention, the mRNA molecule further has PolyA.

在本发明中，所述PolyA结构选自CN202211032978.7或CN202311309226.5中所提到的PolyA中的一种。In the present invention, the PolyA structure is selected from one of the PolyAs mentioned in CN202211032978.7 or CN202311309226.5.

本发明第三方面提供了一种美容或药用组合物，其中，所述的美容或药用组合物包含本发明第一方面或第二方面所述的mRNA分子。The third aspect of the present invention provides a cosmetic or medicinal composition, wherein the cosmetic or medicinal composition comprises the mRNA molecule described in the first aspect or the second aspect of the present invention.

根据本发明的具体实施方案，所述的美容或药用组合物还包括医学、美容和化妆品用可接受的基质材料。According to a specific embodiment of the present invention, the cosmetic or pharmaceutical composition further comprises a base material acceptable for medical, cosmetic and cosmetic use.

在本发明中，所述的基质材料包括目前常见的与核糖核酸有着良好相容性的基质材料，不会引发核糖核酸成份变性或降解，可以是天然高分子材料中的一种或多种，也可以是人工合成的高分子材料中的一种或多种，也可以是天然高分子材料与人工合成的高分子材料的混合体系。常见的基质材料包括但不限于淀粉、植物树胶、动物明胶，透明质酸钠，透明质酸，聚乙烯醇、聚乙烯吡咯烷酮，甲基纤维素、乙基纤维素、羧甲基纤维素钠、羟乙基纤维素以及瓜耳胶及其衍生物，聚乙烯醇、聚乙烯吡咯烷酮、丙烯酸聚合物。In the present invention, the matrix material includes currently common matrix materials with good compatibility with RNA, which will not cause the denaturation or degradation of RNA components, and can be one or more natural polymer materials, one or more artificially synthesized polymer materials, or a mixed system of natural polymer materials and artificially synthesized polymer materials. Common matrix materials include but are not limited to starch, plant gum, animal gelatin, sodium hyaluronate, hyaluronic acid, polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, ethyl cellulose, sodium carboxymethyl cellulose, hydroxyethyl cellulose, guar gum and its derivatives, polyvinyl alcohol, polyvinyl pyrrolidone, acrylic polymers.

根据本发明的具体实施方案，所述的美容或药用组合物还包括化妆品学、食品学或药学上可接受的载体。According to a specific embodiment of the present invention, the cosmetic or pharmaceutical composition further comprises a cosmetically, food or pharmaceutically acceptable carrier.

在本发明中，所述载体选自赋形剂、崩解剂、结合剂、润滑剂中的一种或多种。In the present invention, the carrier is selected from one or more of an excipient, a disintegrant, a binder, and a lubricant.

所述赋形剂包括但不限于微晶纤维素、乳糖、低取代羟基纤维素或其组合。The excipients include, but are not limited to, microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof.

所述崩解剂包括但不限于淀粉乙醇酸钠、无水磷酸氢钙或其组合。The disintegrant includes, but is not limited to, sodium starch glycolate, anhydrous calcium hydrogen phosphate or a combination thereof.

所述结合剂包括但不限于聚乙烯吡咯烷酮、低取代羟丙基纤维素、羟丙基纤维素或其组合。The binder includes, but is not limited to, polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose, hydroxypropyl cellulose or a combination thereof.

所述润滑剂包括但不限于硬脂酸镁、二氧化硅、滑石或其组合。The lubricant includes, but is not limited to, magnesium stearate, silicon dioxide, talc, or a combination thereof.

在本发明的一种实施方案中，所述的美容或药用组合物还包括水、甘油、丙二醇、透明质酸钠、虾青素、柠檬酸、熊果苷、PEG-40、矿油、1.4-丁二醇、棕榈酸异丙酯、辛酸/癸酸甘油三酯、聚二甲基硅氧烷、PEG-60失水山梨醇硬脂酸酯、鲸蜡硬脂醇、聚山梨醇酯-60、甘油硬脂酸酯、苯氧乙醇、羟苯甲酯。In one embodiment of the present invention, the cosmetic or pharmaceutical composition further comprises water, glycerin, propylene glycol, sodium hyaluronate, astaxanthin, citric acid, arbutin, PEG-40, mineral oil, 1.4-butylene glycol, isopropyl palmitate, caprylic/capric triglyceride, dimethicone, PEG-60 sorbitan stearate, cetearyl alcohol, polysorbate-60, glyceryl stearate, phenoxyethanol, and methylparaben.

根据本发明的具体实施方案，所述的美容或药用组合物还进一步包括保存剂、稳定剂、表面活性剂、溶剂、保湿剂、润肤剂、紫外线吸收剂、防腐剂、杀菌剂、抗氧化剂、pH调节剂、有机和无机颜料、香料、冷感剂或止汗剂。在不损害本发明的目的和效果的范围内，本领域技术人员可以容易地选择诸如上述保湿剂等的附加成分的混合量。According to a specific embodiment of the present invention, the cosmetic or pharmaceutical composition further comprises a preservative, a stabilizer, a surfactant, a solvent, a moisturizer, an emollient, an ultraviolet absorber, a preservative, a bactericide, an antioxidant, a pH adjuster, an organic and inorganic pigment, a fragrance, a cooling agent or an antiperspirant. Those skilled in the art can easily select the mixing amount of the additional ingredients such as the above-mentioned moisturizer within the scope of not impairing the purpose and effect of the present invention.

根据本发明的具体实施方案，所述的美容或药用组合物是无菌的。According to a particular embodiment of the present invention, the cosmetic or pharmaceutical composition is sterile.

根据本发明的具体实施方案，所述的美容或药用组合物适于局部、经皮、皮下、真皮内、口服、鼻内、气管内、舌下、经颊、经直肠、经阴道、经尿道、吸入、静脉内、动脉内、肌肉内、心内、骨内、腹膜内、经粘膜、玻璃体内、视网膜下、关节内、关节周、局部或皮上施用。According to a specific embodiment of the present invention, the cosmetic or pharmaceutical composition is suitable for topical, transdermal, subcutaneous, intradermal, oral, intranasal, intratracheal, sublingual, buccal, rectal, vaginal, transurethral, inhalation, intravenous, intraarterial, intramuscular, intracardiac, intraosseous, intraperitoneal, transmucosal, intravitreal, subretinal, intraarticular, periarticular, local or epidermal administration.

作为一种优选地实施方式，所述的美容或药用组合物适于真皮内施用和浅表注射。As a preferred embodiment, the cosmetic or pharmaceutical composition is suitable for intradermal administration and superficial injection.

根据本发明的具体实施方案，所述的美容或药用组合物可以制成非口服施用剂型。非口服施用剂型可以是注射剂或皮肤外用剂。皮肤外用剂可以是乳膏、凝胶、软膏、皮肤乳化剂、皮肤悬浮液、经皮传递性贴片、含药绷带、乳液或其组合。According to a specific embodiment of the present invention, the cosmetic or pharmaceutical composition can be prepared into a non-oral dosage form. The non-oral dosage form can be an injection or a skin topical agent. The skin topical agent can be a cream, a gel, an ointment, a skin emulsifier, a skin suspension, a transdermal patch, a medicated bandage, an emulsion or a combination thereof.

所述皮肤外用剂可以根据需要适当混合通常用于化妆品或药物等的皮肤外用剂的成分，例如，水性成分、油性成分、粉末成分、醇类、保湿剂、增稠剂、紫外线吸收剂、美白剂、防腐剂、抗氧化剂、表面活性剂、香料、着色剂以及各种皮肤营养剂等。The skin external preparation can be appropriately mixed with ingredients commonly used in skin external preparations such as cosmetics or medicines, such as aqueous ingredients, oily ingredients, powder ingredients, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, and various skin nutrients, as needed.

所述皮肤外用剂可以适当混合乙二胺四乙酸二钠、乙二胺四乙酸三钠、柠檬酸钠、多聚磷酸钠、偏磷酸钠、葡萄糖酸等的金属螯合剂、咖啡因、单宁、戊脉安、甘草酸、氨甲环酸及其衍生物或其盐等药剂、维生素C、抗坏血酸磷酸酯镁、抗坏血酸葡糖苷、熊果苷、曲酸、葡萄糖、果糖、海藻糖等的糖类。The skin external preparation can be appropriately mixed with metal chelating agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconic acid, etc., caffeine, tannin, verapamil, glycyrrhizic acid, tranexamic acid and its derivatives or salts thereof, vitamin C, magnesium ascorbyl phosphate, ascorbyl glucoside, arbutin, kojic acid, glucose, fructose, trehalose, etc.

作为一种优选地实施方式，所述的美容或药用组合物为皮肤护理产品。As a preferred embodiment, the cosmetic or medicinal composition is a skin care product.

根据本发明的具体实施方案，所述的美容或药用组合物还包括以下剂型：化妆水(润肤液)、柔肤水、爽肤水、收敛剂(Astringent)、乳液、牛奶润肤露(milk lotion)、保湿乳液、营养液、按摩霜、营养霜、保湿霜、手霜、粉底、精华、营养精华、膜、肥皂、洁面泡沫、洁面乳、洁面霜、身体乳液、身体清洁液、悬浮液、凝胶、粉末、粘膏(paste)、面膜或片装面膜或喷雾组合物。According to a specific embodiment of the present invention, the cosmetic or pharmaceutical composition also includes the following dosage forms: lotion, skin softener, toner, astringent, emulsion, milk lotion, moisturizing lotion, nutrient solution, massage cream, nutrient cream, moisturizing cream, hand cream, foundation, essence, nutrient essence, film, soap, cleansing foam, cleansing milk, cleansing cream, body lotion, body cleansing liquid, suspension, gel, powder, paste, facial mask or sheet mask or spray composition.

作为一种优选地实施方式，本发明所述的美容或药用组合物为用于射频导入仪、负压导入仪、微晶导入仪、微电流导入仪、微针导入仪、或注射器导入皮肤底层的制剂。As a preferred embodiment, the cosmetic or medicinal composition of the present invention is a preparation for introduction into the underlying layer of the skin via a radio frequency introduction device, a negative pressure introduction device, a microcrystal introduction device, a microcurrent introduction device, a microneedle introduction device, or a syringe.

本发明第四方面提供了一种DNA分子，其能用于转录得到本发明第一方面或第二方面所述的mRNA分子。The fourth aspect of the present invention provides a DNA molecule which can be used to transcribe the mRNA molecule described in the first aspect or the second aspect of the present invention.

根据本发明的具体实施方案，所述的DNA分子还包括启动子、3’端连接有UTR序列和PolyA片段，其5’端连接有UTR序列和Cap。According to a specific embodiment of the present invention, the DNA molecule further comprises a promoter, a UTR sequence and a PolyA fragment connected to its 3' end, and a UTR sequence and a Cap connected to its 5' end.

本发明第五方面提供了一种重组质粒或重组细胞，其包含本发明第四方面所述的DNA分子。The fifth aspect of the present invention provides a recombinant plasmid or a recombinant cell, which comprises the DNA molecule described in the fourth aspect of the present invention.

根据本发明的具体实施方案，所述的重组质粒的载体为表达载体。According to a specific embodiment of the present invention, the vector of the recombinant plasmid is an expression vector.

根据本发明的具体实施方案，所述的重组质粒或重组细胞能用于转录得到本发明第一方面或第二方面所述的mRNA。According to a specific embodiment of the present invention, the recombinant plasmid or recombinant cell can be used to transcribe the mRNA described in the first aspect or the second aspect of the present invention.

本发明第六方面提供了一种试剂盒，其包含本发明第一方面或第二方面所述的mRNA分子、本发明第三方面所述的美容或药用组合物、本发明第四方面所述的DNA分子、或本发明第五方面所述的重组质粒或重组细胞。The sixth aspect of the present invention provides a kit comprising the mRNA molecule described in the first or second aspect of the present invention, the cosmetic or pharmaceutical composition described in the third aspect of the present invention, the DNA molecule described in the fourth aspect of the present invention, or the recombinant plasmid or recombinant cell described in the fifth aspect of the present invention.

根据本发明的具体实施方式，所述的试剂盒为用于制备本发明第一方面或第二方面所述的mRNA分子的试剂盒。所述的用于制备本发明第一方面或第二方面所述的mRNA分子的试剂盒包含本发明第三方面所述的DNA分子、或本发明第四方面所述的重组质粒或重组细胞。According to a specific embodiment of the present invention, the kit is a kit for preparing the mRNA molecule described in the first aspect or the second aspect of the present invention. The kit for preparing the mRNA molecule described in the first aspect or the second aspect of the present invention comprises the DNA molecule described in the third aspect of the present invention, or the recombinant plasmid or recombinant cell described in the fourth aspect of the present invention.

根据本发明的具体实施方式，所述的试剂盒为用于皮肤护理或改善皮肤状态的试剂盒。所述的用于皮肤护理或改善皮肤状态的试剂盒包含本发明第一方面或第二方面所述的mRNA分子或本发明第三方面所述的美容或药用组合物。According to a specific embodiment of the present invention, the kit is a kit for skin care or skin condition improvement. The kit for skin care or skin condition improvement comprises the mRNA molecule described in the first aspect or the second aspect of the present invention or the cosmetic or pharmaceutical composition described in the third aspect of the present invention.

作为一种优选地实施方式，本发明所述的用于皮肤护理或改善皮肤状态的试剂盒还包含辅助导入设备。As a preferred embodiment, the kit for skin care or skin condition improvement of the present invention further comprises an auxiliary introduction device.

在本发明中，辅助导入设备促进mRNA分子通过皮肤进入皮肤底层，达到有效递送的目的；其中所述的辅助导入设备根据其作用机制不同主要分为射频导入仪、负压导入仪、微晶导入仪、微电流导入仪、微针导入仪、注射器。In the present invention, the auxiliary introduction device promotes the mRNA molecules to pass through the skin into the underlying skin layer, thereby achieving the purpose of effective delivery; wherein the auxiliary introduction device is mainly divided into a radio frequency introduction device, a negative pressure introduction device, a microcrystal introduction device, a microcurrent introduction device, a microneedle introduction device, and a syringe according to its different mechanisms of action.

作为一种优选的实施方式，所述的辅助导入设备包括负压导入仪、微电流导入仪、微针导入仪。更为优选的，所述的辅助导入设备为微针导入仪。As a preferred embodiment, the auxiliary introduction device includes a negative pressure introduction device, a microcurrent introduction device, and a microneedle introduction device. More preferably, the auxiliary introduction device is a microneedle introduction device.

作为一种优选的实施方式，所述的辅助导入设备为注射器。As a preferred embodiment, the auxiliary introduction device is a syringe.

本发明第七方面提供了一种制备本发明第一方面或第二方面所述的mRNA分子的方法，其包括：The seventh aspect of the present invention provides a method for preparing the mRNA molecule according to the first aspect or the second aspect of the present invention, comprising:

(1)将本发明第四方面所述的DNA分子克隆至表达质粒获得重组质粒；(1) cloning the DNA molecule described in the fourth aspect of the present invention into an expression plasmid to obtain a recombinant plasmid;

(2)将所述重组质粒转入宿主细胞获得重组细胞，从扩增后的重组细胞中提取质粒，以提取获得的质粒为模板进行PCR扩增获得体外表达mRNA的DNA模板；(2) transferring the recombinant plasmid into a host cell to obtain a recombinant cell, extracting the plasmid from the amplified recombinant cell, and performing PCR amplification using the extracted plasmid as a template to obtain a DNA template for in vitro expression of mRNA;

(3)构建包括所述DNA模板的RNA体外合成体系进行mRNA的体外合成获得所述的mRNA分子。(3) constructing an RNA in vitro synthesis system including the DNA template to perform in vitro synthesis of mRNA to obtain the mRNA molecule.

在本发明中，当所述的mRNA分子为包含编码I型胶原蛋白α1链的mRNA序列的mRNA分子和包含编码I型胶原蛋白α2链的mRNA序列的mRNA分子的混合物时，将上述获得的mRNA分子按比例混合即可得到该混合物。In the present invention, when the mRNA molecule is a mixture of an mRNA molecule containing an mRNA sequence encoding type I collagen α1 chain and an mRNA molecule containing an mRNA sequence encoding type I collagen α2 chain, the mixture can be obtained by mixing the mRNA molecules obtained above in proportion.

在本发明中，合成转录所述的mRNA的DNA片段，并将所述DNA片段克隆至表达质粒获得重组质粒。本发明对合成所述的mRNA对应的DNA片段的方法没有特殊限定，采用本领域常规的DNA合成方法即可，在本发明具体实施过程中，优选的委托生物技术公司合成。In the present invention, a DNA fragment of the mRNA is synthesized and transcribed, and the DNA fragment is cloned into an expression plasmid to obtain a recombinant plasmid. The present invention does not specifically limit the method for synthesizing the DNA fragment corresponding to the mRNA, and a conventional DNA synthesis method in the art can be used. In the specific implementation of the present invention, it is preferably commissioned to a biotechnology company for synthesis.

在本发明中，优选的通过酶切连接的方法将所述DNA片段克隆至表达质粒中；在本发明中，所述DNA片段优选的通过BamHI和NheI酶进行双酶切获得酶切DNA片段；所述表达质粒优选的通过BamHI和NheI酶进行双酶切后获得酶切质粒；然后将所述酶切DNA片段和酶切质粒连接获得重组质粒。In the present invention, the DNA fragment is preferably cloned into the expression plasmid by enzyme digestion and ligation; in the present invention, the DNA fragment is preferably double digested by BamHI and NheI enzymes to obtain a digested DNA fragment; the expression plasmid is preferably double digested by BamHI and NheI enzymes to obtain a digested plasmid; and then the digested DNA fragment and the digested plasmid are ligated to obtain a recombinant plasmid.

本发明在获得所述重组质粒后，将所述重组质粒转入宿主细胞获得重组细胞，从扩繁后的重组细胞中提取质粒，以提取获得的质粒为模板进行PCR扩增获得体外表达mRNA的DNA模板。在本发明中，所述宿主细胞优选为大肠杆菌感受态细胞；After obtaining the recombinant plasmid, the present invention transfers the recombinant plasmid into a host cell to obtain a recombinant cell, extracts the plasmid from the expanded recombinant cell, and uses the extracted plasmid as a template to perform PCR amplification to obtain a DNA template for in vitro expression of mRNA. In the present invention, the host cell is preferably an Escherichia coli competent cell;

本发明在获得重组细胞后，优选的进行阳性重组细胞的筛选和菌落测序。在本发明中，所述阳性重组细胞的筛选优选的在amp抗性的固体培养基上进行。在本发明中，挑选所述amp抗性的固体培养基上的单菌落进行菌落PCR，选取菌落PCR结果含目的条带的菌落进行测序。本发明对所述菌落PCR的具体步骤没有特殊限定，采用本领域常规的菌落PCR步骤即可。After obtaining the recombinant cells, the present invention preferably performs screening of positive recombinant cells and colony sequencing. In the present invention, the screening of the positive recombinant cells is preferably performed on an amp-resistant solid culture medium. In the present invention, a single colony on the amp-resistant solid culture medium is selected for colony PCR, and a colony containing a target band in the colony PCR result is selected for sequencing. The present invention does not specifically limit the specific steps of the colony PCR, and the conventional colony PCR steps in the art can be used.

在本发明中，提取测序正确的重组细胞的质粒；本发明对所述质粒的提取方法没有特殊限定，优选的采用质粒提取试剂盒进行。在本发明中，以提取获得的质粒为模板进行PCR扩增获得体外表达mRNA的DNA模板。所述DNA模板的浓度优选为1ng/μl。在本发明中，所述PCR的扩增程序优选的如下：预变性98℃3分钟；变性98℃10秒，退火60℃5秒，延伸72℃2分钟，34个循环；最后延伸72℃，10分钟。In the present invention, the plasmid of the correctly sequenced recombinant cell is extracted; the present invention does not specifically limit the method for extracting the plasmid, and preferably a plasmid extraction kit is used. In the present invention, the extracted plasmid is used as a template for PCR amplification to obtain a DNA template for in vitro expression of mRNA. The concentration of the DNA template is preferably 1 ng/μl. In the present invention, the PCR amplification procedure is preferably as follows: pre-denaturation at 98°C for 3 minutes; denaturation at 98°C for 10 seconds, annealing at 60°C for 5 seconds, extension at 72°C for 2 minutes, 34 cycles; final extension at 72°C for 10 minutes.

在本发明中，所述PCR扩增反应结束后，优选的对扩增产物进行琼脂糖凝胶电泳检测以确定反应是否成功；所述琼脂糖凝胶电泳检测的参数优选的如下：1.5％琼脂糖，5V/min，40分钟。在本发明中，琼脂糖凝胶电泳出现目的大小的条带认为是反应成功。In the present invention, after the PCR amplification reaction is completed, the amplification product is preferably subjected to agarose gel electrophoresis to determine whether the reaction is successful; the parameters of the agarose gel electrophoresis detection are preferably as follows: 1.5% agarose, 5V/min, 40 minutes. In the present invention, the appearance of a band of the target size in agarose gel electrophoresis is considered to be a successful reaction.

本发明在所述PCR扩增反应结束后，优选的将所述扩增产物进行浓缩和纯化。在本发明中，所述浓缩优选的采用Millipore 30Kd超滤管进行；所述纯化优选的采用FPLC进行；本发明在所述纯化后优选的采用Nano Drop检测纯化后模板的浓度，以及260/280、260/230的比值。优选260/280范围为1.8-2.1，260/230范围为大于2.0。After the PCR amplification reaction is completed, the amplification product is preferably concentrated and purified. In the present invention, the concentration is preferably performed using a Millipore 30Kd ultrafiltration tube; the purification is preferably performed using FPLC; after the purification, the present invention preferably uses Nano Drop to detect the concentration of the purified template, as well as the ratios of 260/280 and 260/230. Preferably, the range of 260/280 is 1.8-2.1, and the range of 260/230 is greater than 2.0.

本发明在获得所述DNA模板后，构建包括所述DNA模板的RNA体外合成体系进行mRNA的体外合成获得所述活性成分mRNA。在本发明中，所述RNA体外合成体系以1600μl计，包括以下组分：
After obtaining the DNA template, the present invention constructs an RNA in vitro synthesis system including the DNA template to perform in vitro synthesis of mRNA to obtain the active ingredient mRNA. In the present invention, the RNA in vitro synthesis system, calculated in 1600 μl, includes the following components:

在本发明中，所述RNA体外合成的条件优选的为36～38℃，8～12小时，更优选为37℃，10小时。在本发明中，所述RNA体外合成优选的在恒温反应器中进行；所述RNA体外合成体系优选的置于2ml RNase-free Tube管中，一次同时反应多管；所述RNA体外合成体系中的反应试剂按照上述顺序添加。In the present invention, the conditions for the in vitro synthesis of RNA are preferably 36-38°C, 8-12 hours, more preferably 37°C, 10 hours. In the present invention, the in vitro synthesis of RNA is preferably carried out in a constant temperature reactor; the in vitro synthesis system of RNA is preferably placed in a 2ml RNase-free Tube tube, and multiple tubes are reacted at the same time; the reaction reagents in the in vitro synthesis system of RNA are added in the above order.

本发明在所述RNA体外合成结束后，优选的还包括去除DNA模板、回收mRNA和纯化mRNA的步骤。在本发明中，所述去除DNA模板优选的通过DNase I消化实现；所述消化优选的包括将DNase I与RNA体外合成反应后的溶液混合后进行；所述DNase I与RNA体外合成反应后的溶液的体积比优选为3:40；所述混合优选的通过上下颠倒所述RNase-free Tube管实现，所述上下颠倒的次数优选为8～12次，更优选为10次；本发明在所述混合后，优选的进行离心将溶液收集至RNase-free Tube管底部。在本发明中，所述离心的转速优选为800～1200rpm，更优选为1000rpm；所述离心的时间优选为8～12秒，更优选为10秒。所述消化的温度优选为37℃；所述消化的时间优选为1小时。After the in vitro synthesis of RNA is completed, the present invention preferably also includes the steps of removing the DNA template, recovering mRNA and purifying mRNA. In the present invention, the removal of the DNA template is preferably achieved by DNase I digestion; the digestion preferably includes mixing DNase I with the solution after the RNA in vitro synthesis reaction; the volume ratio of the DNase I to the solution after the RNA in vitro synthesis reaction is preferably 3:40; the mixing is preferably achieved by turning the RNase-free Tube upside down, and the number of upside downs is preferably 8 to 12 times, more preferably 10 times; after the mixing, the present invention preferably performs centrifugation to collect the solution to the bottom of the RNase-free Tube. In the present invention, the speed of the centrifugation is preferably 800 to 1200 rpm, more preferably 1000 rpm; the time of the centrifugation is preferably 8 to 12 seconds, more preferably 10 seconds. The temperature of the digestion is preferably 37°C; the time of the digestion is preferably 1 hour.

本发明在所述消化结束后，优选的进行DNA片段残留检测。在本发明中，所述回收mRNA优选的通过将所述醋酸铵溶液沉淀实现；具体实施方法参见实施例记载；本发明在回收mRNA后，进行mRNA的质量检测；所述质量检测包括mRNA的浓度、mRNA的260/280、260/230的比值，纯mRNA的A260/A280的值为2.0～2.1，A260/A230范围为1.8～2.2。在本发明中，所述纯化mRNA通过HPLC纯化实现。本发明所述纯化mRNA后，优选的对纯化后的mRNA进行分装。The present invention preferably performs residual DNA fragment detection after the digestion is completed. In the present invention, the recovery of mRNA is preferably achieved by precipitation with the ammonium acetate solution; the specific implementation method is described in the examples; after the mRNA is recovered, the present invention performs a quality test on the mRNA; the quality test includes the concentration of the mRNA, the ratios of 260/280 and 260/230 of the mRNA, and the A260/A280 value of the pure mRNA is 2.0 to 2.1, and the A260/A230 range is 1.8 to 2.2. In the present invention, the purification of mRNA is achieved by HPLC purification. After the mRNA is purified according to the present invention, the purified mRNA is preferably packaged.

本发明第八方面提供了一种美容方法，其包括向皮肤施用本发明第一方面或第二方面所述的mRNA分子、或本发明第三方面所述的美容或药用组合物。The eighth aspect of the present invention provides a cosmetic method, which comprises applying the mRNA molecule described in the first aspect or the second aspect of the present invention, or the cosmetic or pharmaceutical composition described in the third aspect of the present invention to the skin.

根据本发明的具体实施方案，所述的方法还包括借由辅助导入设备促进mRNA分子通过皮肤进入皮肤底层，达到有效递送的目的；其中所述的辅助导入设备根据其作用机制不同主要分为射频导入仪、负压导入仪、微晶导入仪、微针导入仪、注射器。According to a specific embodiment of the present invention, the method further includes promoting the mRNA molecules to pass through the skin into the underlying skin by means of an auxiliary introduction device to achieve effective delivery; wherein the auxiliary introduction device is mainly divided into a radio frequency introduction device, a negative pressure introduction device, a microcrystal introduction device, a microneedle introduction device, and a syringe according to its different mechanisms of action.

作为一种优选的实施方式，所述的辅助导入设备包括负压导入仪、微电流导入仪、微针导入仪。更为优选地，所述的辅助导入设备为微针导入仪。As a preferred embodiment, the auxiliary introduction device includes a negative pressure introduction device, a microcurrent introduction device, and a microneedle introduction device. More preferably, the auxiliary introduction device is a microneedle introduction device.

作为一种优选的实施方式，所述的辅助导入设备为注射器。As a preferred embodiment, the auxiliary introduction device is a syringe.

根据本发明的具体实施方式，所述的mRNA分子的工作浓度为0.001ng/ml-10mg/ml。According to a specific embodiment of the present invention, the working concentration of the mRNA molecule is 0.001 ng/ml-10 mg/ml.

作为一种优选的实施方式，所述的mRNA分子的工作浓度为0.01μg/ml-1.0mg/ml。As a preferred embodiment, the working concentration of the mRNA molecule is 0.01 μg/ml-1.0 mg/ml.

作为一种更为优选的实施方式，所述的mRNA分子的工作浓度为0.01-1.0μg/ml。As a more preferred embodiment, the working concentration of the mRNA molecule is 0.01-1.0 μg/ml.

作为一种优选地实施方式，所述的mRNA分子的工作浓度为1.0mg/ml。As a preferred embodiment, the working concentration of the mRNA molecule is 1.0 mg/ml.

作为一种更为优选的实施方式，所述的mRNA分子的工作浓度为0.01μg/ml。As a more preferred embodiment, the working concentration of the mRNA molecule is 0.01 μg/ml.

根据本发明的具体实施方案，所述的美容方法有助于改善皮肤皱纹，所针对的皮肤皱纹是指浅表凹陷所形成的鼻唇皱褶、鱼尾纹、皱眉纹、烦恼纹、疤痕、眉间纹、眉下垂、泪槽、鼻颊纹、兔宝宝纹、面颊/面中部下垂、木偶纹、罂粟陷窝、微笑纹、笑纹、下巴皱痕、颈纹、颈阔肌带以及它们的任何组合。According to a specific embodiment of the present invention, the cosmetic method helps to improve skin wrinkles, and the targeted skin wrinkles refer to nasolabial folds, crow's feet, frown lines, worry lines, scars, glabellar lines, drooping eyebrows, tear troughs, nasal cheek lines, bunny lines, cheek/mid-face sagging, marionette lines, poppy pits, smile lines, laugh lines, chin wrinkles, neck lines, platysma bands and any combination thereof formed by superficial depressions.

本发明第九方面提供了本发明第一方面或第二方面所述的mRNA分子、本发明第三方面所述的美容或药用组合物、本发明第四方面所述的DNA分子、本发明第五方面所述的重组质粒或重组细胞、或本发明第六方面所述的试剂盒在制备用于皮肤护理或改善皮肤状态的产品中的应用。The ninth aspect of the present invention provides the use of the mRNA molecule described in the first or second aspect of the present invention, the cosmetic or pharmaceutical composition described in the third aspect of the present invention, the DNA molecule described in the fourth aspect of the present invention, the recombinant plasmid or recombinant cell described in the fifth aspect of the present invention, or the kit described in the sixth aspect of the present invention in the preparation of a product for skin care or improving skin condition.

根据本发明的具体实施方案，所述的产品为药物、化妆品或试剂盒。According to a specific embodiment of the present invention, the product is a medicine, a cosmetic or a kit.

根据本发明的具体实施方案，所述的改善皮肤状态为促进胶原蛋白的表达。According to a specific embodiment of the present invention, the improvement of skin condition is to promote the expression of collagen.

根据本发明的具体实施方案，所述的改善皮肤状态为刺激胶原纤维增生。According to a specific embodiment of the present invention, the improvement of skin condition is to stimulate the proliferation of collagen fibers.

有益效果：Beneficial effects:

本发明提供了一种mRNA分子及其用途，所述mRNA分子包含编码胶原蛋白的核酸序列。本发明构建的mRNA序列在很大程度上避免了在哺乳动物细胞中的序列非依赖性凋亡，显示出更好的血清稳定性和提高的体内活性。本发明还涉及医学、美容和化妆品组合物的应用，利用脂质体或者其他基因导入技术，将组合物导入皮肤细胞或被皮肤细胞吸收后通过mRNA翻译蛋白质的生物学机理，使得皮肤细胞产生胶原蛋白，通过使皮肤组织中胶原蛋白含量的上调从而将老化皮肤正常化，本发明提供的美容组合物制剂制备方法简单、快速、活性成分表达量高，结合本发明中的药物导入方法能够增强mRNA制剂导入皮肤的效率，提高有效制剂的利用率，从而达到更有效的皮肤透皮递送的目的，达到皮肤修护的效果。The present invention provides an mRNA molecule and its use, wherein the mRNA molecule comprises a nucleic acid sequence encoding collagen. The mRNA sequence constructed by the present invention largely avoids sequence-independent apoptosis in mammalian cells, and shows better serum stability and improved in vivo activity. The present invention also relates to the application of medical, beauty and cosmetic compositions, using liposomes or other gene introduction technologies, the composition is introduced into skin cells or absorbed by skin cells, and then the biological mechanism of mRNA translation of protein is used to make skin cells produce collagen, and the collagen content in skin tissue is increased to normalize aging skin. The preparation method of the cosmetic composition provided by the present invention is simple, fast, and has a high expression of active ingredients. Combined with the drug introduction method in the present invention, the efficiency of mRNA preparation introduction into the skin can be enhanced, and the utilization rate of effective preparations can be improved, thereby achieving the purpose of more effective skin transdermal delivery and achieving the effect of skin repair.

本发明提供的mRNA剂型的化妆品制剂，包括具有刺激胶原蛋白再生的mRNA，将所述制剂导入皮肤细胞后能够促进成纤维细胞中的胶原蛋白的表达，弥补了因衰老、光照、水分流失等造成的皮肤褶皱等情况，具体实施例中数据展示了有效性数据；根据实施例的记载，本发明提供的具有刺激胶原蛋白生长因子mRNA能够在细胞内特异性的高水平表达；本发明在使用过程中结合辅助设备如射频导入仪、负压导入仪、微晶导入仪、微电流导入仪等突破皮肤吸收屏障激活自体胶原蛋白表达，解决其他护肤品不易透过表皮的现象，刺激胶原纤维增生、保障皮肤组织完整性，使皮肤紧致、细纹淡化、美白补水、淡化色素，使皮肤白晳透亮，而且有自体细胞产生胶原蛋白，不会刺激皮肤，因此可安全使用。The mRNA dosage form cosmetic preparation provided by the present invention includes mRNA that stimulates collagen regeneration. After the preparation is introduced into skin cells, it can promote the expression of collagen in fibroblasts, making up for skin wrinkles and the like caused by aging, light exposure, water loss, etc. The data in the specific embodiments show the effectiveness data; according to the records of the embodiments, the mRNA that stimulates collagen growth factor provided by the present invention can be expressed at a high level specifically in cells; during use, the present invention is combined with auxiliary equipment such as a radio frequency introduction instrument, a negative pressure introduction instrument, a microcrystal introduction instrument, a microcurrent introduction instrument, etc. to break through the skin absorption barrier and activate the expression of autologous collagen, solve the problem that other skin care products are not easy to penetrate the epidermis, stimulate collagen fiber proliferation, ensure the integrity of skin tissue, make the skin firm, fade fine lines, whiten and moisturize, fade pigments, make the skin white and translucent, and have autologous cells to produce collagen, will not irritate the skin, so it can be used safely.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

图1是为编码刺激胶原蛋白生长因子mRNA的结构示意图；FIG1 is a schematic diagram of the structure of mRNA encoding collagen growth factor;

图2是SEQ ID No.1-18不同序列单独转染细胞和组合转染细胞后胶原蛋白含量对比结果；Figure 2 is a comparison of the collagen content of cells transfected with different sequences of SEQ ID No. 1-18 alone and in combination;

图3是细胞转染mRNA后细胞水平胶原蛋白含量定量结果；FIG3 is the quantitative result of collagen content at the cellular level after cells were transfected with mRNA;

图4是细胞转染mRNA后细胞胶原蛋白含量上调率对比结果；Figure 4 is a comparison of the up-regulation rates of collagen content in cells after transfection of mRNA;

图5是使用ELISA检测方法测得3D皮肤试验mRNA进入皮肤后，皮肤中I型胶原蛋白含量变化；FIG5 is a graph showing changes in the content of type I collagen in the skin after 3D skin test mRNA enters the skin using the ELISA detection method;

图6是使用ELISA检测方法测得3D皮肤试验mRNA进入皮肤后，皮肤中I型胶原蛋白上调率对比结果；FIG6 is a comparison of the up-regulation rate of type I collagen in the skin after the 3D skin test mRNA enters the skin using the ELISA detection method;

图7是使用免疫荧光法测得3D皮肤试验mRNA进入皮肤后，皮肤切片中I型胶原蛋白免疫荧光结果；FIG7 is the immunofluorescence result of type I collagen in skin sections after 3D skin test mRNA enters the skin using immunofluorescence method;

图8是使用免疫荧光法测得3D皮肤试验mRNA进入皮肤后，皮肤切片中I型胶原蛋白免疫荧光强度上调对比结果；FIG8 is a comparison of the results of the upregulation of the immunofluorescence intensity of type I collagen in skin sections after the 3D skin test mRNA entered the skin using the immunofluorescence method;

图9为结合辅助导入仪器各实验组小鼠皮肤切片，观察荧光蛋白GFP mRNA在皮肤内表达情况的结果；Figure 9 shows the results of observing the expression of fluorescent protein GFP mRNA in the skin of mice in each experimental group using the auxiliary introduction instrument;

图10为大鼠皮肤切片和masson染色结果，观察I型胶原表达情况；Figure 10 shows rat skin sections and Masson staining results, observing the expression of type I collagen;

图11为各组大鼠皮肤切片真皮层图像测绘厚度，以此体现mRNA制剂对I型胶原生产的促进作用。FIG11 is an image of the dermis layer thickness of skin sections of rats in each group, which reflects the promoting effect of the mRNA preparation on the production of type I collagen.

具体实施方式DETAILED DESCRIPTION

在进一步描述本发明具体实施方案之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention.

当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规技术。Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the technical field.

实施例1、mRNA分子的制备Example 1. Preparation of mRNA molecules

本实施例提供一种mRNA分子，其是由mRNA分子与3’端的PolyA连接而形成。其中所述mRNA分子的序列包含5’帽结构、5'UTR、目的基因序列、3'UTR和PolyA。This embodiment provides an mRNA molecule, which is formed by connecting an mRNA molecule to a 3' end Poly A. The sequence of the mRNA molecule comprises a 5' cap structure, a 5' UTR, a target gene sequence, a 3' UTR and Poly A.

如图1所示，所述编码胶原蛋白的mRNA分子采用以下步骤制备得到：As shown in FIG1 , the mRNA molecule encoding collagen is prepared by the following steps:

步骤S1，设计并合成一段带有启动子序列、目的基因序列的质粒载体，质粒载体是公司载体，各部分连接顺序是5’UTR-胶原蛋白基因的核苷酸序列-3’UTR；Step S1, design and synthesize a plasmid vector with a promoter sequence and a target gene sequence. The plasmid vector is a company vector, and the connection order of each part is 5'UTR-collagen gene nucleotide sequence-3'UTR;

步骤S2，以步骤S1的质粒载体为模板进行体外转录，得到mRNA分子，所述mRNA分子的序列包含5’帽结构、5‘UTR、目的基因序列、3'UTR和plolyA；Step S2, using the plasmid vector of step S1 as a template for in vitro transcription to obtain an mRNA molecule, wherein the sequence of the mRNA molecule comprises a 5' cap structure, a 5' UTR, a target gene sequence, a 3' UTR and plolyA;

mRNA分子合成方式按照一步法合成，除帽子结构外，其余元件皆在质粒构建时集成到DNA模板中。The mRNA molecule is synthesized in a one-step process. Except for the cap structure, all other elements are integrated into the DNA template during plasmid construction.

本实施例中，所述胶原蛋白基因的核苷酸序列如SEQ ID No.1～18所示。In this embodiment, the nucleotide sequence of the collagen gene is shown in SEQ ID No.1~18.

5’帽结构为m7G(5’)ppp(5’)(2’OMeA)pGp。The 5’ cap structure is m7G(5’)ppp(5’)(2’OMeA)pGp.

5’UTR序列如SEQ ID No.19所示。The 5’UTR sequence is shown as SEQ ID No.19.

3’UTR序列如SEQ ID No.20所示。The 3’UTR sequence is shown as SEQ ID No.20.

polyA序列如SEQ ID No.21所示。The polyA sequence is shown as SEQ ID No.21.

本实施例中，步骤S2的mRNA分子的具体合成方法如下：In this embodiment, the specific method for synthesizing the mRNA molecule in step S2 is as follows:

1、构建好的表达质粒按如下反应体系进行DNA模板的扩增：1. Amplify the DNA template using the constructed expression plasmid according to the following reaction system:

反应体积，50μl(为单个管的反应体积，一次同时反应多管)；所述PCR扩增的体系(50μl)：Prime STAR Max Premix(2×)25μl、引物F10μmol/L 1.2μl、引物R10μmol/L 1.2μl、DNA模板(1ng/μl)1μl和水21.6μl。所述PCR的扩增程序如下：预变性98℃3min；变性98℃10s，退火60℃5s，延伸72℃2min，34个循环；最后延伸72℃，10min。反应结束后，将反应液合并于1.5ml Tube管中。取10μl进行DNA琼脂糖凝胶电泳(1.5％琼脂糖，5V/min，40min)。根据电泳目的条带的大小对反应成功与否进行确认。Reaction volume, 50μl (reaction volume of a single tube, multiple tubes reacted at the same time); the PCR amplification system (50μl): Prime STAR Max Premix (2×) 25μl, primer F 10μmol/L 1.2μl, primer R 10μmol/L 1.2μl, DNA template (1ng/μl) 1μl and water 21.6μl. The PCR amplification program is as follows: pre-denaturation 98℃ 3min; denaturation 98℃ 10s, annealing 60℃ 5s, extension 72℃ 2min, 34 cycles; final extension 72℃, 10min. After the reaction is completed, the reaction solution is combined in a 1.5ml Tube tube. Take 10μl for DNA agarose gel electrophoresis (1.5% agarose, 5V/min, 40min). Confirm the success of the reaction based on the size of the electrophoresis target band.

合格标准：电泳检测出现单一的条带，且大小正确。Qualification criteria: A single band appears in electrophoresis detection and the size is correct.

2、DNA模板超滤2. DNA template ultrafiltration

利用Millipore 30Kd超滤管浓缩上述获得的DNA模板。The DNA template obtained above was concentrated using Millipore 30Kd ultrafiltration tube.

3、DNA模板FPLC纯化3. FPLC purification of DNA template

将上述超滤得到的DNA，加入等体积的苯酚/氯仿/异戊醇混合液(苯酚/氯仿/异戊醇＝25/24/1)，充分震荡后，12000g离心15min。去掉沉淀，转移上清至新的离心管中，加入上清体积的1/10 3M NaAc(PH5.2)，混匀，然后加入2倍体积的无水乙醇，混匀，至于-20℃静置30min。4℃，12000g离心10min，弃上清。用70％乙醇洗涤沉淀，12000g离心5min，取上清，于超净台晾干5min。用适当的RNase-free水溶解纯化后的DNA模板。用NanoDrop检测纯化后模板的浓度，以及260/280、260/230的比值。取样进行DNA琼脂糖凝胶电泳检测(1.5％琼脂糖，5V/min，40min)。合格标准：260/280介于1.8至2.1之间，260/230在1.6至2.2之间。Add an equal volume of phenol/chloroform/isoamyl alcohol mixture (phenol/chloroform/isoamyl alcohol = 25/24/1) to the DNA obtained by ultrafiltration, shake thoroughly, and centrifuge at 12000g for 15min. Remove the precipitate, transfer the supernatant to a new centrifuge tube, add 1/10 of the supernatant volume of 3M NaAc (PH5.2), mix well, then add 2 times the volume of anhydrous ethanol, mix well, and let stand at -20℃ for 30min. Centrifuge at 12000g for 10min at 4℃, and discard the supernatant. Wash the precipitate with 70% ethanol, centrifuge at 12000g for 5min, take the supernatant, and dry it on the clean bench for 5min. Dissolve the purified DNA template with appropriate RNase-free water. Use NanoDrop to detect the concentration of the purified template and the ratios of 260/280 and 260/230. Take samples for DNA agarose gel electrophoresis (1.5% agarose, 5V/min, 40min). Pass criteria: 260/280 between 1.8 and 2.1, 260/230 between 1.6 and 2.2.

4、FPLC纯化后模板超滤4. Template ultrafiltration after FPLC purification

Millipore 30Kd超滤管浓缩FPLC纯化后的DNA模板，用RNase-free水洗脱溶解。用NanoDrop检测超滤后模板的浓度，以及260/280、260/230的比值。最终用RNase-free水稀释至150ng/μl。The DNA template purified by FPLC was concentrated by Millipore 30Kd ultrafiltration tube and eluted with RNase-free water. The concentration of the template after ultrafiltration and the ratios of 260/280 and 260/230 were detected by NanoDrop. Finally, the template was diluted to 150ng/μl with RNase-free water.

5、mRNA的体外合成5. In vitro synthesis of mRNA

在恒温反应器中，进行mRNA的体外合成。按照如下合成体系进行(反应试剂按照从上至下添加)：In a thermostatic reactor, in vitro synthesis of mRNA was performed according to the following synthesis system (reaction reagents were added from top to bottom):

反应体积，1600μl(置于2ml RNase-free Tube管中，为单个管的反应体积，一次同时反应多管)：RNA-free水440μl、7.5mM的ATP 160μl、7.5mM的UTP 160μl、7.5mM的CTP 160μl、7.5mM的GTP 160μl、7.5mM的M7G(2’OMeA)pG 160μl、150ng/μl的DNA模板40μl、10×Buffer 160μl和Enzyme Mix 160μl。所述RNA体外合成的程序为37℃，10h。Reaction volume, 1600μl (placed in a 2ml RNase-free Tube, the reaction volume of a single tube, multiple tubes reacted at the same time): RNA-free water 440μl, 7.5mM ATP 160μl, 7.5mM UTP 160μl, 7.5mM CTP 160μl, 7.5mM GTP 160μl, 7.5mM M7G (2'OMeA) pG 160μl, 150ng/μl DNA template 40μl, 10× Buffer 160μl and Enzyme Mix 160μl. The RNA in vitro synthesis procedure is 37°C, 10h.

6、DNase I消化去除DNA模板6. DNase I digestion to remove DNA template

向mRNA体外合成后的每个Tube管中各加入120μl DNase I。上下颠倒10次混匀，1000rpm离心10s。重新置于恒温反应器中，37℃，1h。Add 120 μl DNase I to each tube after in vitro synthesis of mRNA. Invert 10 times to mix, centrifuge at 1000 rpm for 10 seconds. Return to the thermostatic reactor at 37°C for 1 hour.

7、mRNA沉淀回收7. mRNA precipitation and recovery

向上一步骤中的每个50ml Tube管中，加入等体积的醋酸铵溶液。上下颠倒10次混匀。置于-20℃2h，沉淀。17000g，4℃离心，30min。去掉上清，用70％乙醇洗涤沉淀。17000g，4℃离心，10min。去掉70％乙醇，于超净台中蒸干，每管加入RNase-free水20ml。静置10min后，用枪头轻吹混匀。用NanoDrop检测回收后的mRNA浓度为5μg/μl，A260/A280为1.90、A260/A230为2.0。取1μl，稀释10倍，进行RNA ScreenTape assay以及琼脂糖凝胶电泳检测其片段完整性。Add an equal volume of ammonium acetate solution to each 50ml Tube in the previous step. Invert 10 times to mix. Place at -20℃ for 2h to precipitate. Centrifuge at 17000g, 4℃, 30min. Remove the supernatant and wash the precipitate with 70% ethanol. Centrifuge at 17000g, 4℃, 10min. Remove the 70% ethanol, evaporate to dryness in a clean bench, and add 20ml of RNase-free water to each tube. After standing for 10min, mix by blowing gently with a gun tip. The concentration of the recovered mRNA detected by NanoDrop is 5μg/μl, A260/A280 is 1.90, and A260/A230 is 2.0. Take 1μl, dilute 10 times, and perform RNA Screen Tape assay and agarose gel electrophoresis to detect the integrity of the fragment.

8、LiCl沉淀纯化mRNA8. LiCl precipitation to purify mRNA

将上一步骤中回收的mRNA按照其1.5倍体积加入Rnase-free水，混匀。加入原mRNA 1.5倍体积-20预冷的LiCl溶液，混匀。然后于-20℃静至2h。16000g离心20min。弃上清，用70％乙醇洗涤沉淀，16000g离心15min。取上清，于超净台晾干5min。用适当的RNase-free水溶解纯化后的mRNA。Add 1.5 times the volume of mRNA recovered in the previous step to RNase-free water and mix well. Add 1.5 times the volume of the original mRNA to -20 pre-cooled LiCl solution and mix well. Then let stand at -20℃ for 2 hours. Centrifuge at 16000g for 20 minutes. Discard the supernatant, wash the precipitate with 70% ethanol, and centrifuge at 16000g for 15 minutes. Take the supernatant and dry it on a clean bench for 5 minutes. Dissolve the purified mRNA with appropriate RNase-free water.

9、mRNA试剂获得9. Obtaining mRNA reagents

通过上述步骤分别得到的mRNA SEQ ID No.1-No.18，SEQ ID No.1-9为表达COL1A1，即I型胶原蛋白α1链，SEQ ID No.10-18为表达COL1A2，即I型胶原蛋白α2链，目前有研究报道单独提高COL1A1蛋白表达就可以提高皮肤内胶原的表达量，但mRNA SEQ ID No.1-No.18无论单独使用还是联合使用均有提高胶原表达的能力，各mRNA组细胞转染结果见图2。在本文下述的实施例中，所有mRNA试验组皆有两种mRNA序列联合使用。The mRNA SEQ ID No.1-No.18 obtained by the above steps, SEQ ID No.1-9 expresses COL1A1, i.e., type I collagen α1 chain, and SEQ ID No.10-18 expresses COL1A2, i.e., type I collagen α2 chain. At present, some studies have reported that increasing the expression of COL1A1 protein alone can increase the expression of collagen in the skin, but mRNA SEQ ID No.1-No.18 has the ability to increase collagen expression whether used alone or in combination. The cell transfection results of each mRNA group are shown in Figure 2. In the embodiments described below, all mRNA test groups have two mRNA sequences used in combination.

实施例2、mRNA细胞试验Example 2, mRNA cell test

S1细胞培养、接种：细胞消化、接种成纤维细胞。倒置显微镜下观察，至大部分细胞变圆并处于悬浮状态时，加入约胰酶体积2～3倍的含血清的DMEM培养基终止消化，并收集至离心管中，1200r/min离心5min。离心结束后，弃掉上清液，向离心管中加入一定体积的细胞培养基，弯头吸管吹打混匀细胞，细胞计数仪进行计数。稀释细胞至接种密度后，接种至96孔板中，每孔液量为200μL。接种结束后，放置于CO2培养箱中培养24h±2h。S1 cell culture and inoculation: cell digestion and inoculation of fibroblasts. Observe under an inverted microscope. When most of the cells become round and suspended, add about 2 to 3 times the volume of trypsin and serum-containing DMEM medium to terminate digestion, collect them into a centrifuge tube, and centrifuge at 1200r/min for 5 minutes. After centrifugation, discard the supernatant, add a certain volume of cell culture medium to the centrifuge tube, mix the cells with an elbow pipette, and count them with a cell counter. After diluting the cells to the inoculation density, inoculate them into a 96-well plate with 200μL of liquid per well. After inoculation, place them in a CO2 incubator and culture for 24h±2h.

S2给药：弃掉96孔板中的培养基，开展给药操作。样品孔中加入1.0μg/mL、0.1μg/mL、0.01μg/mL胶原蛋白mRNA的培养基，因在AI算法筛选过程中，优选的8组序列间相对于野生型都具有有效性，因此本实施例中优选组随机选择SEQ ID No.3和SEQ ID No.12的组合物，混合比例是1:1同体积混合；阳性对照孔中加入250ng/mL TGF的培养基，空白对照孔中加入正常的细胞培养基，每孔200μL。给药完毕后将96孔板放置在CO2培养箱中培养24h±2h。细胞上清收集：孵育培养结束后，离心收集培养液采用ELISA试剂盒测定Ⅰ型胶原蛋白(COL-1)含量。各组测试数据采用SPSS进行单因素方差分析，统计方法采用双尾检验，检验水准＝0.05；按照下述公式计算各组COL-1上调率(％)：S2 Dosing: Discard the culture medium in the 96-well plate and carry out the dosing operation. Add 1.0μg/mL, 0.1μg/mL, and 0.01μg/mL collagen mRNA culture medium to the sample wells. Because the 8 preferred groups of sequences are all effective relative to the wild type during the AI algorithm screening process, the preferred group in this embodiment randomly selects a combination of SEQ ID No.3 and SEQ ID No.12, and the mixing ratio is 1:1 mixed with the same volume; add 250ng/mL TGF culture medium to the positive control well, and add normal cell culture medium to the blank control well, 200μL per well. After the dosing is completed, place the 96-well plate in a CO2 incubator and culture for 24h±2h. Collection of cell supernatant: After the incubation is completed, the culture fluid is collected by centrifugation and the type I collagen (COL-1) content is determined using an ELISA kit. The test data of each group were subjected to one-way analysis of variance using SPSS, and the statistical method used was a two-tailed test with a test level of 0.05; the COL-1 upregulation rate (%) of each group was calculated according to the following formula:

COL-1上调率％＝(试验组含量-对照组含量)/对照组含量×100％COL-1 up-regulation rate % = (test group content - control group content) / control group content × 100%

从图3和图4中的数据可以得出结论，mRNA试验组促进细胞表达胶原蛋白的效率要远高于空白对照组，阳性对照组有佐证细胞实验正确性和体现阳性物质的有效性的作用，与阳性对照组相比mRNA组上调率明显要高，在细胞实验中相比于阳性对照组的上调效率，mRNA组有更高的上调效率。相比于空白对照组细胞表达胶原蛋白含量提高2倍。From the data in Figures 3 and 4, it can be concluded that the efficiency of the mRNA test group in promoting the expression of collagen in cells is much higher than that of the blank control group. The positive control group has the function of supporting the correctness of the cell experiment and reflecting the effectiveness of the positive substance. Compared with the positive control group, the upregulation rate of the mRNA group is significantly higher. In the cell experiment, the mRNA group has a higher upregulation efficiency than the positive control group. Compared with the blank control group, the content of collagen expressed by cells is increased by 2 times.

实施例3、3D皮肤试验Example 3: 3D skin test

试验系统：T-Skin体外人造全层皮肤模型检测盒，购自上海斯安肤诺生物科技有限公司；培养环境：37.0±1℃，5±1％CO2(v/v)，饱和湿度；细胞培养基：T-Skin体外人造全层皮肤模型专用培养基；Test system: T-Skin in vitro artificial full-thickness skin model test kit, purchased from Shanghai Sianfunuo Biotechnology Co., Ltd.; culture environment: 37.0±1°C, 5±1% CO2 (v/v), saturated humidity; cell culture medium: T-Skin in vitro artificial full-thickness skin model special culture medium;

主要试剂：维生素C(上海麦克林生化科技有限公司)、COL-1ELISA检测试剂盒(武汉博士德生物工程有限公司)、DAPI核染液(1mg/mL)、一抗：Anti-Collagen1 antibody(美国Abcam公司)、二抗：Dylight-488(羊抗兔IgG武汉博士德生物工程有限公司)；Main reagents: Vitamin C (Shanghai McLean Biochemical Technology Co., Ltd.), COL-1 ELISA detection kit (Wuhan Boster Biotechnology Co., Ltd.), DAPI nuclear stain (1 mg/mL), primary antibody: Anti-Collagen1 antibody (Abcam, USA), secondary antibody: Dylight-488 (sheep anti-rabbit IgG, Wuhan Boster Biotechnology Co., Ltd.);

主要设备：CO2培养箱、多功能酶标仪、生物安全柜、TS-8型转移脱色摇床、共聚焦显微镜；Main equipment: CO2 incubator, multifunctional microplate reader, biological safety cabinet, TS-8 transfer and decolorization shaker, confocal microscope;

试验步骤：Test steps:

1.受试物制备：因在AI算法筛选过程中，优选的8组序列间相对于野生型都具有有效性，因此本实施例中优选组随机选择SEQ ID No.5和SEQ ID No.14的组合物，混合比例是1:1同体积混合作为受试物。1. Preparation of test substances: During the AI algorithm screening process, the 8 preferred groups of sequences were all effective compared to the wild type. Therefore, in this embodiment, the preferred group randomly selected a combination of SEQ ID No. 5 and SEQ ID No. 14, and the mixing ratio was 1:1 with the same volume as the test substance.

2.T-Skin模型于培养第18天进入实验室，在37℃、5％ CO2的新鲜维持培养基中继续维持48小时。2. The T-Skin model was brought into the laboratory on the 18th day of culture and maintained in fresh maintenance medium at 37°C and 5% CO2 for another 48 hours.

3.将受试物(终浓度为1mg/mL)、阳性对照(200μM维生素C)、空白对照(PBS)，添加到培养基中，孵育5天。隔天将受试物/对照物直接添加于T-Skin模型表面。每天更换一次的培养基，连续处理培养5天。3. Add the test substance (final concentration of 1 mg/mL), positive control (200 μM vitamin C), and blank control (PBS) to the culture medium and incubate for 5 days. Add the test substance/control substance directly to the surface of the T-Skin model every other day. Change the culture medium once a day and continue to treat and culture for 5 days.

4.T-Skin模型组织切片I型胶原蛋白免疫荧光半定量：5天处理结束后，用4％多聚甲醛，固定T-SkinTM组织(4℃)，脱水处理后石蜡包埋，切片。随后将组织切片进行脱蜡和水化，枸橼酸修复液抗原微波热修复，滴加正常山羊血清封闭液室温孵育1h，甩去多余液体，不洗。滴加一抗(稀释15倍)，4℃过夜，然后PBS洗3次每次5min。滴加荧光二抗(稀释100倍)，37℃孵育1h，然后PBS洗1次5min。滴加DAPI核染液(1：1000)，室温孵育2min，PBS洗4次每次5min。抗荧光淬灭剂封片，镜下观察(DAPI：Ex＝358nm，Em＝461nm；I型胶原：Ex＝493nm，Em＝518nm)，采集图像，采用Image J分析荧光强度。4. Semi-quantitative immunofluorescence analysis of type I collagen in tissue sections of T-Skin model: After 5 days of treatment, fix T-Skin TM tissues with 4% paraformaldehyde (4°C), dehydrate, embed in paraffin, and slice. Then dewax and hydrate the tissue sections, microwave-heat-repair the antigens with citric acid repair solution, add normal goat serum blocking solution and incubate at room temperature for 1 hour, shake off excess liquid, and do not wash. Add primary antibody (15-fold dilution), incubate at 4°C overnight, then wash 3 times with PBS for 5 minutes each. Add fluorescent secondary antibody (100-fold dilution), incubate at 37°C for 1 hour, then wash once with PBS for 5 minutes. Add DAPI nuclear staining solution (1:1000), incubate at room temperature for 2 minutes, and wash 4 times with PBS for 5 minutes each. The slides were sealed with an anti-fluorescence quencher and observed under a microscope (DAPI: Ex=358 nm, Em=461 nm; type I collagen: Ex=493 nm, Em=518 nm). Images were collected and the fluorescence intensity was analyzed using Image J.

5.ELISA试剂盒测定Ⅰ型胶原蛋白(COL-1)含量：5天处理结束后，收集模型下方培养液，-80℃保存，采用ELISA试剂盒进行测定。5. Determination of type I collagen (COL-1) content using ELISA kit: After 5 days of treatment, the culture medium below the model was collected, stored at -80°C, and measured using an ELISA kit.

结果分析：计算各组COL-1上调率(％)。Result analysis: The up-regulation rate of COL-1 in each group (%) was calculated.

COL-1上调率(％)＝(试验组含量-对照组含量)/对照组含量×100％COL-1 up-regulation rate (%) = (content in the experimental group - content in the control group) / content in the control group × 100%

各组测试数据采用SPSS进行独立样本T检验，统计方法采用双尾检验，检验水准＝0.05；图5、图6、图7、图8数据显示试验结果：当样品组I型胶原含量>对照组，且经统计学分析有显著性差异时(P<0.05)，认为样品对T-Skin模型组织真皮层成纤维细胞具有促进I型胶原分泌的效果。The test data of each group were subjected to independent sample T test using SPSS, and the statistical method adopted was two-tailed test, with the test level = 0.05; the data in Figures 5, 6, 7, and 8 show the test results: when the type I collagen content in the sample group was greater than that in the control group, and there was a significant difference after statistical analysis (P<0.05), it was considered that the sample had the effect of promoting the secretion of type I collagen in the dermal fibroblasts of the T-Skin model tissue.

实施例4、动物试验验证辅助仪器效果Example 4: Animal experiment to verify the effect of auxiliary instruments

实验前器械准备：在购物商城中采购日常护肤仪，护肤宣称具有增加化妆品皮肤透过率的功能，为了方便理解从机理区分，分别为微电流导入仪，微针导入仪，负压导入仪；上述三种导入仪均可直接从商城采购，属于日常使用仪器，对此本专利中不对导入仪品牌和种类进行限制和权利保护。Equipment preparation before the experiment: Purchase daily skin care devices in the shopping mall. The skin care devices claim to have the function of increasing the skin permeability of cosmetics. For the convenience of understanding, they are distinguished from the mechanism as microcurrent introduction devices, microneedle introduction devices, and negative pressure introduction devices. The above three introduction devices can be purchased directly from the mall and are daily-use instruments. This patent does not restrict or protect the brands and types of introduction devices.

主要试剂准备：本实验中选择使用可以表达绿色荧光蛋白的编码mRNA作为标识物，绿色荧光蛋白(Green fluorescent protein，简称GFP)，是一个由约238个氨基酸组成的蛋白质，从蓝光到紫外线都能使其激发，发出绿色荧光。是生物分子实验中常用的指示蛋白，可以快速定位到表达细胞位置，方便检测mRNA导入皮肤后在皮肤中的分布情况；Preparation of main reagents: In this experiment, we choose to use mRNA that can express green fluorescent protein as a marker. Green fluorescent protein (GFP) is a protein composed of about 238 amino acids. It can be excited by light from blue to ultraviolet light and emit green fluorescence. It is a commonly used indicator protein in biomolecular experiments. It can quickly locate the position of the expressing cells and facilitate the detection of the distribution of mRNA in the skin after it is introduced into the skin.

试验动物准备：选取检疫合格10周龄健康Balb/C雌性小鼠25只，随机分为空白对照组、eGFP mRNA组、eGFP mRNA+微电流导入仪测试组(以下简称mRNA微电流组)，eGFP mRNA+微针导入仪测试组(以下简称mRNA微针组)，eGFP mRNA+负压导入仪测试组(以下简称mRNA负压组)，每组5只。Preparation of experimental animals: 25 healthy Balb/C female mice aged 10 weeks that passed the quarantine were selected and randomly divided into blank control group, eGFP mRNA group, eGFP mRNA + microcurrent introduction instrument test group (hereinafter referred to as mRNA microcurrent group), eGFP mRNA + microneedle introduction instrument test group (hereinafter referred to as mRNA microneedle group), eGFP mRNA + negative pressure introduction instrument test group (hereinafter referred to as mRNA negative pressure group), with 5 mice in each group.

实验步骤：在试验前4～24小时，将小鼠背部脊柱两侧被毛除去(约3cm x 3cm区域)，作为试验和观察部位。为了便于观察和再次试验，可能需反复除毛。Experimental procedures: 4 to 24 hours before the experiment, remove the hair on both sides of the spine of the mouse back (about 3cm x 3cm area) as the test and observation site. In order to facilitate observation and re-testing, it may be necessary to remove the hair repeatedly.

小鼠背部脱毛后，用1.5％异氟醚麻醉小鼠，使用消毒酒精对小鼠背部进行擦拭，待酒精挥发后，将eGFP mRNA溶液以5μg/每只小鼠的剂量，均匀涂抹在小鼠背部，控制涂抹范围在1cm x 1cm方框内，随后按照使用说明书操作，使用辅助导入仪器对小鼠背部进行15min的液体导入处理；空白对照组使用生理盐水作为对照试剂对小鼠进行背部涂抹。After depilating the back of the mice, the mice were anesthetized with 1.5% isoflurane and wiped with disinfectant alcohol. After the alcohol evaporated, the eGFP mRNA solution was evenly applied on the back of the mice at a dose of 5 μg/mouse. The application range was controlled within a 1cm x 1cm square. Then, according to the instruction manual, the auxiliary introduction instrument was used to perform liquid introduction treatment on the back of the mice for 15 minutes. In the blank control group, normal saline was used as a control reagent to apply on the back of the mice.

试验结束后，将小鼠背部残留多余液体擦拭干净后，解除麻醉，观察24小时后，切除皮肤并放置在4％甲醛中固定。随后进行冷冻切片在荧光显微镜下观察。After the experiment, the excess fluid on the back of the mouse was wiped clean, and the anesthesia was removed. After 24 hours of observation, the skin was excised and fixed in 4% formaldehyde, and then frozen sections were observed under a fluorescence microscope.

相比于空白对照组，mRNA组在荧光显微镜下观察均有不同程度的绿色荧光团(如图9中显示)，这证明了mRNA透过皮肤被皮肤细胞摄取并表达出荧光蛋白；相较于单独涂抹mRNA组，使用辅助导入设备的实验组荧光蛋白的表达量均有不同程度提高，其中mRNA微电流组和mRNA负压组在表皮层可以观察到更明显的绿色荧光。Compared with the blank control group, the mRNA group showed varying degrees of green fluorescent groups under a fluorescence microscope (as shown in Figure 9), which proved that the mRNA passed through the skin and was taken up by skin cells to express fluorescent protein; compared with the group that applied mRNA alone, the expression level of fluorescent protein in the experimental group using the auxiliary introduction device increased to varying degrees, among which more obvious green fluorescence could be observed in the epidermis in the mRNA microcurrent group and the mRNA negative pressure group.

实施例5、大鼠皮肤试验验证胶原表达效果Example 5: Rat skin test to verify the collagen expression effect

实验前器械准备：在购物商城中采购日常护肤仪，护肤宣称具有增加化妆品皮肤透过率的功能，为了方便理解从机理区分，分别为微电流导入仪，微针导入仪，负压导入仪；上述三种导入仪均可直接从商城采购，属于日常使用仪器，对此本专利中不对导入仪品牌和种类进行限制和权利保护。Equipment preparation before the experiment: Purchase daily skin care devices in the shopping mall. The skin care devices claim to have the function of increasing the skin permeability of cosmetics. For the convenience of understanding, they are distinguished from the mechanism as microcurrent introduction devices, microneedle introduction devices, and negative pressure introduction devices. The above three introduction devices can be purchased directly from the mall and are daily-use instruments. This patent does not restrict or protect the brands and types of introduction devices.

主要试剂准备：本实验中选择使用可以表达胶原蛋白的编码mRNA作为标识物，因在AI算法筛选过程中，优选的8组序列间相对于野生型都具有有效性，因此本实施例中优选组随机选择SEQ ID No.7和SEQ ID No.16的组合物，混合比例是1:1同体积混合；Preparation of main reagents: In this experiment, mRNA encoding collagen was selected as the marker. During the AI algorithm screening process, the eight groups of sequences selected were all effective relative to the wild type. Therefore, the preferred group in this embodiment randomly selected a combination of SEQ ID No. 7 and SEQ ID No. 16, and the mixing ratio was 1:1 with the same volume.

试验动物准备：选取检疫合格4周龄健康SD雌性大鼠25只，随机分为空白对照组、胶原蛋白mRNA+微电流导入仪测试组(以下简称mRNA微电流组)，胶原蛋白mRNA+微针导入仪测试组(以下简称mRNA微针组)，胶原蛋白mRNA+负压导入仪测试组(以下简称mRNA负压组)，胶原蛋白mRNA皮内注射作为阳性对照组，每组5只。Preparation of experimental animals: 25 healthy 4-week-old SD female rats that passed the quarantine were selected and randomly divided into a blank control group, a collagen mRNA + microcurrent introduction instrument test group (hereinafter referred to as the mRNA microcurrent group), a collagen mRNA + microneedle introduction instrument test group (hereinafter referred to as the mRNA microneedle group), and a collagen mRNA + negative pressure introduction instrument test group (hereinafter referred to as the mRNA negative pressure group). Collagen mRNA intradermal injection was used as the positive control group, with 5 rats in each group.

实验步骤：在试验前4～24小时，将大鼠背部脊柱两侧被毛除去(约3cm x 3cm区域)，作为试验和观察部位。为了便于观察和再次试验，可能需反复除毛。大鼠背部脱毛后，用1.5％异氟醚麻醉大鼠，使用消毒酒精对大鼠背部进行擦拭，待酒精挥发后，将eGFP mRNA溶液以10μg/每只鼠的剂量，均匀涂抹在大鼠背部，控制涂抹范围在1cm x 1cm方框内，随后按照使用说明书操作，使用辅助导入仪器对大鼠背部进行15min的液体导入处理；空白对照组使用生理盐水作为对照试剂，按照同样操作对大鼠背部进行涂抹。试验结束后，将大鼠背部残留多余液体擦拭干净后，解除麻醉，观察48h后取实验处皮肤用4％多聚甲醛过夜固定后制作石蜡切片，切片进行masson染色，拍照分析。Experimental steps: 4 to 24 hours before the experiment, remove the hair on both sides of the rat spine (about 3cm x 3cm area) on the back as the test and observation site. In order to facilitate observation and re-testing, repeated hair removal may be required. After depilation of the rat's back, anesthetize the rat with 1.5% isoflurane, wipe the rat's back with disinfectant alcohol, and after the alcohol evaporates, evenly apply the eGFP mRNA solution to the rat's back at a dose of 10μg/mouse, and control the application range within a 1cm x 1cm box. Then, according to the instruction manual, use the auxiliary introduction instrument to introduce liquid into the rat's back for 15 minutes; the blank control group uses physiological saline as a control reagent, and applies it to the rat's back in the same way. After the experiment, wipe off the excess liquid on the rat's back, remove the anesthesia, and observe for 48 hours. Take the skin of the experimental site and fix it overnight with 4% paraformaldehyde to make paraffin sections, and the sections are masson stained and photographed for analysis.

在图10可以直观对比各组间处理后真皮层中胶原纤维的变化，真皮层主要由胶原纤维、弹力纤维构成，直接决定皮肤的弹性。图11的数据结果中显示阳性对照组通过皮内注射的方式对胶原蛋白上调有促进作用，通过微针导入的方式同样也可以提高皮肤内胶原蛋白的表达提高，与对照组相比有明显差异。Figure 10 shows a direct comparison of the changes in collagen fibers in the dermis after treatment in each group. The dermis is mainly composed of collagen fibers and elastic fibers, which directly determine the elasticity of the skin. The data results in Figure 11 show that the positive control group has a promoting effect on the upregulation of collagen through intradermal injection, and the microneedle introduction method can also increase the expression of collagen in the skin, which is significantly different from the control group.

上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此，本发明不限于上述实施例，本领域技术人员根据本发明的揭示，不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims (10)

一种用于导入皮肤底层以美容的mRNA分子，其包含编码胶原蛋白的核酸序列。An mRNA molecule for introduction into the bottom layer of the skin for beauty treatment, comprising a nucleic acid sequence encoding collagen. 一种mRNA分子，其中，所述的mRNA分子包含编码胶原蛋白的核酸序列，所述的编码胶原蛋白的核酸序列含有SEQ ID NO：1～8、SEQ ID NO：10～17中任一项所示的序列，An mRNA molecule, wherein the mRNA molecule comprises a nucleic acid sequence encoding collagen, and the nucleic acid sequence encoding collagen comprises a sequence shown in any one of SEQ ID NOs: 1 to 8 and SEQ ID NOs: 10 to 17, 优选地，所述的mRNA分子包含至少一条编码I型胶原蛋白α1链的mRNA序列以及至少一条编码I型胶原蛋白α2链的mRNA序列，所述编码I型胶原蛋白α1链的mRNA序列具有SEQ ID NO：1～9中任一所示序列，所述编码I型胶原蛋白α2链的mRNA序列具有SEQ ID NO：10～18中任一所示序列。Preferably, the mRNA molecule contains at least one mRNA sequence encoding type I collagen α1 chain and at least one mRNA sequence encoding type I collagen α2 chain, the mRNA sequence encoding type I collagen α1 chain has a sequence shown in any one of SEQ ID NOs: 1 to 9, and the mRNA sequence encoding type I collagen α2 chain has a sequence shown in any one of SEQ ID NOs: 10 to 18. 根据权利要求2所述的mRNA分子，其中，所述的mRNA分子还具有5'帽结构、5'UTR、3'UTR、和/或PolyA。The mRNA molecule according to claim 2, wherein the mRNA molecule further comprises a 5' cap structure, a 5' UTR, a 3' UTR, and/or Poly A. 一种美容或药用组合物，其中，所述的美容或药用组合物包含权利要求1-3任一项所述的mRNA分子；A cosmetic or medicinal composition, wherein the cosmetic or medicinal composition comprises the mRNA molecule according to any one of claims 1 to 3; 优选地，所述的美容或药用组合物还包括医学、美容和化妆品用可接受的基质材料；Preferably, the cosmetic or pharmaceutical composition further comprises a base material acceptable for medical, cosmetic and cosmetic use; 优选地，所述的美容或药用组合物还包括化妆品学或药学上可接受的载体；Preferably, the cosmetic or pharmaceutical composition further comprises a cosmetically or pharmaceutically acceptable carrier; 优选地，所述的美容或药用组合物为用于射频导入仪、负压导入仪、微晶导入仪、微电流导入仪、微针导入仪、或注射器导入皮肤底层的制剂。Preferably, the cosmetic or medicinal composition is a preparation for introduction into the underlying layer of the skin via a radio frequency introduction device, a negative pressure introduction device, a microcrystal introduction device, a microcurrent introduction device, a microneedle introduction device, or a syringe. 一种DNA分子，其能用于转录得到权利要求1-3任一项所述的mRNA分子。A DNA molecule, which can be used for transcription to obtain the mRNA molecule according to any one of claims 1 to 3. 一种重组质粒或重组细胞，其包含权利要求5所述的DNA分子，A recombinant plasmid or recombinant cell comprising the DNA molecule according to claim 5, 优选地，所述的重组质粒或重组细胞能用于转录得到权利要求1-3任一项所述的mRNA分子。Preferably, the recombinant plasmid or recombinant cell can be used to transcribe the mRNA molecule according to any one of claims 1 to 3. 一种试剂盒，其包含权利要求1-3任一项所述的mRNA分子、权利要求4所述的美容或药用组合物、权利要求5所述的DNA分子、或权利要求6所述的重组质粒或重组细胞。A kit comprising the mRNA molecule according to any one of claims 1 to 3, the cosmetic or pharmaceutical composition according to claim 4, the DNA molecule according to claim 5, or the recombinant plasmid or recombinant cell according to claim 6. 根据权利要求7所述的试剂盒，其中，所述的试剂盒还包含辅助导入设备；优选地，所述的辅助导入设备包括射频导入仪、负压导入仪、微晶导入仪、微电流导入仪、微针导入仪、或注射器。The kit according to claim 7, wherein the kit further comprises an auxiliary introduction device; preferably, the auxiliary introduction device comprises a radio frequency introduction device, a negative pressure introduction device, a microcrystal introduction device, a microcurrent introduction device, a microneedle introduction device, or a syringe. 一种制备权利要求1-3任一项所述的mRNA分子的方法，其包括：A method for preparing the mRNA molecule according to any one of claims 1 to 3, comprising: (1)将权利要求5所述的DNA分子克隆至表达载体获得重组质粒；(1) cloning the DNA molecule described in claim 5 into an expression vector to obtain a recombinant plasmid; (2)将所述重组质粒转入宿主细胞获得重组细胞，从扩增后的重组细胞中提取质粒，以提取获得的质粒为模板进行PCR扩增获得体外表达mRNA的DNA模板；(2) transferring the recombinant plasmid into a host cell to obtain a recombinant cell, extracting the plasmid from the amplified recombinant cell, and performing PCR amplification using the extracted plasmid as a template to obtain a DNA template for in vitro expression of mRNA; (3)构建包括所述DNA模板的RNA体外合成体系进行mRNA的体外合成获得所述的mRNA分子。(3) constructing an RNA in vitro synthesis system including the DNA template to perform in vitro synthesis of mRNA to obtain the mRNA molecule. 权利要求1-3任一项所述的mRNA分子、权利要求4所述的美容或药用组合物、权利要求5所述的DNA分子、权利要求6所述的重组质粒或重组细胞、或权利要求7或8所述的试剂盒在制备用于皮肤护理或改善皮肤状态的产品中的应用，Use of the mRNA molecule according to any one of claims 1 to 3, the cosmetic or pharmaceutical composition according to claim 4, the DNA molecule according to claim 5, the recombinant plasmid or recombinant cell according to claim 6, or the kit according to claim 7 or 8 in the preparation of a product for skin care or improvement of skin condition, 优选地，所述的产品为药物、化妆品或试剂盒，Preferably, the product is a medicine, a cosmetic or a kit. 优选地，所述的改善皮肤状态包括促进胶原蛋白的表达、刺激胶原纤维增生。Preferably, the improvement of skin condition includes promoting the expression of collagen and stimulating the proliferation of collagen fibers.
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