WO2019196420A1 - Polypeptide and application of skin repair function thereof - Google Patents

Abstract

The present invention relates to the field of polypeptide application, in particular relates to the field of polypeptide application in skin repair, and specifically relates to a polypeptide and an application of the skin repair function thereof. ZKSA-P-1001. The research finds that ZKSA-P-1001 has good stability and relatively good skin wound healing promoting ability. ZKSA-P-1001 can be used as a raw material or an auxiliary material for producing drugs, postoperative medical devices, healthcare products, cosmetics and the like with the skin repair function.

Description

Application of a polypeptide and its skin repair function Technical field

The invention belongs to the field of polypeptide application, in particular to the field of skin repair polypeptide application, and particularly relates to the application of a polypeptide and its skin repair function.

Background technique

The skin is the first barrier to contact the outside world. It is the first line of defense against dehydration, damage and infection. It is a barrier to maintain internal environment stability and prevent the invasion of microorganisms and chemicals. Large areas of skin defects and refractory skin ulcers caused by various acute and chronic injuries such as inflammation, ulcers, severe burns, trauma, postoperative tumors, and congenital malformations, causing many local and systemic changes, and bacteria are highly susceptible to invasion and reproduction. It can cause water and electrolyte disorders, septic shock, high metabolic reactions, multiple organ failure, etc., so that the body can not maintain a normal state of self-stability, and even lead to death. It is reported that about 15% of the elderly suffer from chronic skin trauma. As far as the United States is concerned, the annual cost of trauma repair for elderly patients is as high as $25 billion. Approximately 15-27% of all ulcers require surgical osteotomy, but the proportions vary slightly from country to country. The prognosis of chronic skin ulcers is very poor. Because the disease is chronic, prolonged, difficult to cure and consumes a lot, it causes great psychological stress on the patients, seriously affecting the patient's physical condition and quality of life. The treatment of chronic skin ulcers has caused domestic and foreign Scholars have a wide range of concerns. Therefore, the development of safe, low-toxic, low-cost, high-efficiency skin-healing drugs, reducing their incidence and disability, and improving the quality of life of patients has become an urgent problem for pharmaceutical companies and researchers.

Skin wound repair is the most basic model in the healing and healing of various tissues and organs in the human body. In addition, skin wound healing is a very complex biological process, a dynamic process of complex interactions between multiple types of cells, cytokines and extracellular matrices in the skin, a complex and continuous molecule, cell, tissue and A multi-level reaction process between the whole. It involves blood coagulation, the occurrence and development of inflammation, matrix synthesis, angiogenesis, fibrous tissue hyperplasia, re-epithelialization, wound contraction, and tissue remodeling. Skin wound healing can be roughly divided into the following processes: blood coagulation phase, inflammation phase, proliferative phase, and tissue remodeling. The four overlapping periods include cell proliferation, differentiation, epithelialization, migration, and matrix synthesis. Deposition.

Many studies have shown that amphibians contain polypeptides that promote skin repair. These peptides can promote the rapid healing of animal skin to achieve wound healing.

Summary of the invention

In view of the circumstances in the prior art, it is an object of the present invention to provide a polypeptide and its use in skin repair function.

In order to achieve the above and other related objects, the present invention adopts the following technical solutions:

In a first aspect of the invention, there is provided an isolated polypeptide comprising:

(a) a polypeptide consisting of the amino acid represented by SEQ ID NO: 1;

(b) or a polypeptide derived from (a) which is substituted, deleted or added with one or several amino acids and having an equivalent activity in the amino acid sequence shown in SEQ ID NO: 1.

Preferably, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1, specifically: RWRFKWKK, that is, Arg-Trp-Arg-Phe-Lys-Trp-Lys-Lys.

In a second aspect of the invention, an isolated polynucleotide is provided, the isolated polynucleotide encoding the aforementioned polypeptide.

The polynucleotide encoding the aforementioned polypeptide of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded.

Polynucleotides encoding the polypeptides of the invention can be prepared by any suitable technique well known to those skilled in the art. Such techniques are described in the general description of the art, such as the Guide to Molecular Cloning (J. Sambrook et al., Science Press, 1995). These include, but are not limited to, recombinant DNA techniques, chemical synthesis, and the like.

In a third aspect of the invention, a recombinant expression vector comprising the isolated polynucleotide described above is provided.

Methods well known to those skilled in the art can be used to construct the recombinant expression vector. These methods include recombinant DNA techniques, DNA synthesis techniques, and the like. A polynucleotide encoding the polypeptide is operably linked to a multiple cloning site in a vector to direct mRNA synthesis to express a polypeptide, or for homologous recombination.

In a fourth aspect of the invention, a host cell comprising the aforementioned recombinant expression vector or the above-described isolated polynucleotide integrated with an exogenous source in the genome is provided.

According to a fifth aspect of the present invention, there is provided a method for producing the aforementioned polypeptide, which comprises any one of the following:

(1) synthesizing the polypeptide by chemical synthesis;

(2) cultivating the aforementioned host cell under appropriate conditions and expressing the polypeptide, followed by isolation and purification to obtain the polypeptide.

In a sixth aspect of the invention, there is provided the use of the aforementioned polypeptide for the preparation of a dermal fibroblast proliferation promoter.

Preferably, the polypeptide is one of the sole active ingredients or active ingredients of the accelerator.

In a seventh aspect of the invention, the use of the aforementioned polypeptide for the preparation of a vascular endothelial cell proliferation promoter is provided.

Preferably, the polypeptide is one of the sole active ingredients or active ingredients of the accelerator.

In an eighth aspect of the invention, the use of the aforementioned polypeptide for the preparation of a vascular endothelial cell migration promoting agent is provided.

Preferably, the polypeptide is one of the sole active ingredients or active ingredients of the accelerator.

According to a ninth aspect of the present invention, there is provided the use of the aforementioned polypeptide for the preparation of a skin superficial layer or a dermal layer damage repairing agent.

Preferably, the polypeptide is one of the sole active ingredients or active ingredients of the accelerator.

In a tenth aspect of the invention, there is provided the use of the aforementioned polypeptide for the preparation of a skin wound healing promoting medicament.

Preferably, the polypeptide is one of the sole active ingredients or active ingredients of the drug.

Further, the skin wound may be a skin wound, and may further be a superficial cortical wound or a dermis wound.

In an eleventh aspect of the present invention, the use of the aforementioned polypeptide for the preparation of a medicament for treating skin defects and refractory skin ulcers is provided.

Preferably, the polypeptide is one of the sole active ingredients or active ingredients of the pharmaceutical composition.

Further, the skin defect and the refractory skin ulcer may be a large area of skin defect and refractory skin ulcer caused by various acute and chronic injuries such as inflammation, ulcer, severe burn, trauma, tumor surgery and congenital malformation. .

In a twelfth aspect of the invention, the use of the aforementioned polypeptide for the preparation of a medicament for the treatment of traumatic skin damage is provided.

Preferably, the polypeptide is one of the sole active ingredients or active ingredients of the drug.

In a thirteenth aspect of the invention, the use of the aforementioned polypeptide for the preparation of a skin repairing drug, a skin device after skin repair, a skin repair post product, a skin repair health product or a skin repair cosmetic is provided.

Preferably, the polypeptide is one of the sole active ingredients or active ingredients of the drug, post-operative medical device, health care product or cosmetic.

In a fourteenth aspect of the invention, there is provided a pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable carrier, the active ingredient comprising the aforementioned polypeptide.

Preferably, the polypeptide is one of the sole active ingredients or active ingredients of the pharmaceutical composition.

The polypeptide acts as an active ingredient for any of the following functions:

(1) promote the proliferation of skin fibroblasts; (2) promote the proliferation of vascular endothelial cells; (3) promote the migration of vascular endothelial cells; (4) promote the repair of skin superficial or dermal layer damage; (5) promote skin wound healing (6) treatment of traumatic skin damage; (7) repair of skin damage.

The pharmaceutically acceptable carrier is a variety of pharmaceutically acceptable excipients and/or excipients including, but not limited to, saccharides (such as lactose, glucose, and sucrose), starch (such as corn starch and potato starch), cellulose. And derivatives thereof (such as sodium carboxymethylcellulose, ethylcellulose and methylcellulose), tragacanth powder, malt, gelatin, talc, solid lubricants (such as stearic acid and magnesium stearate), Calcium sulphate, vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter, polyols (such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol), alginic acid, emulsifiers (such as Tween, polyoxyethylene castor oil), wetting agent (such as sodium lauryl sulfate), coloring agents, flavoring agents, compressed tablets, stabilizers, antioxidants, preservatives, pyrogen-free water, isotonic saline solutions and phosphates Buffer, etc.; the carrier can improve the stability, activity and bioavailability of the formulation as needed.

When the pharmaceutical composition of the present invention is used, the polypeptide is used as the sole active ingredient, or the polypeptide is one of the active ingredients, and can be mixed with one or more pharmaceutically acceptable carriers or excipients to prepare different administration routes. Pharmaceutical dosage form.

Preferably, the pharmaceutical composition is in the form of a tablet, a capsule, a powder, a granule, a syrup, a solution, an oral solution, an elixir, an elixir, an aerosol, a powder, an injection, and an injection for sterilizing. Powder, or suppository. The above preparation types can be understood in accordance with the relevant definitions in pharmacy (sixth edition, People's Medical Publishing House, Cui Fu), and the preparation of the above preparations can be carried out according to the relevant preparations in pharmacy (sixth edition, People's Medical Publishing House, Cui Fu) Method preparation.

The effective dose of the polypeptide as an active ingredient may vary depending on the mode of administration and the severity of the disease. The specific dosages administered are within the skill of the art.

According to a fifteenth aspect of the invention, there is provided a skin repair product comprising an active ingredient comprising the aforementioned polypeptide.

Further, the product may be a post-operative medical device, a health care product, or a cosmetic product.

For example, the post-operative medical device may be a suture needle, and in use, the polypeptide may be applied as an active ingredient to the suture needle.

Preferably, the polypeptide is one of the sole active ingredients or active ingredients of the product.

In a sixteenth aspect of the invention, a method for treating a skin disease comprising the step of applying the polypeptide of the first aspect of the invention as an active ingredient to the skin.

The polypeptide acts as an active ingredient for any of the following functions:

(1) promote the proliferation of skin fibroblasts; (2) promote the proliferation of vascular endothelial cells; (3) promote the migration of vascular endothelial cells; (4) promote the repair of skin superficial or dermal layer damage; (5) promote skin wound healing (6) treatment of traumatic skin damage; (7) repair of skin damage.

Compared with the prior art, the present invention has the following beneficial effects:

This study found a novel peptide named ZKSA-P-1001. Further research found that ZKSA-P-1001 has good stability and good skin wound healing ability. ZKSA-P-1001 can be used as a raw material or auxiliary material for medicines, postoperative medical devices, health products, cosmetics, etc. that have skin repair functions.

DRAWINGS

Figure 1 is a bar graph of ZKSA-P-1001 samples promoting proliferation of HSF and Huvec cells.

Figure 2 is a bar graph of the migration activity of ZKSA-P-1001 on Huvec cells.

Figure 3 is a graph showing the cell scratching of ZKSA-P-1001 on Huvec cell migration activity.

Figure 4 is a bar graph of the wound healing effects of ZKSA-P-1001 and EGF on a mouse wound animal model.

Figure 5 is a graph of wound healing of ZKSA-P-1001 and EGF in a mouse wound animal model.

detailed description

Before the present invention is further described, it is to be understood that the scope of the present invention is not limited to the specific embodiments described below; It is not intended to limit the scope of the invention. The test methods which do not specify the specific conditions in the following examples are usually carried out according to conventional conditions or according to the conditions recommended by each manufacturer.

When the numerical values are given by the examples, it is to be understood that the two endpoints of each numerical range and any one of the two. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning meaning In addition to the specific methods, devices, and materials used in the embodiments, the methods, devices, and materials described in the embodiments of the present invention may also be used according to the prior art and the description of the present invention by those skilled in the art. Any method, apparatus, and material of the prior art, similar or equivalent, is used to practice the invention.

Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention employ molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields conventional in the art. Conventional technology. These techniques are well described in the existing literature. For details, see Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons , New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (PM Wassarman And AP Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (PBBecker, ed.) Humana Press, Totowa, 1999, and the like.

Example 1 Effect of ZKSA-P-1001 sample on proliferation of skin fibroblastic HSF cells

First, a polypeptide is synthesized in vitro by using the prior art, and its amino acid sequence is: RWRFKWKK (SEQ ID NO. 1), Arg-Trp-Arg-Phe-Lys-Trp-Lys-Lys (SEQ ID NO. 1), Named as: ZKSA-P-1001, purity: 97.96%. Therefore, the ZKSA-P-1001 referred to in the present invention refers to a polypeptide having an amino acid sequence as shown in SEQ ID NO.

Next, the effect of ZKSA-P-1001 on the proliferation of skin fibroblastic HSF cells was examined by cell proliferation assay-MTS assay.

Day0: trypsinize skin fibroblastic HSF cells and count them. Dilute the cell suspension to about 1×10 ⁵ /mL. The cells are seeded in 96-well plates, 100 μL of cell suspension per well, and the cells are cultured overnight.

Day1: ZKSA-P-1001 sample was weighed and dissolved in PBS to prepare 10 mg/mL mother liquor for use; the adherent cells in 96-well plate were treated with ZKSA-P-1001, and the culture was continued for 24 h;

Day2: Incubate for 24 h, add 20 μL of MTS reagent per well, incubate for 1 h at 37 ° C in the dark, read at 490 nm with a microplate reader, and process the data.

Four replicates were set and the cell growth was measured using the MTS method. The results were analyzed using T-test for significant difference, and the results were consistent for each iteration. ZKSA-P-1001 was dissolved in DNEM complete medium, and the concentration was increased to verify whether there was a high concentration inhibition phenomenon. This test excluded the dilution of the culture medium by PBS as a solvent. However, the MTS results at 24 h showed that the ZKSA-P-1001 sample concentration above 2 mg/mL showed a certain inhibitory effect on cell proliferation. As shown in Figure 1, ZKSA-P-1001 proliferating experiments on skin fibroblastic HSF cells showed that ZKSA-P-1001 significantly promoted cell proliferation by acting on skin fibroblastic HSF cells at a concentration of 500-2000 ug/mL for 24 h.

Example 2 Effect of ZKSA-P-1001 sample on proliferation of human umbilical vein Huvec cells

The effect of ZKSA-P-1001 on the proliferation of human umbilical vein Huvec cells was examined by cell proliferation assay-MTS assay.

Day0: trypsinize human umbilical vein Huvec cells and count them. Dilute the cell suspension to about 1×10 ⁵ /mL. The cells are seeded in 96-well plates, 100 μL of cell suspension per well, and the cells are cultured overnight.

Day1: ZKSA-P-1001 sample was weighed and dissolved in PBS to prepare 10 mg/mL mother liquor for use; the adherent cells in 96-well plate were treated with ZKSA-P-1001, and the culture was continued for 24 h;

Day2: cultured for 24 h, add 20 μL LMTS reagent per well, incubate for 1 h at 37 ° C in the dark, read at 490 nm with a microplate reader, and process the data.

Four replicates were set and the cell growth was measured using the MTS method. The results were analyzed using T-test for significant difference, and the results were consistent for each iteration. ZKSA-P-1001 was dissolved in M200 complete medium, and then the concentration was increased to verify whether there was a high concentration inhibition phenomenon. Four replicates were set and the cell growth was measured using the MTS method. The results were analyzed using T-test for significant difference, and the results were consistent for each iteration. This experiment excluded the dilution of the culture medium by PBS as a solvent. The MTS results showed that (Fig. 1) ZKSA-P-1001 significantly promoted cell proliferation by acting on human umbilical vein Huvec cells at a concentration of 500-2000 μg/mL for 24 h.

Example 3 Effect of ZKSA-P-1001 on migration activity of human umbilical vein Huvec cells

This example investigates the effect of ZKSA-P-1001 on the migration activity of human umbilical vein Huvec cells:

Day0: trypsinize human umbilical vein Huvec cells and count them. Dilute the cell suspension to about 1×10 ⁵ /mL. Inoculate the cells in a 12-well plate. Incubate the cells overnight and incubate the cells until the cells are completely confluent. Bottom

Day2-3: When observing the bottom of the cells under the microscope, use a 200 μL yellow sterile tip to centrally dispose and culture the wells from top to bottom in each test well; discard the culture and wash twice with PBS to get out. Cell clusters produced by scratching; ZKSA-P-1001 samples were weighed and dissolved in PBS to a mother liquor of 10 mg/mL for use. The control group was added with fresh medium, and the experimental group was added with different concentrations of ZKSA-P-1001;

Scratch repair rate (%) = (0 hour scratch area - 16h scratch area) / 0 hour scratch area × 100

Four replicates were set, and the resulting data was analyzed for significance using T-test, and the results were consistent for each iteration. ZKSA-P-1001 showed that the human umbilical vein Huvec cells were streaked (as shown in Fig. 2 and Fig. 3), and the cells after scribing were compared with the control group in the case of complete or serum-free medium. When ZKSA-P-1001 concentration was 500 and 1000 ug/mL, the human umbilical vein 16h Huvec cells migrated significantly.

Example 4 Effect of ZKSA-P-1001 on wound healing in mouse whole cortex

Preparation of a skin wound model:

The mice were anesthetized with 1% sodium pentobarbital (1 mg/100 mL); the back hair was removed, and the ipsilateral disinfection was performed on the left and right sides of the back of the mouse to open the same round wound (to remove the full-thickness skin). After the injury model was established, the wounds were cleaned with sterile gauze; the mice were divided into two groups, ZKSA-P-1001 and the epidermal growth factor EGF group (positive control group); ZKSA-P-1001 and PBS group; ZKSA-P-1001 sample (concentration 500μg/mL, dosage 20μL) was added to the left wound of each mouse; EGF (concentration 100μg/mL, dosage 20μL) was added to the right wound of EGF group, PBS group The right side of the wound was added with PBS (20 μL), and the wound was naturally exposed. Each time, the drug was administered once a day in the morning and afternoon, and wound healing was observed. On the 0th to 8th day after the trauma, the wounds were photographed and recorded, as shown in Figure 5. The area of the wound was analyzed by Image J software. The wound area recorded after surgery was compared with 0 days, and the percentage value was plotted as a histogram. The results are shown in Fig. 4. The rate of wound healing is calculated using the following formula:

The Residual wond area(%)=[R(2~8)/R(0)]x100

R(0) represents the wound area at 0 days after surgery; R (2-8) represents the wound area 2 to 8 days after surgery.

The results showed that no physiological or behavioral abnormalities were observed during the entire wound healing experiment in mice. From the wound healing experiment of mice with total cortical defect, we found that the wound surface of ZKSA-P-1001 was significantly accelerated compared with the wound of PBS group, and the wound contraction was significant. We can see from Fig. 4 and Fig. 5, Compared with the wound treated with PBS, the wounds of ZKSA-P-1001 and EGF showed obvious shrinkage on the third day after operation, the difference was significant, and the local application of ZKSA-P-1001 wound was performed. The wounds of the positive control EGF were significantly contracted in the last four days. Through three consecutive traumatic repair processes in mice, we believe that ZKSA-P-1001 can significantly promote the repair of wounds in the whole cortical defect of mice, and it shows better healing ability than the positive control EGF.

The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. It should be noted that those skilled in the art will also A number of improvements and additions may be made which are also considered to be within the scope of the invention. All of the equivalents of the changes, modifications, and evolutions that can be made by the above-disclosed technical content are all those skilled in the art without departing from the spirit and scope of the present invention. Equivalent embodiments; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above-described embodiments in accordance with the essential techniques of the present invention are still within the scope of the technical solutions of the present invention.

Claims (10)

An isolated polypeptide comprising: (a) a polypeptide consisting of the amino acid set forth in SEQ ID NO: 1; (b) or substituted, deleted or added in the amino acid sequence set forth in SEQ ID NO: A polypeptide derived from (a) having several amino acids and having an equivalent activity. An isolated polynucleotide encoding the polypeptide of claim 1. A recombinant expression vector comprising the isolated polynucleotide of claim 2. A host cell comprising the recombinant expression vector of claim 3 or a polynucleotide isolated according to claim 2 integrated in the genome. A method of producing a polypeptide according to claim 1, which is selected from any of the following: (1) synthesizing the polypeptide by chemical synthesis; (2) cultivating the host cell of claim 4 under appropriate conditions and expressing the polypeptide, followed by isolation and purification to obtain the polypeptide. The use of the polypeptide according to claim 1, wherein the use is selected from the group consisting of: (1) use of the polypeptide in the preparation of a skin fibroblast proliferation promoter; (2) preparation of the vascular endothelial cell Use in a proliferation promoter; (3) use of the polypeptide in preparing a vascular endothelial cell migration promoting agent; (4) use of the polypeptide in preparing a skin superficial layer or a skin dermis damage repairing agent; The use of the polypeptide in the preparation of a skin wound healing promoting drug; (6) the use of the polypeptide in the preparation of a medicament for treating skin defects and refractory skin ulcers; (7) the preparation of the polypeptide for the treatment of traumatic skin lesions Use in medicine. The use of the polypeptide of claim 1 for the preparation of a skin restorative drug, a post-skin repair medical device, a skin repair supplement or a skin repair cosmetic. A pharmaceutical composition comprising an active ingredient comprising a polypeptide according to claim 1 and a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 8, wherein the polypeptide acts as an active ingredient to: (1) promote proliferation of skin fibroblasts; (2) promote proliferation of vascular endothelial cells; 3) promote vascular endothelial cell migration; (4) promote the repair of skin superficial or dermal layer damage; (5) promote skin wound healing; (6) treat traumatic skin damage; (7) repair skin damage. A skin repair product comprising an active ingredient comprising the polypeptide of claim 1, the product being a post-operative medical device, a health care product or a cosmetic.

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