KR20190064401A - Pharmaceutical compositions for preventing or treating cartilage diseases - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cartilage disease, which comprises at least one selected from the group consisting of an ITGBL1 (integrin beta-like protein 1) protein and a gene encoding the protein, Or a pharmaceutically acceptable salt thereof.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing or treating cartilage diseases,

The present invention relates to a pharmaceutical composition for preventing or treating cartilage disease comprising at least one selected from the group consisting of an ITGBL1 (integrin beta-like protein 1) protein and a gene encoding the protein as an active ingredient, Lt; RTI ID = 0.0 > pharmaceutically < / RTI >

Degenerative cartilage disease is caused by pain caused by inflammation of the injured cartilage site and loss of additional cartilage. For patients with early cartilage disease, treatment with analgesic anti-inflammatory drugs to suppress inflammation and pain is mainly performed. In the case of mid-term patients, treatment with cyclic anti-inflammatory drugs and hyaluronic acid, and gene therapy or cell therapy And in the terminal period, artificial joint surgery is mainly used.

It is known that TGF-β is a gene therapy agent used in combination with a cell therapy agent that promotes cartilage regeneration. Recently, Invossa product of Kolon Pharmaceutical Co., Ltd. is known to contain TGF-β gene therapy agent.

The treatment methods known to have a cartilage regenerating effect include the use of a stem cell treatment agent or an autologous cell treatment agent. However, the two therapeutic agents fail to successfully enter the market due to insufficient cartilage regeneration effect and high treatment cost.

Therefore, in order to fundamentally treat cartilage diseases, it is necessary to prevent the additional cartilage damage caused by inflammation and regenerate damaged cartilage. However, up to now, there has been proposed a treatment for suppressing additional cartilage damage caused by inflammation and regenerating damaged cartilage There has been no development of a treatment method using a method in which the two drugs are combined.

Accordingly, one object of the present invention is to provide a recombinant vector which is selected from the group consisting of ITGBL1 (Integrin beta-like protein 1) protein, a gene encoding the protein, a recombinant vector containing the gene, and a recombinant vector transformed with the recombinant vector And a pharmaceutical composition for preventing or treating cartilage diseases containing at least one kind thereof as an active ingredient.

Another object of the present invention is to provide a recombinant vector comprising ITGBL1 (Integrin beta-like protein 1) protein, a gene encoding the protein, a recombinant vector containing the gene, and a recombinant cell transformed with the recombinant vector Or more as an active ingredient, and a reagent composition for inhibiting integrin protein activity in vitro.

It is still another object of the present invention to provide a pharmaceutical preparation containing the composition as an active ingredient.

It is still another object of the present invention to provide a method of preventing or treating cartilage diseases comprising administering a pharmaceutically effective amount of the composition.

In one specific example of the present invention, the ITGBL1 protein expressed in the process of differentiation of cartilage cells and secreted around chondrocytes was identified (Example 1, Fig. 1).

In one specific embodiment of the present invention, when the ITGBL1 gene is injected into chondrocytes and the expression of ITGBL1 protein is increased, it is confirmed that the ITGBL1 protein promotes cartilage formation by confirming the increase of cartilage tissue. (Example 2, Example 3, Fig. 2, Fig. 3).

In a specific example of the present invention, it was confirmed that when the ITGBL1 gene is injected into chondrocytes induced by arthritis, the expression is increased, thereby promoting the generation of cartilage (Col2a1, Sox9) (Example 4, Fig. 4 ).

In a specific embodiment of the present invention, when the ITGBL1 gene is injected into cartilage cells to increase the expression of ITGBL1 protein, the production of inflammatory factors (MMP-3, MMP-13, etc.) is reduced in cartilage cells induced by arthritis It was confirmed that the ITGBL1 protein had an effect of suppressing inflammation (Example 5, Fig. 4).

In a specific embodiment of the present invention, degenerative joint disease is induced by inhibiting the expression of ITGBL1 protein by injecting an shRNA vector that inhibits the expression of the ITGBL1 gene into a knee cartilage tissue using a mouse animal model Example 8, Fig. 9).

In a specific example of the present invention, it was confirmed that when the ITGBL1 gene was injected into the joint of the mouse animal model subjected to the meniscus incision, the expression of the ITGBL1 protein was increased, and the regeneration of the joint was promoted (Example 4, Fig. 4 ).

In one specific embodiment of the present invention, the ITGBL1 protein was found to inhibit the activity of integrin proteins, particularly integrin-beta 1 (Example 6 and Figures 5 and 6).

In a specific embodiment of the present invention, it was confirmed that the expression of ITGBL1 in chondrocytes increased the expression of chondrogenic factors (Sox9, Col2a1) in cartilage cells due to the inhibitory function of integrin activity of ITGBL1 protein. (Example 7, Fig. 7).

In a specific embodiment of the present invention, the expression of MMP3 and MMP13, which promote cartilage degradation, is increased by treating Fragmented Fibronectin (Fn-f 29kDa), which is known to cause cartilage degeneration in cartilage cells, 3, reduced the expression of MMP13 and, when re-activate the integrin to handle or Mn ^{2 +} DTT was found to increase the expression of MMP3, MMP13 (example 8, Fig. 8).

In a specific embodiment of the present invention, arthritis is induced by inhibiting the expression of ITGBL1 in the knee joint of a mouse, and when ATN-161, an integrin-beta1 inhibitor, is administered to the knee joint in which ITGBL1 expression is inhibited, arthritis is restored (Example 8, Fig. 9)

Therefore, ITGBL1 (Integrin beta-like protein 1) protein of the present invention has an effect of inhibiting inflammation of cartilage disease and promoting cartilage formation, and thus the composition containing ITGBL1 protein can be used for prevention or treatment of cartilage disease .

Hereinafter, the present invention will be described in more detail.

In one embodiment, the present invention provides a recombinant vector comprising ITGBL1 (integrin beta-like protein 1) protein, a gene encoding the protein, a recombinant vector comprising the gene, and recombinant cells transformed with the recombinant vector. And more particularly, to a pharmaceutical composition for preventing or treating cartilage disease.

The ITGBL1 gene has been conventionally known to play a role in regulating the marker of the metastatic cancer or the WNT / PCP signal.

In the present invention, ITGBL1 protein has an effect of inhibiting inflammation of cartilage disease and promoting cartilage formation, and this effect is proved to be a phenomenon caused by the inhibitory activity of integrin activity of ITGBL1. Therefore, the composition comprising the ITGBL1 protein may have an effect of preventing or treating cartilage diseases, and the ITGBL1 gene and protein may be used as an inhibitor for inhibiting the activity of integrins. Therefore, Lt; / RTI > The ITGBL1 protein may comprise the amino acid sequence of SEQ ID NO: 20, and the gene encoding the protein may comprise the nucleotide sequence of SEQ ID NO: 21.

The term "protein" as used herein refers to a molecule comprising a polymer of amino acids linked together by a peptide bond (s). A protein may be a polypeptide comprising two or more amino acids.

The ITGBL1 (Integrin beta-like protein 1) protein may be derived from a vertebrate animal such as a mammal such as a human, a mouse, or an amphibian such as a frog. For example, human ITGBL1 (eg, GenBank Accession Nos. NP_001258683.1 (cDNA): NM_001271754.1), NP_001258684.1 (coding mRNA (cDNA): NM_01271755.1), NP_001258685.1 (coding mRNA (cDNA): NM_001271756.1), P_004782.1 (coding mRNA 2), XP_005254157.1 (coding mRNA (cDNA): XM_005254100.4)), mouse NP_663442.2 (coding mRNA (cDNA): NM_145467.2), XP_006518984.1 (coding mRNA (cDNA): XM_006518921.3 ), A frog ITGBL1 (eg, GenBank Accession No. NP_001084310.1 (coding mRNA (cDNA): NM_001090841.1)), and the like, but the present invention is not limited thereto.

The term "vector" means means for expressing a gene of interest in a host cell. The vector includes elements for expression of a target gene and may include a replication origin, a promoter, an operator, a transcription termination terminator, and the like into the genome of the host cell. Ribosome binding sites (RBS) for translation into selectable markers and / or proteins to confirm successful introduction into the host cell and / or appropriate enzyme sites for introduction of the IRES (e.g., IRES (Internal Ribosome Entry Site), and the like. The vector may be engineered in a conventional genetic engineering manner to have the fusion polynucleotide (fusion promoter) described above as a promoter. The vector may further comprise a transcription control sequence other than the promoter (e.g., an enhancer, etc.).

The recombinant vector may be a viral or non-viral vector, and the viral vector may be, but is not limited to, adenovirus, adeno-associated virus, helper-dependent adenovirus, retroviral vector, and the like.

The recombinant vector may be constructed by a variety of methods known in the art.

The present invention provides, as yet another embodiment, a cell transformed with the recombinant vector.

The cell may be a cell of a mammal, and the mammalian cell may be a human (for example, a fat-derived stem cell, a bone marrow-derived stem cell, a placenta-derived stem cell, another universal induced stem cell, a chondrocyte, But the present invention is not limited thereto.

The transfer (introduction) of the recombinant vector into a cell can be carried out using a carrier method well known in the art. For example, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, gene bombardment, and the like can be used as the delivery method. For example, liposome-mediated transfection (Lipofector reagent). < / RTI >

The cartilage disease may be, but is not limited to, degenerative arthritis, post traumatic arthritis, or peelable osteochondritis.

In the present invention, the ITGBL1 may be used in the form of DNA, RNA or protein, and may include genes derived from fish, amphibians, algae, and mammalian animals, and may include wild type ITGBL1 nucleotide sequences and amino acid sequence variants have.

The mutant or variant of the amino acid sequence of the ITGBL1 protein means a change in ITGBL1 amino acid sequence, deletion and insertion, and may include a change in chemical properties such as modification of physiological activity and stability of the protein.

The means for increasing the expression of the ITGBL1 gene or protein in the present invention may include all techniques for artificially transferring the ITGBL1 gene into the cell.

In the present invention, it is possible to use only one or more selected from the group consisting of a gene encoding the ITGBL1 protein, a recombinant vector containing the gene, and a recombinant vector transformed with the recombinant vector. However, , A recombinant vector containing the gene, and a recombinant vector transformed with the recombinant vector, in addition to at least one selected from the group consisting of an anti-inflammatory agent, a hyaluronic acid, a cell therapy agent , And stem cell treatment agents can be used in combination.

In the cell treatment agent of the present invention, the cells used for the cell treatment may mean chondrocytes, and may mean various cells associated with cartilage disease, but are not limited thereto.

In the stem cell treatment agent of the present invention, the stem cells may include all kinds of multipotent stem cells including bone marrow-derived stem cells, adipose-derived stem cells, placenta-derived stem cells, induced pluripotent stem cells, and embryo-derived stem cells .

In another aspect, the present invention relates to a pharmaceutical preparation containing the composition as an active ingredient.

The pharmaceutical preparations according to the present invention may be formulated into oral preparations such as powders, granules, tablets, capsules, ointments, suspensions, emulsions, syrups and aerosols or percutaneous preparations such as suppositories and sterilized injection solutions Parenteral formulations, and the like.

The pharmaceutical preparations of the present invention may be pharmaceutically acceptable and additionally contain adjuvants such as physiologically acceptable carriers, excipients and diluents. Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used can be used

When the pharmaceutical preparation according to the present invention is used for parenteral administration, for example, a topical administration such as a liquid preparation, a gel preparation, a cleaning composition, a tablet for insertion, a suppository form, cream, ointment, dressing solution, spray, It may be a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a freeze-dried preparation, a suppository, a cream, an ointment, a jelly, a foam, a cleanser or an insert, May be a skin external agent such as a liquid preparation, a gel preparation, a cleaning composition, and a tablet for insertion. The formulation can be prepared, for example, by adding a solubilizer, an emulsifying agent, a buffering agent for pH control, etc. to the sterilized water. Examples of the non-aqueous solvent or suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like.

More specifically, the pharmaceutical preparation may comprise a carrier for additionally formulating the composition. The carrier may be a binder, a lubricant, a suspending agent, a solubilizer, a buffer, a preservative, a lubricant, an isotonic agent, an excipient, a stabilizer, a dispersant, a suspending agent,

In embodiments where the pharmaceutical preparations of the present invention are applied to humans, the pharmaceutical preparations of the present invention may be administered alone, but are generally administered in a pharmacological manner selected in consideration of the mode of administration and standard phamaceutical practice May be administered in admixture with a carrier. For example, the pharmaceutical preparations of the present invention may be in the form of tablets containing starch or lactose, or in capsules containing the active ingredient alone or as an excipient, or as an elixir or a suspending agent containing a chemical that tastes or colors 0.0 > oral, < / RTI > intraoral, or sublingually.

The pharmaceutical composition for preventing or treating cartilage diseases may be applied to pharmaceutical preparations in the same manner.

In another aspect, the present invention provides a recombinant vector comprising ITGBL1 (integrin beta-like protein 1) protein, a gene encoding the protein, a recombinant vector comprising the gene, and a recombinant vector transformed with the recombinant vector And an integrin protein activity inhibitor containing at least one selected as an active ingredient.

The "integrin protein" is a protein that links a cell and an extracellular matrix (ECM), and is composed of two strands of an alpha chain and a beta chain.

In particular, the integrin protein activity inhibitor of the present invention shows an excellent effect on the inhibition of integrin beta activity. Accordingly, the inhibitor of the present invention can be used for research on the mechanism of disease caused by the activity of integrin beta protein, development of therapeutic agent, and the like. Examples of diseases induced by excessive activity of integrins include, but are not limited to, cancer, irritable bowel syndrome psoriasis, thrombosis, rheumatoid arthritis, osteoporosis.

In another aspect, the present invention relates to a method for preventing or treating a cartilage disease, comprising administering a pharmaceutically effective amount of the composition to a subject in need of prevention or treatment of cartilage disease.

The subject may be an individual (for example, an animal other than a human) or an organ, tissue, cell or culture thereof isolated from a vertebrate including mammals such as humans, rodents such as mice, amphibians such as frogs and the like , Preferably an animal other than a human, but is not limited thereto.

The cartilage disease may be, but is not limited to, degenerative arthritis, post traumatic arthritis, or peelable osteochondritis.

The pharmaceutical composition for preventing or treating cartilage diseases may be applied to a method for preventing or treating cartilage diseases.

The present invention relates to a pharmaceutical composition for preventing or treating cartilage diseases, which contains at least one selected from the group consisting of an ITGBL1 (integrin beta-like protein 1) protein and a gene encoding the protein, an integrin protein activity inhibitor, The present invention relates to a pharmaceutical preparation containing the above-mentioned composition as an active ingredient, which comprises at least one compound selected from the group consisting of ITGBL1 (integrin beta-like protein 1) protein and a gene encoding the protein, The composition has the effect of inhibiting inflammation and promoting cartilage formation according to the inhibitory function of integrin protein activity of ITGBL1 protein, and thus can be used as an effective therapeutic agent for cartilage diseases.

FIG. 1 (A) is a schematic diagram of the experiment to confirm that the ITGBL1 gene is expressed in the cartilaginous tissue of Xenopus laevis embryo. B and C show the ITGBL1 gene in the chondrocytes of Xenopus laevis embryo according to one embodiment of the present invention And expresses the result of confirming whether or not it is expressed. FIG. 1D shows that the ITGBL1 gene is expressed in cartilage tissue by comparing the expression pattern of Col2a1 and ITGBL1, and the expression of ITGBL1 gene is strongly expressed in the stage of cartilage formation (stage 30) .
FIGS. 2A and 2C show that when the ITGBL1 gene is inhibited in the chondrocytes of the Xenopus laevis embryo according to an embodiment of the present invention, the size of the cartilage tissue is reduced, cartilage tissue formation is significantly inhibited, and the expression of the ITGBL1 gene , B and D are graphs of cartilage size in A and C, respectively, as a result of confirming that cartilage is normally formed and the size of cartilage is increased. On the other hand, FIG. 2E shows the results of confirming that the expression of the Sox9 and Col2a1 genes, which are markers of cartilage tissue, is increased in the embryos in which the expression of the ITGBL1 gene is increased.
FIGS. 3A and 3B show that the expression of ITGBL1 is increased in bone marrow-derived stem cell (BM-MSC) chondrocyte differentiation and the expression of ITGBL1 is decreased in bone tissue formation, And that the formation of cartilage tissue is inhibited by decreasing the expression of ITGBL1 protein in cartilage cells of human. FIG. 3E shows that gradually increasing the expression of ITGBL1 in rat cartilage cells leads to a gradual increase in the expression of Col2a1 and Sox9, which promote cartilage formation. F indicates quantitation of E expression by qPCR "He said. G of FIG. 3 confirms that cartilage differentiation is accelerated when ITGBL1 expression of Limb bud mesenchyme differentiates into cartilage cells as a part of the arm and leg of a mouse embryo. H indicates the amount of Glycosaminoglycan (GAG) As a result, it was confirmed that ITGBL1 overexpression promotes cartilage formation.
FIGS. 4A to 4F show that when the expression of ITGBL1 protein is increased in cartilage cells of inflammation-induced mice treated with IL-1β according to an embodiment of the present invention, the expression of Sox9 and Col2a1, , And the expression of MMP-3 and MMP-13, which are inflammatory factors, is decreased. H and I in FIG. 4 show that when ITGBL1 expression is increased by injecting ITGBL1-containing adenovirus into the inside of the knee joints of rats induced by arthritis by damaging the medial meniscus of the mouse, Results are shown.
FIG. 5A and FIG. 5C show the results of inhibition of ITGBL1 expression in the PC3 cell line using ITGBL1-siRNA, resulting in a marked increase in the amount of Focal adhesion complex formed at the site where the cell substrate and integrin bind, Is a graphical representation of the number and intensity of FAK puncta, a marker of the Focal adhesion complex of A, and D is a graph of the number and intensity of integrin-beta 1 puncta, another marker of the Focal adhesion complex of C. FIG. 5E shows the result that when the expression of ITGBL1 is inhibited, the amount of activated integrin-beta1 is increased and, conversely, when the expression of ITGBL1 is increased, the amount of activated integrin-beta1 is decreased. In the case of decreasing the expression of ITGBL1, the increase of the activated integrin-beta1 was confirmed by fluorescence staining. G was confirmed by the co-immunoprecipitation experiment technique that integrin-beta1 and ITGBL1 bind to each other.
FIG. 6A shows that when the ITGBL1 gene expression was increased (Itgbl1-OE), PC3 cells were not adhered well to the surface coated with Fibronectin, but when the activity of integrin was increased by Mn ^{2 +} ion, B is the measured cell area of A, and E and F are the results of the experiment of A in human chondrocytes. 6C and D show that ITGBL1 expression in bone marrow-derived stem cells was inhibited when adhesion of cells was inhibited. E and F in Fig. 6 show that when the expression of ITGBL1 is increased in human chondrocytes, cell adhesion is inhibited, and when the activity of integrin is increased by Mn ^{2 +} ion, it is confirmed to be attached to the surface again.
FIG. 7A shows that when the expression of ITGBL1 gene is increased in chondrocytes, the expression of Sox9 and Col2a1 is increased. However, when the activity of integrin is increased by treatment with Mn ^{2 +} or DTT, B is a quantitative analysis of the results of A using the qPCR technique. FIG. 7C shows that the increase in cartilage size by overexpression of ITGBL1 in the process of making cartilage tissue using ATDC5 cartilage cell line is decreased by addition of Mn ^{2 +} or DTT, (D) and the amount of GAG as a constituent of cartilage (E). F was found to inhibit the expression of integrin alpha and beta as shown in Table 1, and the expression of Sox9 was increased. When ITGBL1 was overexpressed, the expression of Sx9 was further increased by decreasing the expression of integrin alpha and beta .
FIGS. 8A to 8C show that the expression of MMP-3 and MMP-13, which promote the degradation of cartilage in chondrocytes treated with Fragmented Fibronectin (Fn-f 29kDa), which is known to cause cartilage degradation in arthritis patients, It was confirmed that expression of the two genes was increased when the expression of the gene was increased, and then the expression of the two genes was increased by activating the integrin by treatment with Mn ^{2 +} or DTT. D and E showed Fn-f 29 kDa The degree of adhesion to chondrocytes was measured and shown.
9A to 9C show that when the inhibitor that inhibits the expression of the ITGBL1 gene and then inhibits only one type of integrin is treated, the expression of MMP-3 and MMP-13 is not inhibited. However, when several kinds of integrin inhibitors are treated together , The expression of both genes is inhibited, and ATN-161 among the integrin inhibitors is most effective. 9, D and E show that when ITGBL1 expression is inhibited by injecting adenovirus containing ITGBL1-shRNA into the inside of the knee joint of a mouse, articular cartilage deteriorates and integrin- Injection of ATN-161, a beta 1 inhibitor, results in recovery of knee cartilage.
FIG. 10 is a graphical representation showing that the expression of the ITGBL1 gene inhibits cartilage disease inflammation and inhibits cartilage formation by inhibiting integrin activity.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Reference Example 1 . Xenopus laevis  Incubation of embryo

Female Xenopus laevis is cultured in a 16 ° C incubator (JSR, JSBI-150C), and 800 units of hCG hormone (Daesung HCG) is injected into female Xenopus laevis to induce egg laying. When eggs are started by hormones, And spermatozoa were inoculated with male testes and waited for 30 minutes. After confirming the presence or absence of fertilization with the naked eye, the jelly layer was dejeled with 3% (w / v) cysteine (pH 7.8, Sigma: C7880), and then Xenopus in Marc's Modified Ringers laevis embryos were cultured.

Reference Example 2 . Cell culture

Human PC3 cells, HEK293T cells or hBMSC were cultured in 1% L-glutamine, 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, RPMI 1640 medium, Dulbecco's Modified Eagle Medium (DMEM) and? A pellet, micromass or Transwell culture system was used to assess cartilage formation of hBMSCs.

Articular cartilage cells were separated from the femoral condyles and tibial plateaus of mice 5 days after birth, and the cartilage tissue was disassembled with 0.2% type II collagenase. Chondrocyte cells were maintained in DMEM containing 10% FBS, penicillin and streptomycin.

Human chondrocytes were obtained from Cell Application, Inc. (San Diego, CA, USA), and human bone marrow-derived stem cells were purchased from ATCC.

Mesenchymal cells from fetuses of ICR mice were resolved with 1% trypsin and 0.2% type II collagenase and maintained to induce cartilage formation and non-growth maturation. A total of 2 × 10 ⁷ cells / ml was suspended in DMEM / F-12 medium (2: 3) containing 10% (v / v) FBS. Cells were dropped in a 20 μl drop into the culture dish and maintained for 6 days to induce cartilage formation.

Example 1 . ITGBL1  Confirming chondrocyte expression of genes

1-1. ITGBL1  Identification of gene expression site

The following experiments were carried out to analyze the genes expressed in the pharyngeal arches of the Xenopus laevis embryo.

Specifically, the embryo stage 37 Xenopus laevis embryo, which is the stage in which the embryo's face is formed, is extracted and divided into three equal parts in the head-tail direction and two equal parts in the back- Sigma), and a Library for RNAseq was prepared from the extracted RNA using the Illumina TruSeq RNA Library Prep Kit. Sequencing analysis was then submitted to the Stanford Sequencing Center, USA.

The results of the above analysis are shown in Fig. 1 (A), and the results of analysis are shown in Table 1.

SeqID log2FC Arch1 Arch2 Arch3 ArchD ArchV LOC100491886.L | HS = TLL2 | 40 | Xelaev18021897m 7.943 32.731 0.133 0.238 2.657 4.3 sst.S | HS = SST | 100 | Xelaev18029613m 7.869 43.723 0.611 0.187 1.929 8.099 clec19a.S | HS = CLEC19A | 93 | Xelaev18047527m 7.825 0.012 0.073 2.722 0.013 1.884 fstl3.L | HS = FSTL3 | 86 | Xelaev18006463m 7.311 0.065 1.509 10.318 0.383 8.008 LOC100493098.L | HS = DNASE1L2 | 94 | Xelaev18045463m 7.21 5.923 0.04 0.124 0.138 1.051 unnamed | HS = CA14 | 77 | Xelaev18042787m 7.102 10.163 0.425 0.074 0.131 0.89 unnamed | HS = HGFAC | 84 | Xelaev18005068m 6.664 0.179 0.184 18.148 19.627 14.491 unnamed | HS = C4orf48 | 46 | Xelaev18005037m 6.647 3.908 0.111 0.039 0.24 0.179 unnamed | HS = FETUB | 74 | Xelaev18029648m 6.635 0.115 1.254 11.427 46.345 10.328 unnamed | HS = FETUB | 66 | Xelaev18027506m 6.349 0.144 1.23 11.735 56.671 8.782 dct.L | HS = DCT | 97 | Xelaev18013152m 6.202 4.713 0.196 0.064 3.466 0.307 tyr.S | HS = TYR | 100 | Xelaev18016340m 6.135 2.178 0.048 0.031 1.507 0.117 unnamed | HS = TLL2 | 42 | Xelaev18021894m 5.989 26.165 0.608 0.412 1.347 3.602 fgfbp3.L | HS = FGFBP3 | 86 | Xelaev18034766m 5.668 6.202 0.122 0.593 0.229 0.735 c17orf67.L | HS = C17orf67 | 80 | Xelaev18043803m 5.557 2.543 0.253 0.054 0.544 0.415 serpinc1.L | HS = SERPINC1 | 93 | Xelaev18023232m 5.493 0.746 2.766 33.603 28.227 27.363 unnamed | HS = C4orf48 | 46 | Xelaev18008874m 5.407 3.395 0.228 0.08 0.352 0.233 Xetrov90010415m.S | HS = NA | 00 | Xelaev18024389m 5.366 0.079 3.257 2.948 1.784 3.945 LOC101733976.S | HS = EDN3 | 55 | Xelaev18046257m 5.293 6.859 0.175 0.198 1.025 1.698 unnamed | HS = CRISP3 | 77 | Xelaev18028198m 5.239 2.115 0.056 0.19 0.222 0.451 unnamed | HS = AGR3 | 85 | Xelaev18044817m 5.171 31.098 5.1 0.863 2.592 19.559 gbp6.1 | HS = GBP1 | 70 | Xelaev18001671m 5.037 0.095 0.636 3.119 0.55 2.308 LOC100490489.L | HS = NA | 00 | Xelaev18027216m 4.89 7.381 0.756 0.249 0.983 0.864 pcdh8.2.S | HS = PCDH8 | 70 | Xelaev18015747m 4.856 0.334 2.276 9.672 3.254 6.536 npb.L | HS = NPB | 84 | Xelaev18043747m 4.783 3.552 0.285 0.129 0.153 0.208 slurp1l.L | HS = NA | 00 | Xelaev18023882m 4.659 5.888 0.233 0.287 0.34 1.482 LOC100485272-like.S | HS = ANGPT2 | 97 | Xelaev18015134m 4.625 0.169 2.106 4.171 0.465 4.39 Xetrov90021952m.L | HS = TLL2 | 43 | Xelaev18040194m 4.555 27.59 1.174 3.595 1.248 5.305 gdf7.L | HS = GDF6 | 85 | Xelaev18028105m 4.507 0.239 1.846 5.436 6.902 0.795 pmel-like.S | HS = PMEL | 58 | Xelaev18015875m 4.109 22.369 4.832 1.296 7.076 4.256 tyrp1.L | HS = TYRP1 | 97 | Xelaev18006806m 4.076 3.525 0.562 0.209 2.119 0.427 vwde.L | HS = VWDE | 78 | Xelaev18030909m 3.993 4.473 1.18 0.281 0.756 1.698 mcam-like.1 | HS = MCAM | 96 | Xelaev18000065m 3.986 1.822 28.877 4.827 12.698 5.902 sfrp1.L | HS = SFRP1 | 88 | Xelaev18018658m 3.951 3.629 14.22 56.137 21.051 53.337 apoc1-like.L | HS = NA | 00 | Xelaev18036025m 3.926 396.083 124.121 26.058 112.828 124.842 mxra5.L | HS = MXRA5 | 43 | Xelaev18011748m 3.876 3.993 1.212 0.272 0.62 1.325 unnamed | HS = TLL1 | 44 | Xelaev18021890m 3.871 80.83 17.585 5.526 18.007 37.359 fstl3.S | HS = FSTL3 | 87 | Xelaev18009939m 3.871 0.41 1.178 6 0.503 5.87 hebp2.L | HS = HEBP2 | 79 | Xelaev18026611m 3.862 7.083 0.976 0.487 0.71 1.197 apoe.L | HS = APOE | 85 | Xelaev18036024m 3.792 53.004 16.025 3.827 5.81 16.975 slc8a1-like.S | HS = SLC8A1 | 100 | Xelaev18028910m 3.791 0.553 0.749 7.653 0.558 6.617 cpn1.S | HS = CPN1 | 87 | Xelaev18037048m 3.728 20.386 2.279 1.539 6.064 4.589 LOC100496022.1 | HS = PRSS27 | 92 | Xelaev18001642m 3.708 21.708 7.858 1.661 3.825 13.988 c8b.L | HS = C8B | 93 | Xelaev18023028m 3.61 0.502 1.028 6.13 14.667 7.128 unnamed | HS = RBP4 | 89 | Xelaev18037044m 3.608 17.786 15.571 1.459 9.927 4.092 itga7-like.L | HS = ITGA7 | 95 | Xelaev18013342m 3.573 0.694 0.799 8.258 0.479 5.348 lrrn4.S | HS = LRRN4 | 97 | Xelaev18028812m 3.506 0.337 0.967 3.828 1.595 5.497 LOC100487362.L | HS = LY9 | 32 | Xelaev18040478m 3.505 2.838 1.656 0.25 0.178 0.367 rspo2.L | HS = RSPO2 | 100 | Xelaev18032283m 3.424 6.911 2.565 0.644 1.476 1.476 pyy.S | HS = NPY | 96 | Xelaev18046061m 3.418 0.355 0.218 2.33 0.794 0.257 f2.S | HS = F2 | 98 | Xelaev18024485m 3.414 0.39 0.933 4.157 4.675 3.267 sel1l3.S | HS = SEL1L3 | 95 | Xelaev18008974m 3.29 2.298 0.685 0.235 0.336 1.208 unnamed | HS = LCN15 | 99 | Xelaev18038205m 3.213 19.124 3.417 2.063 4.538 6.52 unnamed | HS = ANGPT1 | 101 | Xelaev18032282m 3.155 0.28 0.597 2.494 1.191 1.104 nrn1.S | HS = NRN1 | 75 | Xelaev18033414m 3.09 2.291 0.584 0.269 0.787 0.153 fibin.S | HS = FIBIN | 100 | Xelaev18024379m 3.049 1.484 12.282 3.127 6.681 3.054 olfml2a.S | HS = OLFML2A | 96 | Xelaev18041818m 3.046 7.192 1.606 0.871 1.836 2.419 tgfb2.S | HS = TGFB2 | 100 | Xelaev18028671m 3.035 1.73 6.055 14.175 6.469 18.302 unnamed | HS = GP1BB | 57 | Xelaev18010547m 3.032 0.354 0.441 2.895 3.358 2.148 unnamed | HS = TECPR1 | 7 | Xelaev18036521m 3 4.055 2.121 0.507 0.423 2.221 LOC100489571.S | HS = MMP8 | 97 | Xelaev18016259m 2.96 1.139 8.863 4.084 0.027 3.147 itgbl1.S | HS = ITGBL1 | 95 | Xelaev18015636m 2.952 2.569 0.842 0.332 0.565 0.789 unnamed | HS = SCN4B | 90 | Xelaev18035439m 2.871 0.958 2.188 7.008 2.412 5.981 unnamed | HS = TMEM213 | 58 | Xelaev18002504m 2.865 3.455 13.317 25.166 26.303 16.039 unnamed | HS = TNNT3 | 67 | Xelaev18024361m 2.857 1.944 6.248 14.083 4.678 8.964 unnamed | HS = CRISP3 | 79 | Xelaev18030177m 2.852 6.094 1.635 0.844 1.303 2.18 unnamed | HS = ANGPTL5 | 64 | Xelaev18021227m 2.835 5.622 1.554 0.788 0.778 1.391 unnamed | HS = LOXL4 | 96 | Xelaev18034711m 2.823 2.066 0.468 0.292 0.698 0.539 prrt3-like.L | HS = PRRT3 | 67 | Xelaev18024053m 2.819 3.727 1.313 0.528 0.91 0.589 Xetrov90018420m.1 | HS = ROBO4 | 99 | Xelaev18003883m 2.79 2.325 3.695 16.082 8.331 10.299 Xetrov90024887m.L | HS = NA | 00 | Xelaev18043270m 2.786 6.387 1.799 0.926 0.743 2.384 fgf3.S | HS = FGF3 | 78 | Xelaev18024490m 2.763 4.625 0.833 5.655 8.273 3.668 unnamed | HS = APELA | 100 | Xelaev18003936m 2.747 3.911 8.978 26.249 11.39 22.66 Xetrov90018420m.L | HS = ROBO1 | 17 | Xelaev18035307m 2.712 2.853 3.26 18.696 13.616 8.351 nov.S | HS = NOV | 88 | Xelaev18033972m 2.691 0.99 3.13 6.393 6.832 2.166 unnamed | HS = NELL2 | 93 | Xelaev18017595m 2.688 3.539 0.549 1.623 1.913 1.051 cdh15.S | HS = CDH15 | 96 | Xelaev18024865m 2.654 6.662 15.01 2.384 5.413 7.229 igdcc3.L | HS = IGDCC3 | 89 | Xelaev18018436m 2.64 4.418 2.005 0.709 2.334 1.009 fgf8.L | HS = FGF8 | 79 | Xelaev18034665m 2.609 19.454 3.188 9.32 11.654 11.125 unnamed | HS = ATP6AP1 | 92 | Xelaev18017668m 2.608 0.643 1.909 3.919 4.359 2.667 c6.2.L | HS = C6 | 100 | Xelaev18008334m 2.588 0.925 1.463 5.56 6.348 4.378 unnamed | HS = CA4 | 90 | Xelaev18010514m 2.569 2.391 0.566 0.403 0.673 0.462 clec19a.L | HS = CLEC19A | 83 | Xelaev18045207m 2.558 0.525 1.552 3.091 0.9 2.85 unnamed | HS = CACNA2D4 | 93 | Xelaev18003010m 2.556 1.986 1.461 8.592 6.055 3.817 unnamed | HS = NELL2 | 93 | Xelaev18021119m 2.541 2.218 0.381 0.39 1.124 0.573 unnamed | HS = KDR | 96 | Xelaev18009068m 2.525 1.047 5.879 6.025 3.959 7.705 fam132a.S | HS = FAM132A | 107 | Xelaev18037500m 2.514 0.426 0.895 2.433 0.411 2.035 LOC101733976.L | HS = EDN3 | 53 | Xelaev18043542m 2.449 2.544 1.992 0.466 2.728 1.195 nog2.S | HS = NOG | 95 | Xelaev18047671m 2.448 2.93 1.76 0.537 0.86 0.411 npr3-like.L | HS = NPR3 | 90 | Xelaev18008385m 2.429 8.499 3.773 1.578 2.862 2.916 unnamed | HS = LY86 | 82 | Xelaev18031614m 2.396 2.379 0.482 0.452 0.929 0.547 Xetrov90000623m.L | HS = SHISA3 | 93 | Xelaev18005242m 2.381 7.436 6.743 1.428 1.119 4.449 ramp2.L | HS = RAMP2 | 61 | Xelaev18043825m 2.377 1.44 3.696 7.482 5.008 3.22 igfbpl1.L | HS = IGFBPL1 | 74 | Xelaev18006653m 2.372 4.945 8.355 1.614 6.356 4.638 stc2.L | HS = STC2 | 104 | Xelaev18017248m 2.37 42.763 18.002 8.272 11.249 12.744 LOC100496170-like.1 | HS = IZUMO1R | 68 | Xelaev18000799m 2.37 11.898 2.97 2.301 3.944 4.662 prtn3-like.1.L | HS = PRTN3 | 85 | Xelaev18006264m 2.367 0.524 1.797 2.704 0.556 1.014 fgfbp2.L | HS = FGFBP2 | 99 | Xelaev18005148m 2.359 15.019 14.741 2.928 0.936 12.958 unnamed | HS = CFI | 105 | Xelaev18005652m 2.32 3.237 7.08 16.167 28.023 12.637 mmp11.S | HS = MMP11 | 92 | Xelaev18010511m 2.316 3.304 2.639 13.137 1.768 11.177

In addition, the following experiment was conducted to confirm whether the ITGBL1 gene was expressed in chondrocytes of Xenopus laevis embryo.

Specifically, the probe RNA of the nucleotide sequence complementary to the ITGBL1 mRNA was prepared using the Ambion Megastar T7 RNA Transcription kit (Promega, P2077), and then extracted and fixed in the same manner as described above. 37. Xenopus laevis The sections were treated with prehybridization buffer (formamide 1.13g / ml, Biosesang, F1014) at 65 ° C for 12 hours and the treated sections were incubated at 65 ° C in 2X SSC solution (0.3M NaCl-Biosesang, 30mM sodium citrateBiosesang, C1029) (Sigma) for 12 h, washed three times with TBST, and then treated with BM purple (Sigma). The cells were washed three times with anti-digoxigenin antibody To induce color development.

Primer sequences for making probe RNAs are listed in Table 2 below.

denomination The sequence (5 '- > 3') SEQ ID NO: ITGBL1 5 ' augcacgcuggagccuu One ITGBL1 3 ' aggauauucgcuuccaagcca 2

The results of the analysis are shown in B and C in Fig.

As a result, it was confirmed that the ITGBL1 gene was expressed in chondrocytes of Xenopus laevis embryo as shown in B and C of FIG. 1.

1-2. Col2a1  Gene expression pattern and ITGBL1  Comparison of expression patterns of genes

Fixed Embryonic Stage 37 Xenopus laevis is sectioned to 100 μm thickness using a vibratome (Leica, VT 1000S) machine. The probe RNAs complementary to the ITGBL1 mRNA and the complementary sequences of Col2a1 mRNA were prepared and fixed in the embryonic stage 37 Xenopus laevis head tissue sections at 65 ° C for 12 hours in prehybridization buffer (formamide 1.13 g / ml, Biosesang, F1014) (w / v) were diluted in prehybridization and treated at 65 ℃ for one day. The treated sections were washed twice with a 2X SSC solution (0.3M NaCl-Biosesang, 20mM sodium citrate-Biosesang, C1029) for 30 minutes, treated with RNase, washed twice with 0.2X SSC solution for 30 minutes The anti-Digoxigenin antibody (Sigma) was then treated for 12 hours, washed 3 times with TBST, and the color developed using BM purple (Sigma). As another method, DAB staining, immunosurfactant treatment with Col2a1 antibody (DSHB, II-II6B3), which can detect cartilage specific substrate expression in a 100-μm-thick section, was performed.

As shown in Fig. 1D, the expression pattern of the Col2a1 gene and the expression pattern of the ITGBL1 gene were similar, and it was confirmed that the ITGBL1 gene was expressed in cartilage tissue.

1-3. ITGBL1  Identify the timing of gene expression

RNA was extracted from the embryos of each step 20, 24, 27, 30, 33, 35 and 41 using modified PureLink RNA Mini Kit (Invitrogen, 12183018A), and then transformed with cDNA using GoScript Reverse Transcriptase (Promega, A5004A) Were synthesized. The synthesized cDNA was subjected to RT-PCR (BIO RAD, T100 Thermal Cycler) with Taq polymerase (Coregen, CE-500U).

As shown in FIG. 1E, it was confirmed that the expression level of the ITGBL1 gene was most strongly expressed in the stage 30, which is the period of generation of cartilage tissue.

Example 2 . ITGBL1  Function of promoting the generation of cartilage tissue of gene

2-1. ITGBL1  When gene expression is suppressed

In order to confirm the function of promoting the formation of cartilage tissue of ITGBL1 gene, the expression of ITGBL1 gene was inhibited by the following method.

First, splicing blocking anti-sense Morpholino was ordered from Gene Tools, LLC for use in inhibiting the expression of the ITGBL1 gene in chondrocytes of Xenopus laevis embryos, and the sequence thereof is shown in Table 3 below.

denomination The sequence (5 '- > 3') SEQ ID NO: ITGBL1 MO AGTAGGGAAGATATACAGACCTGCA 3

The above splicing blocking anti-sense Morpholino was injected into the back-axis of Xenopus laevis embryos in the second stage, and the morpholino-implanted embryo was cultured until embryonic stage 45 After fixation with MEMFA (4% Formaldehyde, Biosesang, F1012), cartilage was stained specially with Alcian blue (1% Alcian Blue 8GX, Georgiachem, AB1082) and tissues other than cartilage were removed using trypsin. Non-specific staining was removed using a% (v / v) methanol solution. This was observed using an Olympus (SZX16-ILLB) microscope.

The control was carried out in the same manner as described above except that morpholino was not injected to inhibit the expression of ITGBL1 gene in chondrocytes of Xenopus laevis embryo.

The experimental results are shown in Figs. 2A and 2B.

As a result, when the ITGBL1 gene expression was inhibited in chondrocytes of Xenopus laevis embryo as shown in A and B of FIG. 2, the size and shape of the cartilage were abnormally reduced, compared with the control group which did not inhibit the expression of ITGBL1 gene, It was confirmed that it occurred.

2-2. ITGBL1  Increased gene expression

In order to confirm the function of promoting the formation of cartilage tissue of the ITGBL1 gene, the ITGBL1 gene expression was increased by the following method.

Specifically, mRNA of the ITGBL1 gene was synthesized by applying the cDNA obtained from the facial cartilage cells of Xenopus laevis embryos to mMESSAGE mMACHINE SP6 kit (Ambion, AM1340), and the ITGBL1 mRNA was expressed in the Xenopus laevis embryo And the expression of ITGBL1 was increased in chondrocytes. Alcian blue staining was performed in the same manner as in Example 2-1, and the expression of Sox9 and Col2a1 genes was confirmed by WISH technique.

The control was carried out in the same manner as described above except that the ITGBL1 gene mRNA was not injected to increase the expression of the ITGBL1 gene in chondrocytes of the Xenopus laevis embryo.

The experimental results are shown in Fig. 2C, D and E.

As a result, when ITGBL1 mRNA (mMESSAGE mMACHINE SP6 kit, Ambion, AM1340) was injected into the facial cartilage cells of Xenopus laevis embryos as shown in C and D of FIG. 2, the expression of ITGBL1 gene The increase of cartilage size was 1.2 to 1.3 times that of the control group which did not increase the number of cells. As shown in E, gene expression of Sox9 and Col2a1, which are cartilage tissue markers, was increased compared with the control group.

Example  3. In human and rat cartilage cells ITGBL1  Promotion of cartilage formation by protein

3-1. Bone marrow origin  In the differentiation process of stem cells, in cartilage cells and bone tissue ITGBL1  Gene expression comparison

In order to confirm the promotion of cartilage formation by ITGBL1 protein in human bone marrow-derived stem cell (hBMSC) differentiation, experiments were carried out as follows. Specifically, isolated and cultured human bone marrow-derived stem cells (ATCC) were cultured in α-MEM (Welgene, LM008-01, supplemented with 10% (v / v) FBS and 1% , The cells were made into a lump form by a micromass method, and cartilage tissue formation was induced by treating the cell mass with the culture medium with a cartilage formation inducer (TGF-beta, dexamethasome, Ascorbate-2-phosphate). After inducing the formation of cartilage tissue for 12 days, RNA was extracted using PureLink RNA Mini Kit (Invitrogen, 12183018A) and cDNA was synthesized with GoScript Reverse Transcriptase (Promega, A5004A). The synthesized cDNA was subjected to qRT-PCR using QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). All reactions were performed in 96-well plates and the mean values were used to calculate mRNA expression. The results of the above experiment are shown in Figs. 3A and 3B.

As shown in FIG. 3A, it was confirmed that the expression of ITGBL1 in chondrogenic differentiation of bone marrow-derived stem cells was increased and the expression of ITGBL1 was decreased in the bone tissue formation process of bone marrow-derived stem cells as shown in B .

3-2. Bone marrow origin  During the process of cartilage differentiation of stem cells ITGBL1  Expression of the gene Suppressed  In case and cartilage cells Of ITGBL1  Increased expression

In order to confirm the promotion of cartilage formation by ITGBL1 protein in human chondrocytes, experiments were carried out as follows.

Specifically, human chondrocytes (Human chondrocyte, Cell Application, Inc. San Diego, Calif., USA) were cultured in α-MEM (Welgene, LM008-01, 10% / v) antibiotics) medium. The cells were transfected with the ITGBL1 siRNA (Genome 1) by the protocol provided by the manufacturer, and the transfected cells were lump-formed by the micromass method TGF-beta, dexamethasone, and ascorbate-2-phosphate were added to the cell mass to induce cartilage formation. The cartilage tissue was fixed with 4% (w / v) paraformaldehyde to induce the formation of cartilage tissue for 7 days, and the fixed cartilage tissue was placed in OCT solution (Cell Path, KMA-0.00-00A) Bright, OTF5000), and immunofluorescent staining was performed to confirm expression of Col2a1 gene. Alcian blue staining was performed in the same manner as in Example 2-1 in order to confirm whether cartilage formation was promoted.

As a control in which the expression of ITGBL1 protein was inhibited, ITGBL1 siRNA was not transfected.

RNA was extracted from chondrocytes transfected with ITGBL1 DNA using a PureLink RBA Mini Kit (Invitrogen, 12183018A). The cDNA was synthesized using GoScript Reverse Transcriptase (Promega, A5004) PCR system (Applied Biosystems, Foster City, Calif., USA) was used for qRT-PCR. All reactions were performed in 96-well plates and the mean values were used to calculate mRNA expression. The primer sequences used in qRT-PCR are shown in Table 4 below.

denomination The sequence (5 '- > 3') Tm (占 폚) SEQ ID NO: SOX9-F AAGGAGAGCGAGGAGGACAAGTTC 62 4 SOX9-B TGTTCTTGCTGGAGCCGTTG 57.4 5 MMMP3-F GATGCGCAAGCCCAGGTGTG 64.13 6 MMP3-B GCCAATTTCATGAGCAGCAACGA 59.57 7 MMP13-F AGGAGCATGGCGACTTCTACCC 62.88 8 MMP13-B TTTGTCTGGCGTTTTTGGATGTTT 56.23 9 Col2al-F CAGTTGGGAGTAATGCAAG 58 10 Col2al-B GCCTTGAGCAGTTCACCTTC 58 11

The experimental results are shown in Fig. 3C, D and E.

As a result, it was confirmed that formation of cartilage tissue was inhibited when the expression of ITGBL1 protein was inhibited, as shown in C and D of Fig.

As shown in E and F of FIG. 3, when the expression of ITGBL1 protein was gradually increased, the expression of Sox9 and Col2a1 genes, which are cartilage-producing factors, gradually increased, confirming that the formation of cartilage tissue was promoted I could.

Limb bud mesenchyme, which differentiates into chondrocytes, was isolated from the limb bud of the mouse embryo. The adenovirus vector (Ad-ITGBL1) (Vector Biolabs, Malvern, PA 19355 USA, custom made to the company) to increase the expression of ITGBL1, to induce cartilage differentiation, and then to accelerate cartilage differentiation using the Alcian blue staining technique. The results of the above experiment are shown in G and H in Fig.

As shown in G and H in FIG. 3, the amount of Glycosaminoglycan (GAG) was increased in cartilage cells with increased ITGBL1 expression, and cartilage formation was promoted.

Example  4. In arthritis-induced mice ITGBL1  Promoting cartilage formation of proteins

The following experiment was conducted to confirm whether ITGBL1 protein promotes cartilage formation in arthritis-induced mice.

Specifically, chondrocytes isolated from 5-day-old mouse knee cartilage were cultured for 2 days, treated with IL-1β (GenScript, 201-LB) at a concentration of 5 ng / ml for 72 hours to induce inflammation, After transfection of the Ad-ITGBL1 adenovirus vector into the inflammation-induced mouse cartilage cells, qRT-PCR was carried out by the same method as in Example 3 to change the expression of the cartilage forming factor (Sox9, Col2a1). The primer sequences used in qRT-PCR are shown in Table 5 below.

The control group was subjected to the same experiment as above except that no adenovirus vector was transduced and IL-1? Was not treated (None).

denomination The sequence (5 '- > 3') Tm (占 폚) SEQ ID NO: Col2al-F CACACTGGTAAGTGGGGCAAGA 55 16 Col2al-B GGATTGTGTTGTTTCAGGGTTCG 55 17 SOX9-F CATCAGCAGCACCGCACCCA 58 18 SOX9-B CGGGTGATGGGCGGGTAGGA 58 19

The results of the experiment are shown in Figs. 4A to 4D.

As a result, as shown in Fig. 4A, the expression of ITGBL1 was markedly decreased when IL-1? Was treated to induce inflammation in chondrocytes isolated from mouse knee cartilage. As shown in Figs. 4B to 4D, The expression of Sox9 and Col2a1 was increased when the expression level was increased.

In addition, the Ad-ITGBL1 adenovirus vector was transduced into the joints of mice induced by arthritis by dissection of the medial meniscus (Medial meniscus) and induction of arthritis using a destabilization of medial meniscus (DMM) After the expression of the protein was increased, mouse joints were isolated after 6 weeks, and the expression levels of safranin-O and Col2a1 and Sox9 were compared by immunohistochemistry. The results of the above experiment are shown in H and I in Fig.

The control was performed in the same manner as above, except that the meniscus was not used (Sham).

As a result, as shown in H and I in FIG. 4, when the expression of ITGBL1 protein in the joint of mice induced arthritis is increased, the expression level of Osteoarthritis Research Society International (OARSI) is decreased and the expression of Col2a1 and Sox9 is increased And it was confirmed that arthritis was suppressed.

Example  5. In arthritis-induced mice ITGBL1  Inflammation inhibitory effect of protein

The following experiment was carried out to confirm the inflammation inhibitory effect of ITGBL1 protein in arthritis-induced mice.

Specifically, as described in Example 4, in order to induce inflammation in the cartilage cells of mice, IL-1? Of 5 ng / ml was treated for 72 hours, and the ITGBL1 gene was inserted into the inflamed mouse chondrocytes After the transfected adenoviral vector, the expression of MMP-3 and MMP-13, which are inflammatory factors, was changed by qRT-PCR in the same manner as in Example 3. [ The primer sequences used in qRT-PCR are shown in Table 6 below.

The control group was subjected to the same experiment as above except that the adenovirus vector without the ITGBL1 gene was transduced (Mock), the adenovirus vector was not transduced and the IL-1β was not treated (None) Respectively.

denomination The sequence (5 '- > 3') Tm (占 폚) SEQ ID NO: MMP-3-F TCCTGATGTTGGTGGCTTCAG 58 12 MMP-3-B TGTCTTGGCAAATCCGGTGTA 58 13 MMP-13-F ACCACATCGAACTTCGA 58 14 MMP-13-B CGACCATACAGATACTG 58 15

The experimental results are shown in E and F in Fig.

As a result, as shown in E and F of FIG. 4, when the expression of ITGBL1 protein was increased in cartilage cells of mice in which arthritic conditions were induced by treating with IL-1β, inflammatory factors (MMP-3, MMP-13) The expression was remarkably inhibited.

In addition, Destabilization of medial meniscus (DMM) The expression of ITGBL1 protein was increased by transfection of the Ad-ITGBL1 adenovirus vector into the joints of mice that induced arthritis. Six weeks later, mouse joints were isolated and immunohistochemistry was performed to measure the expression of MMP3 and MMP13. The results are shown in H and I in Fig.

The control was performed in the same manner as above, except that the meniscus was not used (Sham).

As a result, as shown in H and I in FIG. 4, it was confirmed that when the expression of ITGBL1 protein is increased in the joint of mice induced by arthritis, joint degeneration and inflammatory factors MMP3 and MMP13 decrease.

Example 6 . ITGBL1  Protein Integrin  Protein activity inhibitory effect

6-1. ITGBL1  Analysis of changes in Focal adhesion complexes by inhibition of gene expression.

In order to confirm the change of the Focal adhesion complex by the ITGBL1 protein, the following experiment was carried out.

Specifically, separately cultured PC3 cells were cultured in RPMI 1640 (gibco 22400-099, 10% (v / v) FBS, 1% (v / v) antibiotics supplemented) medium and 2x10 ⁵ cells After that, the cells were transfected with ITGBL1 siRNA (Gen 1) or ITGBL1 DNA using jetPRIME (Polyplus, 114-15) according to the protocol provided by the manufacturer and incubated with 0.25% Trypsin EDTA (gibco, 25200-072 ), The transfected cells are removed from the bottom, and the cells are fixed on a coverslip coated with Fibronectin for 4 hours and fixed in 4% (w / v) paraformaldehyde. Immunofluorescent staining analysis was performed using anti-FAK (abcam, ab40794) and anti-β1 integrin (DSHB, AIIB2) to detect changes in the focal adhesion complex. The results of the experiment are shown in FIGS. 5A to 5D.

As shown in Figs. 5A to 5D, ITGBL1 expression was inhibited by ITGBL1-siRNA in the PC3 cell line. As a result, it was confirmed that the amount of the Focal adhesion complex formed at the site where the cell substrate and integrin bind was markedly increased I could.

6-2. ITGBL1  Expression of genes and activated Integrin's correlation  analysis

The following experiment was conducted to confirm the correlation between ITGBL1 protein and integrin activity.

Specifically, separately cultured PC3 cells were cultured in RPMI 1640 (gibco 22400-099, supplemented with 10% (v / v) FBS and 1% (v / v) antibiotics) and cells were plated at ⁶ × 10 ⁵ , ITGBL1 siRNA (Genzyme 1) or ITGBL1 DNA was transfected into the cells by jetPRIME (Polyplus, 114-15) according to a protocol provided by the manufacturer, the transfected cells were removed with 4 mM EDTA, and FACS (MILLIPORE, MAB2079Z) binding to activated β1-integrin in PBS (1XPBS, 2% FBS) and analyzed using BD LSRFORTESSA.

As shown in E to F of FIG. 5, when the expression of ITGBL1 was suppressed, the amount of the activated integrin was increased. On the contrary, when the expression of ITGBL1 was increased, the amount of the activated integrin was decreased.

In addition, a co-immunoprecipitation experiment was performed to confirm the mutual binding between ITGBL1 and integrin beta1.

Specifically, the ITGBL1-HA vector and the ITGB1-flag (integrin beta1) vector were transfected into HEK 293T cells and the cellular proteins were extracted. Co-immunoprecipitation experiments were performed using paramagnetic beads (Dynabeads, ThermoFisher) with HA antibody. The results of the above experiment are shown in G of Fig.

As shown in FIG. 5G, it was confirmed that ITGBL1 binds ITGB1 in the presence of calcium ion.

6-3. ITGBL1  Increased gene expression Integrin  Determine activity and cell attachment

Specifically, the cultured PC3 cells were cultured in a medium supplemented with RPMI 1640 (gibco 22400-099, 10% v / v FBS, 1% (v / v) antibiotics) The cells were cultured on a 6-well plate at a concentration of 2 × 10 ⁵ cells, and the ITGBL1 siRNA (Gen 1) or ITGBL1 DNA was injected into the cells by jetPRIME (v / v) FBS and 1% (Polyplus, 114-15), and the transfected cells were removed from the bottom using 0.25% Trypsin EDTA (gibco, 25200-072). Then, Fibronectin The cells are plated on a coverslip of each cell with a concentration of Mn ^{2 +} in a concentration of 4% (w / v) paraformaldehyde. This was observed using an Olympus (IX73) microscope.

As shown in Fig. 6, the expression of ITGBL1 in both PC3 cells (A and B in Fig. 6) and human mesenchymal stem cells (C and D in Fig. 6) and human chondrocytes (E and F in Fig. 6) Cell adhesion was inhibited. However, when Mn ^{2 +} was treated to increase the activity of integrin protein, the degree of reattachment was increased. This demonstrates that the ITGBL1 protein inhibits integrin activity and inhibits cell adhesion.

Example 7 . ITGBL1  Protein Integrin  Active inhibition and chondrocyte Cartilage forming factor  Correlation analysis of expression regulation

7-1. ITGBL1  Increasing gene expression

The following experiment was conducted to analyze the effect of ITGBL1 integrin inhibition on cartilage formation during cartilage formation. Chondrocytes were cultured in α-MEM (Welgene, LM008-01, supplemented with 10% (v / v) FBS and 1% (v / v) antibiotics) medium and 2 × 10 ⁵ cells were plated on a 6-wells plate. Cells are transfected with ITGBL1 DNA using jetPRIME (Polyplus, 114-15) by the protocol provided by the manufacturer, and the medium is treated with Mn ^{2 +} and DTT and incubated at 37 ° C. Then, the cells were extracted with RNA using a PureLink RNA Mini Kit (Invitrogen, 12183018A), and cDNA was synthesized using GoScript Reverse Transcriptase (Promega, A5004A). The synthesized cDNA was amplified with Taq polymerase (Coregen, CE-500U ), And qRT-PCR was performed using QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) -well plate, and the average value was used in the calculation of mRNA expression. The results of the above experiment are shown in Figs. 7A and 7B.

As also shown in A and B of 7, one when in chondrocytes increased the expression of ITGBL1 gene increases the expression of Sox9 and Col2a1, Mn ^{2 +,} or by handling the DTT by increasing the activity of an integrin reduces the expression of two genes, again .

This demonstrates that the inhibitory function of integrin activity of ITGBL1 is a functional group that promotes the expression of cartilage forming gene.

In addition, the following micromass culture experiment was conducted to analyze the effect of ITGBL1 integrin inhibition on cartilage formation during cartilage formation. The chondrocytes (Sigma, 402-05f) were cultured in DMEM / F12 (gibco, 10565-018, supplemented with 10% (v / v) FBS and 1% (v / v) antibiotics) , ITGBL1 DNA was transfected by jetPRIME (Polyplus, 114-15) according to the protocol provided by the manufacturer, and the transfected cells were made into a lump form by a micromass method, and the cell lumps TGF-β, dexamethasone, and ascorbate-2-phosphate were treated with Mn ^{2 +} and DTT to induce cartilage formation. The cartilage tissue was fixed with 4% (w / v) paraformaldehyde to induce the formation of cartilage tissue for 7 days, and the fixed cartilage tissue was placed in OCT solution (Cell Path, KMA-0.00-00A) Bright, OTF5000) and then stained with Alcian blue. The amount of Glycosaminoglycan (GAG) was measured. The results of the above experiment are shown in C to E of Fig.

As shown in FIGS. 7E to 7E, the size of the cartilage increased by the overexpression of ITGBL1 was decreased by the addition of Mn ^{2 +} or DTT, and the size of the cartilage and the amount of GAG as a cartilage component decreased .

This demonstrates that the inhibitory activity of integrin activity of ITGBL1 is a functional group that promotes cartilage formation.

7-2. Integrin  Alpha < / RTI > and beta, Of ITGBL1  Expression of cartilage-forming gene when expression is increased at the same time

The following experiment was conducted to confirm the correlation between integrin and ITGBL1 in the process of cartilage formation. Chondrocyte cells were cultured in a medium supplemented with α-MEM (Welgene, LM008-01, 10% (v / v) FBS, 1% (v / v) antibiotics) and 2 × 10 ⁵ cells were plated on a 60 mm plate. 7, F, and G in the following table using the method of the protocol provided by the manufacturer using integrin beta 1, alpha 1, alpha 3, alpha 5, alpha 10 siRNA using jetPRIME (Polyplus, 114-15) As well as inhibiting the expression of the indicated integrin. In order to simultaneously perform the expression of ITGBL1 and the expression of integrin, the ITGBL1 DNA was transfected and inhibited at 37 < 0 > C. Then, the cells were extracted with RNA using a PureLink RNA Mini Kit (Invitrogen, 12183018A), and cDNA was synthesized using GoScript Reverse Transcriptase (Promega, A5004A). The synthesized cDNA was amplified with Taq polymerase (Coregen, CE-500U ), And qRT-PCR was performed using QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) -well plate, and the mean value was used to calculate mRNA expression.

As shown in F and G of FIG. 7, when integrin alpha and beta were simultaneously inhibited, expression of Sox9 was increased. When ITGBL1 expression was increased, expression of integrin alpha and beta was decreased and expression of Sox9 was decreased It was confirmed that it increased further.

The results of the experiments shown in Examples 6 and 7 show that the ITGBL1 protein inhibits the activity of the integrin protein and that the ITGBL1 inhibitory activity is a molecular mechanism promoting the expression of chondrogenic factors and cartilage formation of chondrocytes .

Example 8 . Of ITGBL1 Integrin  Correlation analysis between inhibitory activity and inflammatory and cartilage regression inhibition

8-1. ITGBL1  Increase the expression of the gene Integrin Activate  Occation Of cartilage regressing factor  Expression analysis

Chondrocyte cells were cultured in α-MEM medium (Welgene, LM008-01, supplemented with 10% (v / v) FBS and 1% (v / v) antibiotics) and 2 × 10 ⁵ cells were plated on a 60 mm plate. Fn-f 29kDa, a disruption product of Fibronectin, which is known to promote inflammation, has been treated. After using the jetPRIME (Polyplus, 114-15) ITGBL1 the vector in the cell by the manufacturer illustration how the transfection protocol provided by the process as Mn ^{+ 2} or DTT to activate the integrin to incubation for 37 ℃. Then, the cells were extracted with RNA using a PureLink RNA Mini Kit (Invitrogen, 12183018A), and cDNA was synthesized using GoScript Reverse Transcriptase (Promega, A5004A). The synthesized cDNA was amplified with Taq polymerase (Coregen, CE-500U ), And qRT-PCR was performed using QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) -well plates, and the mean values were used in the calculation of mRNA expression. The results of the above experiments are shown in Figures 8A-C.

As shown in FIGS. 8A to 8C, when the expression level of the MMP-3 and MMP-13 genes increased by increasing the expression of the ITGBL1 gene by treatment with Fn-f 29 kDa, Mn ^{2 +} or DTT was further treated The expression of MMP-3 and MMP-13 genes was increased when the integrin was activated. This means that the inhibitory effect of the ITGBL1 gene on integrin activity is a molecular functional group that reduces MMP-3 and MMP-13 gene expression that promote cartilage degeneration and inflammation.

In addition, the following experiment was conducted to confirm whether the ITGBL1 protein inhibited the binding between Fn-f 29kDa and chondrocytes, which promotes cartilage degradation. Cells were plated on a 60 mm plate at 2 × 10 ⁵ cells, and ITGBL1 siRNA or DNA was transfected into the cells by jetPRIME (Polyplus, 114-15) according to the protocol provided by the manufacturer. Then, The cells were fixed with 4% paraformaldehyde after treatment with a Fn-f 29kDa (sigma, F9911). The cells were observed using a Confocal (Zeiss, LSM880) microscope. The results of the above experiment are shown in D and E of FIG.

As shown in D and E of FIG. 8, when the expression of ITGBL1 was increased, the binding of Fn-29 kDa and chondrocytes was remarkably decreased, and this increased again when Mn ^{2 +} or DTT was treated. Therefore, it can be said that ITGBL1 inhibits the binding of Fn-29 kDa to chondrocytes by inhibiting integrin activity and suppresses the expression of cartilage regressing factors.

8-2. ITGBL1  Inhibiting the expression of the gene, Integrin's  Analysis of changes in cartilage regressors when inhibiting activity

In order to confirm the correlation between the inhibitory activity of integrin activity of ITGBL1 protein and the expression of cartilage regression factor, the following experiment was carried out. Chondrocyte cells were cultured in a medium supplemented with α-MEM (Welgene, LM008-01, 10% (v / v) FBS, 1% (v / v) antibiotics) and 2 × 10 ⁵ cells were plated on a 60 cm plate. ITGBL1-siRNA was transfected by jetPRIME (Polyplus, 114-15) according to the protocol provided by the manufacturer, followed by the addition of Factor Bio 1211 (TOCRIS, 3910), Obtustatin (TOCRIS, 4664) and ATN-161 (TOCRIS, 6058). Then, the cells were extracted with RNA using a PureLink RNA Mini Kit (Invitrogen, 12183018A), and cDNA was synthesized using GoScript Reverse Transcriptase (Promega, A5004A). The synthesized cDNA was amplified with Taq polymerase (Coregen, CE-500U ), And qRT-PCR was performed using QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) -well plate, and the mean value was used in the calculation of mRNA expression. The results of the above experiment are shown in Figures 9A-C.

As shown in FIGS. 9A to 9C, when the ITGBL1 gene was inhibited, the expression of MMP-3 and MMP-13 genes was increased. When these cells were treated with various kinds of integrin inhibitors, MMP-3, Expression of the MMP-13 gene was inhibited and ATN-161 of the integrin inhibitor was found to be effective in the home.

In addition, when knockdown of ITGBL1 is inhibited, cartilage degeneration and inflammatory reaction of the knee joint are confirmed, and the state of knee cartilage is examined when ATN-161, an integrin-beta1 inhibitor, is administered to the knee joints with suppressed ITGBL1 expression The following experiment was carried out.

 ITGBL1-shRNA-containing adenovirus vector (Ad-ITGBL1 shRNA) was injected into the knee joint to inhibit the expression of ITGBL1, and degeneration and inflammation of the knee joint cartilage were confirmed. In addition, ATN-161, an integrin-beta1 inhibitor, was injected into the joint muscles in which the expression of ITGBL1 was inhibited by injecting an adenovirus vector containing ITGBL1-shRNA (Ad-ITGBL1 shRNA) into the knee joint, The expression of Col2a1 and Sox9 was confirmed by Safranin-O staining and immunohistochemistry.

At this time, the control group was subjected to the same experiment as above, except that an adenovirus vector (Ad-C) not containing ITGBL1-shRNA was transduced. The results of the above experiment are shown in D and E of Fig.

As a result, as shown in D and E of FIG. 9, when the ITGBL1 expression was inhibited by injecting adenovirus containing ITGBL1-shRNA into the knee joint of the mouse, the health condition of the knee cartilage deteriorated and ITGBL1-shRNA The injection of ATN-161, an integrin-beta1 inhibitor, into joints that inhibited the expression of ITGBL1 by injection of adenovirus containing the compound resulted in restoration of knee cartilage health.

<110> UNIST (ULSAN NATIONAL INSTITUTE OF SCIENCE AND TECHNOLOGY) <120> Pharmaceutical compositions for preventing or treating cartilage          diseases <130> APC-2018-0367 <150> KR 10-2017-0163527 <151> 2017-11-30 <160> 21 <170> KoPatentin 3.0 <210> 1 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> ITGBL1 5 ' <400> 1 augcacgcug gagccuu 17 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ITGBL1 3 ' <400> 2 aggauauucg cuuccaagcc a 21 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> ITGBL1 MO <400> 3 agtagggaag atatacagac ctgca 25 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SOX9-F <400> 4 aaggagagcg aggaggacaa gttc 24 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SOX9-B <400> 5 tgttcttgct ggagccgttg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MMMP3-F <400> 6 gatgcgcaag cccaggtgtg 20 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> MMP3-B <400> 7 gccaatttca tgagcagcaa cga 23 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> MMP13-F <400> 8 aggagcatgg cgacttctac cc 22 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> MMP13-B <400> 9 tttgtctggc gtttttggat gttt 24 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Col2al-F <400> 10 cagttgggag taatgcaag 19 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Col2al-B <400> 11 gccttgagca gttcaccttc 20 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> MMP-3-F <400> 12 tcctgatgtt ggtggcttca g 21 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> MMP-3-B <400> 13 tgtcttggca aatccggtgt a 21 <210> 14 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> MMP-13-F <400> 14 accacatcga acttcga 17 <210> 15 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> MMP-13-B <400> 15 cgaccataca gatactg 17 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Col2al-F <400> 16 cacactggta agtggggcaa ga 22 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Col2al-B <400> 17 ggattgtgtt gtttcagggt tcg 23 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SOX9-F <400> 18 catcagcagc accgcaccca 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SOX9-B <400> 19 cgggtgatgg gcgggtagga 20 <210> 20 <211> 353 <212> PRT <213> Homo sapiens <400> 20 Met Cys Lys Asn Ser Gln Asp Ile Ile Cys Ser Asn Ala Gly Thr Cys   1 5 10 15 His Cys Gly Arg Cys Lys Cys Asp Asn Ser Asp Gly Ser Gly Leu Val              20 25 30 Tyr Gly Lys Phe Cys Glu Cys Asp Asp Arg Glu Cys Ile Asp Asp Glu          35 40 45 Thr Glu Ile Cys Gly Gly His Gly Lys Cys Tyr Cys Gly Asn Cys      50 55 60 Tyr Cys Lys Ala Gly Trp His Gly Asp Lys Cys Glu Phe Gln Cys Asp  65 70 75 80 Ile Thr Pro Trp Glu Ser Lys Arg Arg Cys Thr Ser Pro Asp Gly Lys                  85 90 95 Ile Cys Ser Asn Arg Gly Thr Cys Val Cys Gly Glu Cys Thr Cys His             100 105 110 Asp Val Asp Pro Thr Gly Asp Trp Gly Asp Ile His Gly Asp Thr Cys         115 120 125 Glu Cys Asp Glu Arg Asp Cys Arg Ala Val Tyr Asp Arg Tyr Ser Asp     130 135 140 Asp Phe Cys Ser Gly His Gly Gln Cys Asn Cys Gly Arg Cys Asp Cys 145 150 155 160 Lys Ala Gly Trp Tyr Gly Lys Lys Cys Glu His Pro Gln Ser Cys Thr                 165 170 175 Leu Ser Ala Glu Glu Ser Ile Arg Lys Cys Gln Gly Ser Ser Asp Leu             180 185 190 Pro Cys Ser Gly Arg Gly Lys Cys Glu Cys Gly Lys Cys Thr Cys Tyr         195 200 205 Pro Pro Gly Asp Arg Arg Val Tyr Gly Lys Thr Cys Glu Cys Asp Asp     210 215 220 Arg Arg Cys Glu Asp Leu Asp Gly Val Val Cys Gly Gly His Gly Thr 225 230 235 240 Cys Ser Cys Gly Arg Cys Val Cys Glu Arg Gly Trp Phe Gly Lys Leu                 245 250 255 Cys Gln His Pro Arg Lys Cys Asn Met Thr Glu Glu Gln Ser Lys Asn             260 265 270 Leu Cys Glu Ser Ala Asp Gly Ile Leu Cys Ser Gly Lys Gly Ser Cys         275 280 285 His Cys Gly Lys Cys Ile Cys Ser Ala Glu Glu Trp Tyr Ile Ser Gly     290 295 300 Glu Phe Cys Asp Cys Asp Asp Arg Asp Cys Asp Lys His Asp Gly Leu 305 310 315 320 Ile Cys Thr Gly Asn Gly Ile Cys Ser Cys Gly Asn Cys Glu Cys Trp                 325 330 335 Asp Gly Trp Asn Gly Asn Ala Cys Glu Ile Trp Leu Gly Ser Glu Tyr             340 345 350 Pro     <210> 21 <211> 4901 <212> DNA <213> Homo sapiens <400> 21 actctccact gaggggtttg gcaccagcac cccgcccaga gcagtgccgc tgcccaaatc 60 ctcgcaggca gctcatcaac gcaattgcaa ctccggctgg agccccggac ctgcaagcct 120 gggtgtccgt gggtccgtct gcccagccat ctgctggtgg cacctctccc tcctgccgcc 180 tccctcggtg aaccccacct tgcagaagtg cagctcgccc ggagcagccc aggagctcag 240 catgcgtccc ccaggcttca ggaacttctt gctgctggcg tcctcccttc tctttgctgg 300 gttgtcagct gttcctcaaa gcttctcgcc atctctgagg ccatggtaag tgtgactgtg 360 gcaagtgcaa gtgtgaccag ggatggtatg gggatgcttg ccagtaccca actaactgtg 420 acttgacaaa gaagaaaagt aaccaaatgt gcaagaattc acaagacatc atctgctcta 480 atgcaggtac atgtcactgt ggcaggtgta agtgtgataa ttcagatgga agtggacttg 540 tgtatggtaa attttgtgag tgtgacgata gagaatgcat agacgatgaa acagaagaaa 600 tatgtggagg ccatgggaag tgttactgtg gaaactgcta ctgcaaggct ggttggcatg 660 gagataaatg tgaattccag tgcgatatca ccccctggga aagcaagcga agatgcacgt 720 ctccagatgg caaaatctgc agtaacagag ggacttgtgt atgtggtgaa tgtacctgtc 780 acgatgttga tccgactggg gactggggag atattcatgg ggacacctgt gaatgtgatg 840 agagggactg tagagctgtc tatgaccgat attctgatga cttctgttca ggtcatggac 900 agtgtaattg cggaagatgt gactgcaaag caggctggta tgggaagaag tgtgagcacc 960 cacagtcctg cacgctgtca gctgaggaga gcatcaggaa gtgccaggga agctcggatc 1020 tgccttgctc tgggaggggt aaatgtgaat gtggcaaatg cacctgctat cctccaggag 1080 atcgccgggt gtatggcaag acttgtgagt gtgatgatcg ccgctgtgaa gacctcgatg 1140 gtgtggtctg tggaggccac ggcacatgtt cctgtggtcg ctgtgtttgt gagagaggat 1200 ggtttggaaa gctctgccaa catccgcgga agtgtaacat gacggaagaa caaagcaaga 1260 atctgtgtga atcagcagat ggcatattgt gctcggggaa gggttcttgt cattgtggga 1320 agtgcatttg ttctgctgaa gagtggtata tttctgggga gttctgtgac tgtgatgaca 1380 gagactgcga caaacatgat ggtctcattt gtacagggaa tggaatatgt agctgtggaa 1440 actgtgaatg ctgggatgga tggaatggaa atgcatgtga aatctggctt ggctcagaat 1500 atccttaaca attacatgag agagcaattt tgatgacgta ttcgctgatg cacagacatt 1560 ggaattcatt acattatcta gccatcgtaa tacaaatgac ctttggattt ctctatcacg 1620 gcgcttatcc tcctattaag gatcagaaag ttaaacttta cggaccacgt acgtccctgt 1680 ctcatattct tctttgttgg tttctgttta atactttaaa gctgtaaaaa aaatttaaaa 1740 aattccttct gagctcccag gccatacaaa agcaacttaa gggtggattt ggctggcagc 1800 tgggcagtac tttgtcagcc tcagctccac tggaactgcc cttgattctc tatagctcat 1860 cacaaaacct caggtatgtc ttatttttct tataatcctc tgtatgttat tatccttttt 1920 attaacaaca acaactacaa caaaagtgct gctctttttc atagtctcat gtaagaagag 1980 tgtcaaaaat gtcctcggca tttgttttct cagaaaaaga atttagatat tggagaaatt 2040 atcataccat aaaaagttca aaatgtgatt catctaaaat tttggacaaa gatgaaaata 2100 tagtcagctc tgttctagtg ttcatttagt tagaaaccat agtaccggaa ctgtattata 2160 gtagaagtta atggtaccta aactctaagg aatgcttttt ctttgaaaac agaaatattt 2220 ttacttattt ctgtccacta tgtttgtgtg tatgtgtgtg tttgtgtgtg tatgtgtggt 2280 tttgatgcca gcaatgcaaa caccaatata tctattaaga agtgattgaa tttagattgg 2340 aaaaaataaa atagttacat gtaaagacat aaactctttc cttcttaaat gtgattttca 2400 actctatgat agaaactccc tctaaaaaac aagcagggca agaacaaact tacttgtaat 2460 gggtgagaga aaataagcaa atctcattgg ctcattcctg tttttaggaa gttgcagtgg 2520 gattaatgtc aattggaagg gatgcagcaa gtttagagaa ggtgttggca ctaacgctgc 2580 ttgtttggca aatcatcatc actgaggtat tccacccaga gactttttca aaaaagtcaa 2640 ctaaagtgct aagtcataag aggagagcca ttatacccgt tgtctttctg ggctccttga 2700 gtttatctgg attccaacag cacttggaaa gtaccgccct ccactacctc aaatgcaaac 2760 acaatctctg ccagtagaca ttggaattag acttagtgaa aaccaggaaa aaaaaaaaaa 2820 actaaaatat aactcagcct tagtttgcag actcaactgt caacactttg aaaccctact 2880 ccatattcaa gagagtcaac aggccctatg tcacattgta ggctaagaaa cctcagctat 2940 gactactatt tcactcaaag taagaccatg tacatcctgg attttgatat gcctgcattt 3000 tggaaaaaaa aattgaggat ttagcccagt cgctttccct tgatatagcc ttgttctgaa 3060 ttaaaatgga aaaaatattt caattatttt catcttgttc tcaactggat gttagtcaat 3120 gtggtctctt tttgaatatg tatcaaatat taatgatcca taatattagg gagataattt 3180 taaatgaagt gatacgtgtc taacttactt aaagaataag tttttggttt ttgcttttaa 3240 agacaaccaa atctgatatt gttcatcctg ataaaaataa caacttttag tgcttaaagc 3300 attaattaag caagtggcta ggtatgataa agaacttctg cttgctcccc aagaggcaaa 3360 ctattagaag aactggagcg ggagtccttt ggacccatcg tggatctctt taagccactg 3420 ctacccaaaa acattcagga caagcaaaca tttagagcaa gaatctccaa attcttcagg 3480 atttgtaatg aaatgatgtt cagttccatt ttgctcttta catagggtgg agaattgtca 3540 tgtcttctct aatttttcca agtaaagtgg tagcaaaatg ttttaaaaag caatcttata 3600 ttagaaaaca aaaatgttgt cacttgaaat accaaaacaa catttctgag cgttgttgag 3660 ggactggcaa agcaatcagc tactataaca aatcagtaga aataaccctc ccacaccaga 3720 tatgcatgca gaaggaatgg agtattatag agacttgata caatggacat atgcacatgg 3780 aggtacaaaa cacacagtct aaatacaaat gaattccatc agatttacta tacggaacat 3840 cagtagtgac agattgcact tcttacttaa taacagcaaa cttaatttct gaggggaaaa 3900 aaatggcgaa gtcttatccc aaacaaatag caagagaggt atcatcaaag agctaaaatt 3960 ttctttggca tggtaaaggg ggaaattgag tttaccaact tatttacatg acatttctct 4020 atattggtga gtaatgcaat gccattttgt tacataaagt tgtttgatgt tttttaatat 4080 gccttcatat aaatatttta ttcaatatgt tgtatttgtg aatttaacaa atgatattaa 4140 acacaaacta caatgcagac agacaaactc tttgtatgca aattagcaat acataccaac 4200 agttcttgat acacaggtac ctactacatg cagctcatca ttgctgtcct cttcccatgc 4260 tacaggtgag accagacaca aagtaaatga cctaactcaa ttacaaataa caaggacccc 4320 tctgcaatat gtctaaacat atattagaag aaagtatttg acatccttca tagggataaa 4380 tgcccttata gacaccccat atataaaaca caacagagat acacatttac ataaatctca 4440 gtcatttaca aaaataaatc ttgtcttaat ttaaaccaat aaacagactt gcaggggaaa 4500 aagaaaccag caagtagggc taattatcag taacaaatag atgggggtgt ttgctctgtg 4560 tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tatatgtgtg tgtttgtgtg aagtgaagtg 4620 ttgctgctgt aagtagtgtc cataagccca tttgactgta ttacaagtta gttaattact 4680 catatagttg gccacatatt atgggatcta tgttttctga aaataattgg ttaatggaag 4740 ttatctaata tattttaact gttcctgtta aaaacaatag gcttcaagat gacataacac 4800 caaatcaaaa atgaccaaag gaatcatttt gtttgttaga tttgtaattt agcatcattg 4860 gcaataaatc tactcaaacg ttcaaaaaaa aaaaaaaaaa a 4901

Claims (16)

An integrin beta-like protein 1 (ITGBL1) protein, a gene encoding the protein, a recombinant vector comprising the gene, and a recombinant cell transformed with the recombinant vector. A pharmaceutical composition for preventing or treating cartilage diseases.
The pharmaceutical composition for preventing or treating cartilage diseases according to claim 1, wherein the ITGBL1 protein comprises the amino acid sequence of SEQ ID NO: 20.
The pharmaceutical composition for preventing or treating cartilage diseases according to claim 1, wherein the gene encoding the protein comprises the nucleotide sequence of SEQ ID NO: 21.
The pharmaceutical composition according to claim 1, wherein the cartilage disease is degenerative arthritis, post traumatic arthritis, or peelable osteochondritis.
The pharmaceutical composition according to claim 1, wherein the recombinant vector is a viral or non-viral vector.
The pharmaceutical composition according to claim 1, wherein the viral vector is selected from the group consisting of adenovirus, adeno-associated virus, and helper-dependent adenovirus and retrovirus vector.
The pharmaceutical composition for preventing or treating cartilage diseases according to claim 1, wherein the cell is a cell of a mammal.
The pharmaceutical composition for preventing or treating cartilage diseases according to claim 1, wherein the ITGBL1 gene or protein inhibits the interaction of ECM and integrin.
The pharmaceutical composition for preventing or treating cartilage diseases according to claim 1, wherein the composition inhibits inflammation of cartilage diseases.
The pharmaceutical composition for preventing or treating cartilage diseases according to claim 1, wherein the composition promotes cartilage formation.
A pharmaceutical preparation containing the composition of claim 1 as an active ingredient.
12. The pharmaceutical preparation according to claim 11, wherein the pharmaceutical preparation further comprises at least one member selected from the group consisting of a carrier, an excipient and a diluent.
The pharmaceutical composition according to claim 11, wherein the pharmaceutical preparation is in the form of tablets, pills, powders, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, Wherein the pharmaceutical formulation is a formulation selected from the group consisting of sterile disintegrants.
An integrin beta-like protein 1 (ITGBL1) protein, a gene encoding the protein, a recombinant vector comprising the gene, and a recombinant cell transformed with the recombinant vector. , Integrin protein activity inhibitor.
A method for the prophylaxis or treatment of cartilage diseases, comprising administering a pharmaceutically effective amount of the composition of claim 1 to an animal other than a human.
16. The method of claim 15, wherein the cartilage disease is degenerative arthritis, post traumatic arthritis, or peelable osteochondritis.

Images