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WO2015088162A1 - Composition permettant de favoriser la différenciation des cellules souches en chondrocytes - Google Patents

Composition permettant de favoriser la différenciation des cellules souches en chondrocytes Download PDF

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Publication number
WO2015088162A1
WO2015088162A1 PCT/KR2014/011329 KR2014011329W WO2015088162A1 WO 2015088162 A1 WO2015088162 A1 WO 2015088162A1 KR 2014011329 W KR2014011329 W KR 2014011329W WO 2015088162 A1 WO2015088162 A1 WO 2015088162A1
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stem cells
composition
expression
mir
chondrocytes
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Korean (ko)
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이진우
이슬기
윤동석
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Industry Academic Cooperation Foundation of Yonsei University
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Industry Academic Cooperation Foundation of Yonsei University
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    • 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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0655—Chondrocytes; Cartilage
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/65—MicroRNA

Definitions

  • the present invention relates to a composition for promoting differentiation from stem cells to chondrocytes comprising an inhibitor of microRNA.
  • Stem cells are cells that can differentiate the organisms that make up the tissue into a variety of cells. These stem cells are used to differentiate the undifferentiated cells of the embryonic and prenatal stages. Collectively. Stem cells are differentiated into specific cells by differentiation stimulation (environment), and unlike the differentiated cells in which cell division is stopped, they can proliferate because they can produce the same cells as themselves by cell division (proli ferat). It has the property of ion expansion, and can be differentiated into other cells by different environment or differentiation stimulus, so it has plasticity in differentiation.
  • Stem cells are largely derived from embryos and have itipotent embryonic stem cells (ES cells) with the potential to differentiate into all cells and multipotents obtained from each tissue ( It is divided into adult stem cells of mult ipotency.
  • Embryonic stem cells are undifferentiated cells capable of unlimited proliferation and can be differentiated into all cells. Unlike adult stem cells, embryonic stem cells can be generated and can be inherited to the next generation.
  • embryonic stem cells have been proposed as an alternative to overcome these problems.
  • Mesenchyme Stem cells are pluripotent cells capable of differentiation into adipocytes, bone cells, chondrocytes, muscle cells, neurons, and cardiomyocytes, and have been reported to have a function of regulating immune response.
  • m i croRNA micro RNA
  • mi croRNA regulates target genes by digesting the target mRNA with a small NA of 20-24 nucleotides or by inhibiting post-transcriptional processes.
  • Many studies have reported that mi croRNA plays an important role in various biological processes including cell proliferation, death and differentiation.
  • Degenerative arthritis is a representative senile disease that nearly 100% of people over 65 years of age are 70-80%.
  • more than 10% of the population in Korea suffers from degenerative arthritis, and the prevalence is increasing very rapidly due to the rapid aging of Korean society.
  • the cartilaginous tissue which is a structural complete layer, is surrounded by a membrane at the end of the joint within the joint. It prevents pain and bone abrasion that may occur when the bones directly contact each other.
  • Chondrocytes are the only cellular components in cartilage tissues, which are responsible for synthesizing and secreting collagen and proteoglycan matrices, which are essential for the normal function of cartilage tissues, and decomposing them at appropriate rates, thereby maintaining functional homeostasis of articular cartilage tissue It plays an essential role in giving. Therefore, maintaining the activity of these chondrocytes is directly related to the structural functional preservation of joint tissue.
  • the chondrocytes In order for the function of chondrocytes in the chondrocytes to function normally, the chondrocytes should be properly formed and differentiated, the survival of the chondrocytes generated should be well protected, and the calcification of the chondrocytes existing is continuously suppressed. Hardening should be prevented. The most common cause of degenerative arthritis is the loss of the intrinsic functionality of these articular chondrocytes with age.
  • micro RNA-495 plays a role in inhibiting cartilage differentiation of human stem cells, and when the expression thereof is inhibited, specific differentiation into chondrocytes is induced to treat cartilage tissue through regeneration.
  • the present invention has been completed by discovering that the present invention can be usefully applied to diseases such as degenerative arthritis.
  • an object of the present invention is to provide a composition for promoting differentiation from stem cells to chondrocytes.
  • Another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of cartilage damage disease.
  • composition for promoting differentiation from stem cells to chondrocytes comprising a nucleic acid molecule that inhibits the expression of micro R A-495. As an active ingredient.
  • micro RNA-495 plays a role in inhibiting cartilage differentiation of human stem cells, and when inhibiting its expression, specific differentiation into chondrocytes is induced and can be treated through regeneration of cartilage tissue. It has been found that it can be usefully applied to diseases such as degenerative arthritis.
  • stem cell refers to a cell capable of differentiating into various cells constituting biological tissue, and refers to undifferentiated cells that can be reproduced without limitation to form specialized cells of tissues and organs. .
  • Stem cells are developable pluripotent or pluripotent cells. Stem cells can divide into two daughter stem cells, or one daughter stem cell and one derived (trans) cell, and then proliferate into mature, fully formed cells of the tissue.
  • stem cells capable of promoting differentiation into chondrocytes with the composition of the present invention are mesenchymal stem cells. More specifically, the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells.
  • the term “mesenchymal stem cell” refers to a stem cell having a multipotency capable of differentiation into adipocytes, bone cells, chondrocytes and muscle cells, neurons, and cardiomyocytes. Mesoderm stem cells are identified by their swirling morphology and expression of the basic cell surface markers CD73 (+), CD105 (+), CD34 (-), and CD45 (-).
  • the term “nucleic acid molecule” is meant to encompass DNMgDNA and cDNA) and RNA molecules inclusively, and the nucleotides that are the basic structural units in nucleic acid molecules are naturally occurring nucleotides, as well as analogs in which sugar or base sites are modified. also included (analogue) (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)).
  • nucleic acid molecule that inhibits expression refers to any nucleic acid-based molecule that has a complementary sequence to the target miRNA, ie, the sequence of miR-495, and can form a duplex with niiRNA. Include. Thus, the term “nucleic acid molecule that inhibits expression” may also be expressed as "complementary nucleic acid-based inhibitor ' '.
  • the nucleic acid molecule used in the present invention is siRNA, shRNA, miRNA, ribozyme (r ibozyme), peptide nucleic acids (PNA) or antisense oligonucleotides.
  • siRNA refers to a short double-chain NA that can induce RNAKRNA interference through cleavage of specific mRNAs. It consists of a sense RNA strand having a sequence homologous to the mRNA of the other gene and an antisense RNA strand having a complementary sequence. siRNA is provided as an efficient gene knockdown method or gene therapy because it can suppress the expression of the target gene. siRNAs are not limited to fully paired double-stranded RNA portions of RNA, but are paired by mismatches (the corresponding bases are not complementary), bulges (there are no bases corresponding to one chain), etc. May be included.
  • the total length is 10 to 100 bases, preferably 15 to 80 bases, most preferably 20 to 70 bases.
  • the siRNA terminal structure can be either smooth or cohesive, as long as the expression of the target gene can be suppressed by the RNAi effect.
  • the cohesive end structure is possible in both a three-terminal protruding structure and a five-terminal protruding structure.
  • the number of protruding bases is not limited. For example, the number of bases may be 1 to 8 bases, preferably 2 to 6 bases.
  • siRNA is, for example, one side to the extent that can maintain the expression inhibitory effect of the target gene
  • the protruding portion of the terminal may include low molecular RNA (eg, natural RNA molecules such as tRNA, rRNA, viral RNA or artificial RNA molecules).
  • the siRNA terminal structure does not need to have a cleavage structure at both sides, and may be a stem loop type structure in which one terminal portion of the double chain RNA is connected by a linker RNA.
  • the length of the linker is not particularly limited as long as it does not interfere with pairing of stem portions.
  • shRNA refers to a nucleotide composed of 50-70 single strands, and forms a stem-loop structure in vivo.
  • Long R A of 19-29 nucleotides complementary to both loop regions of 5-10 nucleotides form base pairs to form a double stranded stem.
  • miRNA modulates gene expression and includes full length 20-50 nucleotides, preferably 20-45 nucleotides, more preferably 20-40 nucleotides, even more preferably 20- Refers to a single stranded RNA molecule consisting of 30 nucleotides, most preferably 21-23 nucleotides miRNAs are oligonucleotides that are not expressed intracellularly and have a short stem-loop structure. homologous (messenger RNA) to the whole or in part, and by complementary binding to the mRNA inhibits the target gene expression.
  • Ribozyme refers to an RNA molecule having a function such as an enzyme that recognizes and cleaves itself by a specific sequence of a base as a kind of RNA. Ribozyme is a complementary nucleotide sequence of a target messenger RNA strand consisting of a region that binds with specificity and a region that cleaves the target RNA.
  • PNA protein nucleic acid
  • PNA protein nucleic acid
  • PNA is a molecule having both nucleic acid and protein properties, and means a molecule capable of complementarily binding to DNA or RNA.
  • PNA was first reported in 1999 as analogous DNA with nucleobases linked by peptide bonds (Nielsen PE, Egholm M, Berg RH, Buchardt 0, “Sequence-select ive recognition of DNA by strand displacement with a thymine -subst i tuted polyamide ", Science 1991, Vol. 254: ppl497-1500]).
  • PNA is not found in nature Artificially synthesized by chemical method. PNAs hybridize with native nucleic acids of complementary base sequences to form double strands.
  • PNA / DNA double strands are more stable than DNA / DNA double strands and PNA / RNA double strands are more stable than DNA / RNA double strands. It is most commonly used as a peptide basic skeleton that N- (2-aminoethyl) glycine is repeatedly linked by amide bonds. In this case, the backbone of the peptide nucleic acid is electrically different from that of the negatively charged natural nucleic acid. As neutral.
  • the four nucleic acid bases present in the PNA have approximately the same spatial size and distance between the nucleic acid bases as for natural nucleic acids.
  • PNA is not only chemically stable than natural nucleic acids, but also biologically stable because it is not degraded by nucleases or proteases.
  • antisense oligonucleotide refers to DNA or RNA or derivatives thereof that contain a nucleotide sequence complementary to a sequence of a particular mRNA, which binds to a complementary sequence within the mRNA and translates the mRNA into a protein.
  • Antisense nucleotide sequence of the present invention refers to a DNA or RNA sequence complementary to the mRNA of the target gene and capable of binding to the mWA of the target gene, and the translation of the target gene into mRNA and translocation into the cytoplasm. cation), maturation ions or any other essential biological activity can be inhibited.
  • the antisense oligonucleotides are 6 to 100 bases in length, preferably 10 to 40 bases.
  • the antisense oligonucleotides can be modified at the position of one or more bases, sugars or backbones to enhance efficacy (De Mesmaeker et al., Curr Op in Struct Biol., 5 (3): 343-55, 1995). Oligonucleotide backbones can be modified with phosphorothioates, phosphoesters, methyl phosphonates, short chain alkyls, cycloalkyl short chain heteroatomics, heterocyclic intersaccharide bonds, and the like. In addition, antisense nucleic acids may include one or more substituted sugar moieties. Antisense oligonucleotides may comprise modified bases.
  • Modified bases include hypoxanthine, 6-methyladenine, 5-methyl pyrimidine (particularly 5 ′ methylcytosine), 5-hydroxymethylcytosine (HMC).
  • HMC 5-hydroxymethylcytosine
  • Glycosyl HMC gentobiosil HMC, 2-aminoadenine, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminonucleosil) adenine, 2, 6-diaminopurine and the like.
  • the nucleic acid molecule used in the present invention is an antisense oligonucleotide.
  • the antisense oligonucleotides used in the present invention have a length of 15 to 40 nucleotides.
  • the .1 sense oligonucleotide comprises a sequence complementary to the 15th to 21st nucleotide sequence of SEQ ID NO: 1.
  • the antisense oligonucleotide comprises the nucleotide sequence of SEQ ID NO: 2.
  • the composition of the present invention increases the expression of Sox9 in stem cells.
  • mi R-495 inhibits the expression of Sox9, which is an important transcription factor in the whole process of cartilage differentiation
  • composition of the present invention which inhibits mi R-495 inhibits the expression of Sox9 in stem cells.
  • the present invention provides a pharmaceutical composition for the prevention or treatment of cartilage damage diseases comprising the composition of the present invention as an active ingredient.
  • the cartilage injury disease prevented or treated with the composition of the present invention is degenerative arthritis.
  • the term "ost eoar thr itis” means a disease in which joint tissue necessary for joint movement is damaged due to quantitative loss of cartilage tissue.
  • the composition of the present invention promotes regeneration of cartilage tissue in the joint by inducing specific differentiation of endogenous stem cells or transplanted therapeutic stem cells into chondrocytes.
  • a symptomatic approach such as conventional inflammation control, results in an inflammation of the joint tissue caused by abnormal immune function.
  • the compositions of the present invention provide a fundamental treatment for degenerative arthritis.
  • the invention provides a method for screening a composition for promoting differentiation from stem cells to chondrocytes comprising the following steps:
  • the sequence listing first sequence is a nucleotide sequence of m iR-495.
  • test material refers to an unknown material used in screening to examine whether it affects the expression level of miR-495.
  • test defects include, but are not limited to, chemicals, nucleotides, antisense-RNAs, small interference RNAs (siRNAs), and natural extracts.
  • siRNAs small interference RNAs
  • the expression level of iiiiR-495 is measured in the cells treated with the sample. The measurement of the expression level can be made through various methods known in the art, for example, can be measured through a micro array or the like.
  • the test substance when the expression of niiR-495 is suppressed, the test substance may be determined as a composition for promoting differentiation into chondrocytes.
  • the present invention provides a method for screening a composition for preventing or treating cartilage damage disease comprising the following steps:
  • the sample is determined as a composition for preventing or treating cartilage damage diseases.
  • Stem cells, nucleotides, and specific screening methods and procedures used in the present invention have already been described above, so the description thereof is omitted to avoid excessive duplication.
  • Substances discovered through the expression changes of mi R-495 using the method of the present invention promote differentiation of endogenous stem cells or transplanted therapeutic stem cells into chondrocytes, resulting in degeneration of cartilage tissues. It can be used as a therapeutic composition, such as arthritis.
  • the present invention provides a method for promoting differentiation into chondrocytes comprising contacting stem cells with a composition comprising a nucleic acid molecule that inhibits the expression of microRNA-495 as an active ingredient.
  • the method for promoting differentiation into chondrocytes of the present invention is achieved by contacting stem cells with a composition comprising a nucleic acid molecule that inhibits the expression of microRNA-495 as an active ingredient, which is another aspect of the present invention.
  • the content will be omitted in order to avoid excessive complexity of the description.
  • the present invention provides a method for preventing or treating cartilage damage disease comprising administering to a subject (subj ec t) a composition comprising a nucleic acid molecule that inhibits the expression of microRNA-495 as an active ingredient. Provide a method.
  • the cartilage damage disease is degenerative arthritis.
  • the prophylactic or therapeutic method of the present invention is performed by administering to a subject a composition comprising a nucleic acid molecule that inhibits the expression of microRNA-495 as another active aspect of the present invention. Omitted to avoid excessive complexity of the description.
  • the present invention provides a composition for promoting differentiation of stem cells, specifically, mesenchymal stem cells into cartilage cells, a composition for preventing or treating cartilage damage diseases and a screening method thereof.
  • the present invention newly identified miR-495 as a miRNA that inhibits the differentiation of stem cells into chondrocytes, and it was confirmed that the stem cells having pluripotency can be efficiently differentiated into chondrocytes through the inhibition thereof. .
  • the present invention can be usefully used for the fundamental prevention and treatment of cartilage damage-related diseases including degenerative arthritis through regeneration of lost cartilage tissue.
  • FIG. 1 is a diagram showing the expression patterns of Sox9 and miRNA during chondrocyte differentiation.
  • La is a diagram showing that the mRNA expression of Sox9 gradually increases as the mesenchymal stem cells differentiate into cartilage.
  • FIG. Lb is a diagram showing the microRNAs showing a difference of 1.5 times or more from the control group by microR A microarray analysis using a one-way AN0VA as a hierarchical clustering map ( D H).
  • FIG. 2 shows the expression patterns of miR-495 and niiR-431 during chondrocyte differentiation.
  • miR-495 and miR-431 were expected to bind to Sox9 3 ′ UTR (FIG. 2A) and confirmed by real-time PCR that expression of miR-495 and miR-431 decreased during cartilage differentiation of mesenchymal stem cells. (FIG. 2B).
  • FIG. 3 shows the results of miR-495 at the mRNA (FIG. 3A) and protein (FIG. 3B) levels, respectively, of the effect of miR-495 on Sox9 expression in human chondromacoma cell SW1353.
  • FIG. 3A shows the results of miR-495 at the mRNA (FIG. 3A) and protein (FIG. 3B) levels, respectively, of the effect of miR-495 on Sox9 expression in human chondromacoma cell SW1353.
  • FIG. 4 is a diagram showing the results of confirming that miR-495 specifically binds to the 3 'UTR of Sox9.
  • Figure 4a shows the 3'UTR site of Sox9 to which miR-495 binds.
  • 4B is a schematic diagram of a vector structure including Sox9 3'UTR.
  • Figure 4c is a diagram showing the results of performing a luciferase analysis after the vector containing the miR-495 and Sox9 3'UTR introduced into the cell.
  • Figure 4d shows the results of performing luciferase analysis after mutating the 15-21st nucleotide sequence that miR-495 can bind to Sox9 3 ' UTR The figure shows that the 15-21st sequence of miR-495 is important for regulating Sox9 expression.
  • Figure 5 is treated with miR-495 in mesenchymal stem cells after increasing the expression of Sox9 using TGF-I 3 and then quantification of Western blotting (Fig. 5a) and Western blotting results of the expression of Sox9, respectively
  • Figure 5b shows the results of the investigation through.
  • FIG. 6 is a diagram showing the effect of miR-495 on the difference in the expression of chondrocyte markers.
  • FIG. 7 is a diagram showing the effect of ' overexpression of miR-495 on the differentiation of human mesenchymal stem cells into cartilage.
  • Figure 7a is a diagram showing the results of measuring the GAG (glycosaminoglycan) on the 10th day after induction of cartilage differentiation after treating miR-sc and miR-495 to mesenchymal stem cells.
  • Figure 7b is a diagram showing the results of safranin ⁇ staining of miR- sc and miR-495 treatment group.
  • BM aspirates were obtained from the posterior iliac crest of 12 adult donors aged 19-63 years with the approval of the Inst itut ional Review Board (IRB).
  • Human bone marrow-derived mesenchymal stem cells (hMSCs) were selected based on their ability to adsorb to plastic cell culture flasks.
  • BM aspirates were 10% FBSCGibco, Grand Island, NY, USA) and 1% Incubated for 7 days in DMEM-LG supplemented with antibiotic-antifungal solution (Gibco), low-glucose Dulbecco's modified Eagle's medium; Wei gene, Daegu, Korea.
  • SW1353 a human chondrosarcoma cell line, was cultured in DMEM-HG (DMEM-high glucose, Welgene) supplemented with 10% FBS and 1% antibiotic-antifungal solution.
  • Micromass culture methods were used to differentiate hMSCs into cartilage in vitro. Trypsinized hMSCs were resuspended in lMEMO 7 cells / inL concentration in DMEM-LG containing 10% FBS, of which only 1 ⁇ L was added to each well of 24-well pleated (lxl0 5 cells per well). Dotting was done.
  • Cartilage differentiation medium consisting of DMEM-HG supplemented with Invitrogen), 50 y / mL ascorbic acid (Invitrogen) and 10 ng / mL TGF - P3 (R & D systems, Minneapolis, MN, USA) was added.
  • Cartilage differentiation cultures containing 10 ng / mL TGF-3 were replaced every two days during the in vitro differentiation period.
  • the primer set was purchased from Bioneer (Dae j eon, South Korea), and the primers used were as follows: GAPDH (P267613), Sox9 (P232240). There were no proven primers for Col2al and aggr'ecnan ' designed as follows: Col2al, 5'-GTCCTCTCCCAAGTCCACACAG-3' (sense) and 5 1 -GGGCACGAAGGCTCATCATTC-3 '(antisense); aggrecai 5'-CCACTGTTACCGCCACTT— 3 '(sense) and 5' GTAGTCTTGGGCATTGTTGT 3 '(antisense).
  • PCR process initiated 30 sec at 95 ° C After that, 40 cycles of heat cycle for 5 seconds at 95 ° C., followed by 20 seconds at 60 ° C. SYBR fluorescence was detected in the annealing / extension step, and all real time PCR products finally had a size of about 100 bp. The measurements of each sample were normalized to the internal control GAPDH.
  • Synthesis kit (Clontech, Mountain View, CA, USA) was used according to the manufacturer's instructions, and cDNA synthesized from ni i cro RNA was quantified by SYBR® qRT-PCR (Clontech). Specific Maturity—A list of primer sets used for microRNA amplification was obtained from Genolution (Genolu ion, Seoul, South Korea) and Bioneer Inc.
  • RNA of undifferentiated hMSCs (0-10 days culture) and chondrogenic hMSCs derived from the same donor were isolated using RNA iso Plus (Takara) according to the manufacturer's instructions. The overall quality of total RNA was confirmed using a spectrophotometer. To 1.0 Genechip® miRNA array (Mfymetrix, Santa Clara, CA, USA) was investigated, the expression pattern of each microRNA. Pearson correlation was performed to identify microRNAs specifically expressed in cartilage differentiation-induced mesenchymal stem cells. Briefly, differently expressed genes were selected using routine equations subtracting control values from differentiated groups: applying a strict cut-off reference point with at least 1.5-fold difference in expression for screening genes. It was. Data was obtained by analyzing two donors, and the results of the two donors were crossed. MicroRNA transformation
  • Selected microRNAs were purchased from Genolution Inc. to analyze the function of the screened microRNAs and their effect on Sox9 expression.
  • the purchased microRNA mimics are prepared in the form of double strands, and once transformed, they undergo an internal mechanism of producing mature microRNAs. Since the anti-microRNA consists of a single strand that targets the complementary sequence of the miRNA, the scrambled oligonucleotide of the anti-microRNA was used as a negative control separately from the scramble oligonucleotide of the microRNA mimic.
  • Anti-miR—SC and anti-miR-495 were purchased from Bioneer inc. And designed with the following sequence: anti-miR-495: 5′-AAGMGUGCACCAUGUUUGUUU-3 ′ (sense), anti-miR-SC: 5 '-UCACAACCUCCUAGA GAGUAGA-3' (sense).
  • the constructed microRNA was transformed into SW1353 and hMSC using Lipofectamin LTX & Plus reagent (Invi trogen) according to the instructions.
  • Western blotting analysis Western blotting analysis
  • the membrane was washed repeatedly with IX TBST and then incubated with the appropriate HRP horseradish peroxidase-conjugated secondary antibody (Aniershani Pharmacia) for 1 hour.
  • GAPDH horseradish peroxidase-conjugated secondary antibody
  • PEZX-S0X9 3 'UTR and pEZX-MTOl vectors were purchased from Genecopoeia (Rockvi 1 le, MD, USA) to determine if miR-495 binds to Sox9 3' UTR.
  • pEZX-S0X9 3 'UTR contains the firefly luciferase gene and the Renilla luciferase gene with S0X9 3' UTR, whereas pEZX-MTOl is the firefly luciferase and Renilla lucifer without S0X9 3 'UTR Laase gene.
  • PEZX-S0X9 3 ′ UTR or pEZX-MTOl was co-transformed into miR-495 or negative microRNA and HeLa cells using Lipofectaniin LTX & Plus reagent (Invi trogen).
  • Firefly and Renilla luciferase activity was measured using a reporter assay system (Promega, Madison, Wisconsin, USA) 24 hours after the dual-luciferase microRNA transformation, and firefly psiferase activity was determined by Renilla expression. Standardized three times for. Immunohistochemistry
  • Paraffin embedded sections were deparaffinized, rehydrated and washed twice with PBS. To reduce nonspecific background staining by endogenous peroxides, sections were incubated in hydrogen peroxide blocks for 10 minutes and washed twice with PBS. Sections were incubated overnight at 4 ° C with rabbit anti-C0L2A1 (Santa Cruz) or mouse anti-agrycan (Santa Cruz) and washed with PBS.
  • PE Physicalerythrin conjugated goth anti-rabbit secondary antibody
  • GFP reen fluorescent protein
  • the mass pellet was washed twice with PBS and fixed in 10% formalin solution for 24 hours. After fixation, the pellets were dehydrated in ethane and the dehydrated pellets were embedded in paraffin and sectioned. For safranin 0 staining, the sectioned micromass pellets were deparaffinized and rehydrated. Rehydrated pellets were washed with PBS and stained with 1% safranin 0 solution (Sigma, St. Louis, MO, USA) dissolved in 1% acetic acid for 30 minutes. Safranin 0-of dyed pellets Hematoxylin solution (Sigma) was used to stain the background. Stained pellet sections were observed under a microscope.
  • the amount of sul fated GAG in culture obtained from niicroRNAs-transformed cartilage micromass was measured according to the manufacturer's instructions using Blyscan 3 ⁇ 4 (Bioc () lor, New ownabbey, Northern Ireland, UK). Briefly, a total of 500 yL culture is gently shaken and mixed with 1 nil Blyscan staining reagent for 30 minutes to complete the binding of the GAG-dye. After centrifugation, the dye bound to GAG was dissolved in the separation reagent. The recovered dye concentration was measured at 656 nm and a standard curve was generated using Chondroitin 4-Sulfate Standard Solution (Biocolor). All samples were run three times. Statistical analysis
  • microRNA microarrays were performed using CT10 and TGF-I33, which did not induce differentiation of mesenchymal stem cells, using CH10 and CT10 samples that did not induce cartilage differentiation for 10 days. Sox9, an important transcription factor for cell cartilage differentiation, increased expression during cartilage differentiation (FIG. La). The microRNA microarrays were analyzed using one-way AN0VA, and microRNAs showing 1.5 times or more expression difference compared to the control group were represented by a hierarchical clustering map (FIG. lb). Expression of iR-495 and miR-431 during Chondrocyte Differentiation Period
  • Luciferase assays were performed using a luciferase vector incorporating Sox9 3 ′ UTR, and the luciferase activity was reduced in proportion to the increased concentration of niiR-495 (FIG. 4C).
  • the seed sequence of miR-495 was confirmed by confirming that luciferase activity did not change when mut-miR-495, which mutated the seed sequence (seed seqeunce) predicted to bind miR-495, did not change ( 4d). It was confirmed that miR-495 directly binds to Sox9 and affects it. miR-4957 ⁇ Effect on endogenous Sox9 expression in human mesenchymal stem cells

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Abstract

La présente invention concerne une composition permettant de favoriser la différenciation à partir des cellules souches, plus spécifiquement des cellules souches en chondrocytes, une composition pour la prévention ou le traitement des lésions ostéochondrales, et un procédé de dépistage associé. La présente invention a récemment identifié miR-495 comme le miARN inhibant la différenciation des cellules souches en chondrocytes et il a été confirmé que des cellules souches pluripotentes peuvent être efficacement différenciées en chondrocytes par l'inhibition de miR-495. La présente invention peut être utilisée à bon escient pour la prévention ou le traitement de base de maladies associées à une lésion ostéochondrale ainsi que de l'arthrose par le biais de la reproduction du tissu cartilagineux perdu.
PCT/KR2014/011329 2013-12-13 2014-11-24 Composition permettant de favoriser la différenciation des cellules souches en chondrocytes Ceased WO2015088162A1 (fr)

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KR1020130155690A KR20150069375A (ko) 2013-12-13 2013-12-13 줄기세포로부터 연골세포로의 분화 촉진용 조성물
KR10-2013-0155690 2013-12-13

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WO2019078587A1 (fr) 2017-10-18 2019-04-25 서울대학교 산학협력단 Composition contenant, en tant que principe actif, une culture de cellules progénitrices ostéochondrales dérivées de moelle osseuse de poulet pour favoriser l'ostéogenèse ou induire une différenciation chondrogénique
KR101992538B1 (ko) 2017-10-18 2019-06-24 서울대학교산학협력단 닭 골수 유래 골·연골전구세포 배양액을 유효성분으로 포함하는 골 생성 촉진용 조성물
WO2019231299A1 (fr) * 2018-05-31 2019-12-05 고려대학교 산학협력단 Acide nucléique modifié inhibant un micro arn et son utilisation

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WO2010139812A1 (fr) * 2009-06-05 2010-12-09 Febit Holding Gmbh Empreinte de micro-arn utilisée dans le diagnostic de maladies
KR101223660B1 (ko) * 2010-05-20 2013-01-17 광주과학기술원 ＨＩＦ－２α 억제제를 유효성분으로 포함하는 관절염 예방 또는 치료용 약제학적 조성물
KR101286154B1 (ko) * 2011-08-10 2013-07-15 연세대학교 산학협력단 안티센스 올리고뉴클레오타이드를 포함하는 줄기세포로부터 연골세포 분화 촉진용 및 항암 약제학적 조성물

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KR101223660B1 (ko) * 2010-05-20 2013-01-17 광주과학기술원 ＨＩＦ－２α 억제제를 유효성분으로 포함하는 관절염 예방 또는 치료용 약제학적 조성물
KR101286154B1 (ko) * 2011-08-10 2013-07-15 연세대학교 산학협력단 안티센스 올리고뉴클레오타이드를 포함하는 줄기세포로부터 연골세포 분화 촉진용 및 항암 약제학적 조성물

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