WO2018169158A1 - Composition, for preventing or treating degenerative arthritis, comprising dicam as active ingredient - Google Patents

Abstract

The present invention relates to a composition, for preventing or treating degenerative arthritis, comprising as an active ingredient dual Ig domain containing cell adhesion molecule (DICAM) protein or nucleic acid molecules coding DICAM protein. According to the present invention, degenerative arthritis treatment effects with respect to DICAM treatment have been specifically confirmed, and more fundamental treatment is expected in preventing or treating degenerative arthritis.

Description

Composition for the prevention or treatment of degenerative arthritis containing DICAM as an active ingredient

The present invention relates to a composition for the prevention or treatment of degenerative arthritis, comprising a dual lg domain containing cell adhesion molecule (DICAM) protein or a nucleic acid molecule encoding a DICAM protein as an active ingredient.

Osteoarthritis is a disease characterized by degeneration and loss of articular cartilage, the most common chronic degenerative joint disease. Osteoarthritis is the leading cause of disability in the elderly population, with joints accompanied by pain, stiffness, swelling, and decreased function.

The prevalence rate continues to increase due to an aging society and an increase in obesity, and according to a 2010 National Health and Nutrition Survey report, the prevalence of osteoarthritis in adults over 50 years of age is 28.2%, with more than one in four having osteoarthritis. . Of these, 5,5% were male and 22.7% were female, and women were four times higher than men, and osteoarthritis is expected to increase as the elderly population and life expectancy affect the prevalence of arthritis due to the characteristics of arthritis that occurs frequently in the elderly. The social and economic losses are also expected to increase.

According to the 2011 National Health Insurance Corporation's data, the average life expectancy has increased, and the number of reoperations has increased, and the cost of knee joint surgery has more than doubled since five years ago. Accordingly, research is being actively conducted to develop effective treatments.

According to a recent report entitled Global Osteoarthritis Therapeutics Market: Pipeline Analysis and Market Forecasts, the 2011 global osteoarthritis market is estimated at $ 4.52 billion, and has grown at a CAGR of 3.7%. It is expected to reach $ 6,161 million by 2019.

Osteoarthritis is a chronic disease with pain, and there is no curative therapy.Therefore, it is necessary to improve pain treatment which is essential for maintaining the quality of life of patients.Now, in the market for OA pain medicine, nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics, corticosteroids, Opioids and the like have effectively alleviated mid-term OA pain. However, this does not alleviate the pain of late OA during cartilage degeneration, and NSAID may cause side effects when used for a long time. Therefore, it is expected to develop a safe new drug to treat the pain of late OA.

The study of the role of adhesion proteins in the proliferation and differentiation of chondrocytes has given us new opportunities to broaden our understanding of the pathogenesis of osteoarthritis as well as new academic approaches. Currently, the treatment of osteoarthritis focuses on symptomatic drug treatment and pain relief, and there are no drugs that have been shown to be effective in preventing the degeneration of chondrocytes. If the molecular pathology is identified as a key pathological mechanism, it can be used not only to broaden the pathological understanding of osteoarthritis, but also to develop drugs based on the mechanism.

The differentiation process of chondrocytes and the pathogenesis of osteoarthritis go through a similar process of proliferation, differentiation and apoptosis and calcification. There is little research on the role of adhesion proteins in the proliferation and differentiation of chondrocytes. Condensation is essential for differentiation of mesenchymal stem cells into chondrocytes. In this process, intercellular interactions by adhesion proteins are essential, but there are no studies on the role of adhesion proteins. Although there is a study on Vascular cell adhesion molecule-1 (VCAM-1) among the adhesion proteins related to osteoarthritis (Korean Patent Publication No. 2007-0115761), this is not an explanation for its function but is diagnosed due to an increase in the secretion of osteoarthritis. It is a study on the possibility of use as a marker.

The present invention has been made to solve the above problems, the present inventors have confirmed the effect of treating degenerative arthritis according to the treatment of Dual Ig domain containing cell adhesion molecule (DICAM) to complete the present invention.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of degenerative arthritis, comprising DICAM (Dual Ig domain containing cell adhesion molecule) as an active ingredient.

It is another object of the present invention to provide a composition for chondrocyte proliferation or differentiation comprising a dual lg domain containing cell adhesion molecule (DICAM) as an active ingredient.

In addition, another object of the present invention is the step of treating the test material to the DIG (Dual Ig domain containing cell adhesion molecule) expression cell line (S1);

Measuring the expression or activity level of DICAM in the cell line of step S1 (S2); And

It is to provide a method for screening a candidate degenerative arthritis therapeutic agent, comprising the step (S3) of selecting a test substance whose level of DICAM expression or activity of the step S2 is increased compared to the control group not treated with the test substance.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention is a pharmaceutical composition for the prevention or treatment of degenerative arthritis comprising a dual lg domain containing cell adhesion molecule (DICAM) protein or a nucleic acid molecule encoding a DICAM protein as an active ingredient To provide.

In one embodiment of the invention, the DICAM protein is characterized in that consisting of the amino acid sequence represented by SEQ ID NO: 1.

In another embodiment of the present invention, the nucleic acid molecule encoding the DICAM protein is characterized by consisting of a nucleotide sequence represented by SEQ ID NO: 2.

In another embodiment of the present invention, the nucleic acid molecule encoding the DICAM protein is characterized in that it is included in a vector.

In another embodiment of the invention, the vector is characterized in that at least one member selected from the group consisting of linear DNA, plasmid DNA and recombinant viral vector.

In another embodiment of the present invention, the recombinant viral vector is characterized in that AAV (Adeno-associated virus).

In another embodiment of the present invention, the composition is characterized in that it can proliferate or differentiate into chondrocytes.

In addition, the present invention can provide a composition for chondrocyte proliferation or differentiation comprising a DICAM protein or a nucleic acid molecule encoding a DICAM protein as an active ingredient.

In addition, the present invention comprises the steps of treating the test material to the DIIG (Dual Ig domain containing cell adhesion molecule) expression cell line (S1);

Measuring the expression or activity level of DICAM in the cell line of step S1 (S2); And

It provides a method for screening a candidate degenerative arthritis therapeutic agent, comprising the step (S3) of selecting the test substance whose level of DICAM expression or activity of the step S2 is increased compared to the control group not treated with the test substance.

In one embodiment of the present invention, the method for measuring the expression level is reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (competitive RT-PCR), real time transcriptase polymerase reaction (real time quantitative RT-PCR), RNase protection method (RNase protection method), Northern blotting (Northern blotting) and DNA chip technology (DNA chip technology) is characterized in that at least one method selected from the group consisting of.

In another embodiment of the present invention, the method for measuring the activity level is Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunoasiffusion (radioalimififfusion), oak Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunofluorescence It is characterized in that at least one method selected from the group consisting of immunochromatography (FAMO), FACS (fluorescenceactivated cell sorter analysis) and protein chip technology (protein chip technology).

The present invention also provides a method of treating degenerative arthritis, comprising administering the pharmaceutical composition to a subject.

In addition, the present invention provides a treatment for degenerative arthritis of a composition comprising a dual lg domain containing cell adhesion molecule (DICAM).

The composition according to the present invention comprises a DICAM (Dual Ig domain containing cell adhesion molecule) protein as an active ingredient, IHH-PTHrP which is an essential factor for increasing expression of chondrocyte matrix proteins and regulating the proliferation and differentiation of chondrocytes according to the DICAM treatment. Increased expression of was confirmed. In addition, the anti-inflammatory and angiogenesis-inhibiting function of DICAMs in previous studies is expected to provide a more fundamental target therapeutic method in the prevention or treatment of degenerative arthritis.

1 is a diagram showing the results of confirming the expression pattern of DICAM through immunofluorescence staining.

Figure 2 shows the results of evaluating the proliferation of chondrocytes by Alcian blue staining after treatment with Ad-LacZ and Ad-DICAM.

3 is a view showing the results of confirming the synergistic effect of DICAM and IHH.

4 is a view showing the results confirmed by tibial tissue culture that the function of DICAM occurs through the IHH signal.

5 is a view showing the results of confirming the formation of primary cilia by DICAM and signal transmission of IHH through the primary cilia.

Figure 6 is a diagram showing the results confirmed by tibial tissue culture primary ciliogenesis by DICAM.

7 is a diagram showing the results of confirming the expression after the injection of a DICAM overexpression transformation vector, and the result of confirming the Genotype of Tg.

8 is a view showing the results of comparing the total bone and cartilage formation pattern of normal mice and Col2-DICAM Tg.

9 is a view showing the results confirmed by immunofluorescence staining the expression level of DICAM in Wild type and Col2-DICAM Tg.

10 is a diagram showing the results of comparing the expression level of the PCNA immunofluorescence staining to determine the proliferation and apoptosis of chondrocytes by DICAM.

11 is a view showing the results of confirming the expression of DICAM after inducing osteoarthritis through the surgical osteoarthritis model (DMM).

12 is injected with adenovirus (Ad-Dicam) and Ad-LacZ, a control group that overexpresses DICAM in the knee joint cavity of osteoarthritis induced normal osteoarthritis by the above method, in order to confirm the role of DICAM in the pathogenesis of osteoarthritis. Figure 4 shows the result of comparing the severity of osteoarthritis after the four weeks, the bottom is a diagram showing the result of comparing the degree of osteoarthritis in Wild type with Col2-Dicam Tg.

The present inventors confirmed the differentiation effect of chondrocytes in DICAM overexpressing mice, and confirmed that they can increase the expression of genes and proteins associated with the differentiation and proliferation of chondrocytes, and completed the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the prevention or treatment of degenerative arthritis, which comprises a dual lg domain containing cell adhesion molecule (DICAM) protein or a nucleic acid molecule encoding a DICAM protein as an active ingredient.

The DICAM (Dual Ig domain containing cell adhesion molecule) protein preferably includes a protein having an amino acid sequence represented by SEQ ID NO: 1 or a protein encoded by a nucleotide sequence represented by SEQ ID NO: 2 and a functional equivalent of the protein. . The term "functional equivalent" means at least 70%, preferably 80%, more preferably 90%, even more preferably at least 70% of the amino acid sequence represented by SEQ ID NO: 1 as a result of the addition, substitution, or deletion of the amino acid. Preferably, it refers to a protein having a sequence homology of 95% or more, and having a substantially homogeneous physiological activity with a protein having an amino acid sequence represented by SEQ ID NO: 1.

DICAM proteins of the present invention also include proteins having their native amino acid sequences, as well as amino acid sequence variants thereof, are also within the scope of the present invention. A variant of a DICAM protein refers to a protein in which the DICAM native amino acid sequence and one or more amino acid residues have a different sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. Amino acid exchanges in proteins and peptides that do not alter the activity of the molecule as a whole are known in the art. The DICAM protein or variant thereof can be extracted from nature or synthesized (Merrifleld, J. Amer. Chem. Soc. 85: 2149-2156, 1963) or by genetic recombination methods based on DNA sequences (Sambrook et al. al, Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd edition, 1989).

The nucleic acid molecule encoding the DICAM protein may be preferably composed of the nucleotide sequence represented by SEQ ID NO: 2, variants of the sequence are included within the scope of the present invention. Specifically, the nucleic acid molecule has a base sequence having at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% homology with the nucleotide sequence of SEQ ID NO: 1, respectively. It may include. The "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).

The nucleic acid molecule encoding the DICAM protein of the present invention may be in a form included in the vector. The vector comprising a nucleic acid molecule encoding the DICAM protein is a linear DNA, a plasmid vector, a vector comprising a viral expression vector, or a recombinant retrovirus vector, a recombinant adenovirus expressed in human or animal cells. It is preferably a recombinant viral vector comprising a vector, a recombinant adeno-associated virus (AAV) vector, a recombinant herpes simplex virus vector or a recombinant lentivirus vector, and preferably a recombinant adeno-associated virus. (adeno-associated virus, AAV) vectors are more preferred, but are not limited thereto.

As used herein, the term "prevention" means any action that inhibits or delays the development of degenerative arthritis by administration of a pharmaceutical composition according to the present invention.

As used herein, the term "treatment" refers to any action in which symptoms for degenerative arthritis improve or beneficially change by administration of the pharmaceutical composition according to the present invention.

The pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient. At this time, the pharmaceutically acceptable carrier is commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose , Polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. In addition to the above components, it may further include a lubricant, wetting agent, sweetener, flavoring agent, emulsifier, suspending agent, preservative and the like.

The pharmaceutical compositions of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is determined by the condition and weight of the patient, Depending on the extent, drug form, route of administration, and time, it may be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level refers to the type of disease, the severity, the activity of the drug, It may be determined according to the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of treatment, factors including the drug used concurrently and other factors well known in the medical field. The pharmaceutical compositions according to the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the age, sex, condition, weight of the patient, the absorption of the active ingredient in the body, the inactivation rate and excretion rate, the type of disease, the drug used in general 100 to 500 mg / kg body weight may be administered daily or every other day, or divided into 1 to 3 times a day. However, since the dose may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., the above dosage does not limit the scope of the present invention by any method.

As used herein, the term " improvement " means any action that at least reduces the parameters associated with the condition being treated, such as the extent of symptoms. In this case, the pharmaceutical composition may be used simultaneously or separately with a medicament for treatment before or after the onset of the disease in order to prevent or improve degenerative arthritis.

In addition, the present invention comprises the steps of treating the test material to the DIIG (Dual Ig domain containing cell adhesion molecule) expression cell line (S1);

Measuring the expression or activity level of DICAM in the cell line of step S1 (S2); And

It provides a method for screening a candidate degenerative arthritis therapeutic agent, comprising the step (S3) of selecting the test substance whose level of DICAM expression or activity of the step S2 is increased compared to the control group not treated with the test substance.

The term "candidate" refers to a substance for testing degenerative arthritis therapeutic activity, and may include any molecule such as extracts, proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides and a wide variety of compounds. Such candidates also include natural as well as synthetic materials.

Methods for measuring the expression level are reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (competitive RT-PCR), real time quantitative RT-PCR, RNase protection assay (RNase protection method), Northern blotting (Northern blotting) and DNA chip technology (DNA chip technology) may be one or more methods selected from the group consisting of, but is not limited thereto.

The method for measuring the activity level is Western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunoasiffusion (radial immunodiffusion), Ouchterlony immunodiffusion (Ouchterlony immunodiffusion) Rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography, immunochromatography, immunochromatography, It may be one or more methods selected from the group consisting of FACS analysis (fluorescenceactivated cell sorter analysis) and protein chip technology, but is not limited thereto.

In another aspect of the present invention, the present invention provides a method for treating degenerative arthritis, comprising administering the pharmaceutical composition to a subject. As used herein, "individual" means a subject in need of treatment of a disease, and more specifically, a mammal, such as a primate, mouse, dog, cat, horse, and cow, which is human or non-human. do.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

EXAMPLE

Example 1. Confirmation of the mechanism of regulation of proliferation and differentiation of chondrocytes by DICAM

Prior to the experiment, recombinant DICAM protein was overexpressed in E. coli strain (BL21), isolated, purified and injected subcutaneously in rabbits to prepare anti-DICAM polyclonal antibodies.

In this example, the expression pattern of DICAM was first confirmed by immunofluorescence staining at 15.5 days and 18.5 days of gestation. Tibia of 15.5 days and 18.5 days of gestation were separated, paraffin sections were used, and expression sites were identified using the DICAM antibody. In Tibia at 15.5 and 18.5 days of gestation, DICAM was found to be expressed mainly in chondrocytes at resting and proliferative phases. As the differentiation progressed, the expression decreased in the late hypertrophic cells where calcification proceeded. It was confirmed that expression did not occur. The expression of DICAM was similar to that of the collagen type 2, the main cartilage matrix protein (Fig. 1). In primary cultured chondrocyte experiments, it was confirmed that the expression of type 2 collagen is increased by DICAM. Therefore, it is necessary to confirm the detailed mechanism of the expression control of type 2 collagen by DICAM.

To confirm the role of DICAM in the proliferation and differentiation of chondrocytes, DICAM was overexpressed using Adenovirus during microculture and primary culture of chondrocytes. For microculture, the mothers were sacrificed to the cervical vertebra after 11 days of gestation (11.5 days of gestation), and fetuses were separated from the placenta. Limb buds of fetuses were isolated and then treated with collagenase to separate them into single cells, and cultured to add 2.5 x 10 ⁷ cells per ml, followed by 10-ul dot culture. During incubation, Ad-DICAM was treated with 100moi and 100moi Ad-LacZ was used as a control to confirm cartilage differentiation through Alcian blue staining after 7 days. When the chondrocyte differentiation was markedly increased, the Alcian blue stain was dissolved with 6M GnHCl, and measured by ELISA Reader. In primary cultured chondrocytes, muscles and soft tissues were removed from the fetal limbs of 15.5 days of gestation, and the remaining cartilage was separated into single cells using collagenase, and seeded at 5 x 10 ⁵ cells / well on a 6 well plate. The next day, the control group Ad-LacZ and Ad-DICAM was treated with 100moi and 7 days later, the proliferation of chondrocytes was evaluated by Alcian blue staining. DICAM overexpression in primary cultured chondrocytes increases the expression of IHH (Abcam (ab51919)), Sox9 (Abcam (ab26414)) and type 2 collagen (Abcam (ab34712)), which are factors regulating proliferation and differentiation of fetal chondrocytes The expression of collagen degrading enzyme, MMP13 (AbCam (ab39012)), was confirmed by Western blot. In addition, the expression of type 2 collagen and Aggrecan (Forward: 5'-CCAGCCTACACCCCAGTG3 ', Reverse: 5'-GAGGGTGGGAAGCCATGT-3' / CosmoGentech Co., Ltd.) was increased by Realtime PCR, and Mmp3 (Forward: 5＇-TTGTTCTTTGATGCAGTCAGC) , Reverse: 5'-GATTTGCGCCAAAAGTGC 3 '/ Cosmotechtech Co., Ltd.) and Mmp13 (Forward: 5'-GCCAGAACTTCCCAACCAT-3', Reverse: 5'-TCAGAGCCCAGAATTTTCTCC-3 '/ Cosmotech Tech Co., Ltd.) It was confirmed that (Fig. 2).

To determine whether the increase in chondrocyte proliferation and differentiation by DICAM occurs through any of the major differentiation regulators of chondrocytes, Id1 (Forward: 5'-GCGAGATCAGTGCCTTGG-3 ', Reverse: 5) in LacZ and DICAM overexpressing primary chondrocytes BMP signal through the expression of '-CTCCTGAAGGGCTGGAGT-3' / Cosmotech Tech Co., Ltd. Hhip (5'-CCCAGACTCACAATGGAAAACTCT-3 ', 5'-GGTGTAGTAGCCGTTTCGACAGA-3' / Cosmotech Inc.), Ptch1 (Forward: 5'-GGAAGGGGCAAAGCTACAGT-3 ', Reverse: 5'-TCCACCGTAAAGGAGGCTTA-3' /) ITHH signal through the expression of Cosmojintech Co., Ltd., PTHrP signal through the expression of Zfp521 (5'-AAGCAAGCGAAACCGAGAT-3 ', Reverse: 5'-TTCTGGCCTCTTCTTGCAGT-3' / CosmoGentech) FGF signal was expressed through expression of Dusp6 (Forward: 5'-TTTTCCCTGAGGCCATTTCTT-3 ', Reverse: 5'-GATACCTGCCAAGCAATGCA-3' / CosmoGentech Co., Ltd.). Reverse: 5'-TTCTTACTCCCCATGCGGTAA-3 '/ Cosmojintech Co., Ltd. Wnt signal, Hes1 (Forward: 5'-CCAGCCAGTGTCAACACGA-3', Reverse: 5'-AATGCCGGGAGCTATCTTTCT-3 '/ Cosmogenetech) was confirmed the Notch signal through the expression. Among the six major signaling systems, the IHH target gene, Hhip, increased the most by DICAM, and Western blot confirmed the expression of the lower IHH signal protein by DICAM. It was confirmed. In addition, in order to confirm synergistic effects of DICAM and IHH, IHH recombinant protein was treated with DICAM overexpressing primary chondrocytes, and the expression of IHH lower protein was highest in the group treated with DICAM and IHH recombinant protein. It was confirmed that the expression of Pthlh (PTHrP) with growth and degenerative arthritis suppression mechanism showed the highest synergistic effect. The above result is to understand that DICAM is involved in the proliferation and differentiation of chondrocytes through synergistic effect with IHH (Fig. 3).

4 shows the results of confirming the role of DICAM in the proliferation and differentiation of chondrocytes and the tibial tissue culture that the function of DICAM occurs through IHH signaling. Tibial muscle and soft tissue was removed from the fetal hind limbs of 15.5 days of gestation and DICAM was overexpressed by treatment with Ad-DICAM while culturing for 6 days in culture medium. In addition, IHH inhibitor Cyclopamine was treated to inhibit the expression of IHH by DICAM identified in the previous experiment. During tissue culture, DICAM overexpression was confirmed to increase the length of the tibia, and especially to increase the hypertrophic chondrocyte layer. At this time, when treated with Cyclopamine, it was confirmed that the difference in the length of the tibia increased by DICAM was eliminated, and the length of the hypertrophic cartilage layer was also reduced (FIG. 4).

Most cells in the body have cell organelles that protrude out of the cell called primary cilia. Primary cilia have been regarded as by-products of motionless and non-functional evolution unlike normal cilia, but recent studies have shown that primary cilia act as signal hubs that control various signaling pathways that regulate cell proliferation and differentiation. This turns out. Chondrocytes have primary cilia like other cells. Recently, Ihh is known to transmit signals through primary cilia, and the increase of IIHH signal by DICAM can be the basis for promoting the function of primary cilia. The formation of primary cilia by and the signaling of IHH through the primary cilia were confirmed. First, the expression of DICAM in primary cilia was confirmed by immunofluorescence staining. As a result, DICAM and Acetylated Tubulin, a primary cilia marker, were expressed at the same position in primary cultured chondrocytes. To confirm the function of DICAM in primary cilia formation, expression of Acetylated Tubulin and IFT88, the markers of primary cilia, was observed in the tibial sections of DICAM overexpressing primary chondrocytes and Col2-DICAM Tg at 15.5 days. Overexpression of DICAMs in primary cultured chondrocytes increased the number of Acetylated Tubulin positive cells, and in the tibia of Col2-DICAM Tg, the number of Acetylated Tubulin and IFT88-expressing cells also increased. Based on the results, it was confirmed that DICAM increased the formation of primary cilia in vitro and in vivo (FIG. 5).

Increased primary cilia production by DICAM was again confirmed in tibial tissue culture at 15.5 days of gestation. After tibial separation of the 15.5-day-old fetus, the expression of IFT88 was suppressed to overexpress DICAM with adeno-associated virus and to inhibit the production of primary cilia. The number of Acetylated Tubulin-expressing cells, the markers of primary cilia, increased in DICAM overexpressed tibia and Acetylated Tubulin expression decreased in tibia that inhibited IFT88 expression. However, by increasing the expression of DICAM in the IFT88 inhibitory tibia, it was confirmed that the number of Acetylated Tubulin expressing chondrocytes increased again (FIG. 6). This is again a result of increasing the production of primary cilia by DICAM.

Example 2 Study on the Role of DICAM in Proliferation and Differentiation of Chondrocytes by Col2-DICAM Transgenic Mouse Assay

To confirm in vivo the role of DICAM in the proliferation and differentiation of cartilage, mice overexpressed by DICAM by type 2 collagen promoter (Col2-DICAM Tg) were analyzed. In the transformation vector, the ColN promoter was cloned by inserting the Col2 promoter into the pNASSb vector, and then DICAM was inserted at the Not I position to make a DICAM overexpression transformation vector, which was injected into HeLa cells to confirm the expression of DICAM by Western blot. It was. DICAM Tg mice were selected by injecting Col2-DICAM Tg vector with DICAM expression into the sperm derived nucleus of fertilized egg 1 day after fertilization and re-injecting fertilized egg into fallopian tube of surrogate mother. In order to further increase the expression of DICAM, mice that had the Col2-DICAM vector in both pairs of chromosomes were selected by re-crossing the Tg from the Col2-DICAM Tg and the wild type. Genotype of born Tg was confirmed by PCR and genomic DNA PCR (Fig. 7).

The total bone and cartilage formation patterns of the 18.5 days of normal mice and Col2-DICAM Tg were confirmed by Alcian blue / Alizarin red staining. After 18.5 days of birth, normal mice and fetuses of Col2-DICAM Tg were separated, and skin and viscera were removed and fixed in 95% EtOH. Cartilage was stained with Alcian blue, soft tissue was removed with 2% KOH, and bone was stained with Alizarin red to compare total cartilage and bone formation. Overall bone and cartilage formation pattern of Col2-DICAM Tg was not different as compared to normal mice (FIG. 8).

However, Alcian blue / Alizarin red staining of the vertebrae, femurs, and tibias showed no increase in the overall length of the Col2-Dicam Tg. The expression level of DICAM in wild type and Col2-DICAM Tg was confirmed by immunofluorescence staining. Tibia of 15.5 days of gestation were isolated and paraffin sections and immunofluorescent stained using DICAM antibody. It was confirmed that the expression of DICAM is expressed in chondrocytes of the resting and proliferative phase, and the expression level of Col2-DICAM Tg was increased compared to the wild type. The degree of cartilage matrix formation and cartilage differentiation was confirmed by Safranin-O staining. It was confirmed that the femur and tibia increased in Col2-DICAM Tg were mainly due to the increase of the resting and proliferative chondrocytes, not the hypertrophic cartilage layer. In addition, in the proliferative chondrocytes, the proliferative chondrocytes of Col2-DICAM Tg had irregular overlapping patterns and sporadically scattered single or two or three cells compared to the normal mice in which several cells were regularly overlapped. It was also confirmed that the total growth plate cartilage length and the proliferative cartilage length were slightly longer than normal mice (FIG. 9).

To compare the proliferation and apoptosis of chondrocytes by DICAM in normal mice and 15.5 days of tibia of Col2-DICAM Tg, and immunofluorescence staining of PCNA on E15.5-day tibial paraffin sections of normal mice and Col2-DICAM Tg. The degree of expression was compared. Compared to normal mice, PCNA expression can be observed not only in resting and proliferative chondrocytes but also in hypertrophic chondrocytes in the tibia of Col2-DICAM Tg, and in the results of measuring the percentage of PCNA positive cells (Fig. 10). Apoptosis of chondrocytes was confirmed by TUNEL staining. Paraffin-sectioned 15.5 days of wild type and Col2-DICAM Tg Femur slides were rehydrated, washed with PBS, permubilized for 3 minutes with 20ug / ml Proteinase K, and then washed twice with PBS. Buffer was treated for 1 minute and TdT Enzyme for 1 hour. Then washed with PBS, treated with Anti-Digoxigenin conjugate for 30 minutes and then stained with DAB. Comparing the degree of staining, there was no difference in apoptosis of chondrocytes in Wild type and Col2-DICAM Tg (FIG. 10).

Example 3 Confirmation of the Severity of Osteoarthritis Decreased by DICAM

In order to confirm the function of DICAM in the development of osteoarthritis, the expression of DICAM was confirmed in the osteoarthritis model of mice and the incidence and severity of osteoarthritis due to DICAM overexpression was evaluated. First, the osteoarthritis model was selected as the destabilization of medial meniscus (DMM) method, which is the most commonly used surgical osteoarthritis model with symptoms similar to those of human osteoarthritis. After anesthetizing 8-12 weeks old mice, the medial meniscus ligament of the left knee joint was cut with a surgical knife and destabilized to induce osteoarthritis. Four or eight weeks later, the mice were sacrificed and the knee joints were separated. To determine the severity of osteoarthritis. The isolated knee joint was fixed, demineralized, and paraffin sections in the same manner as above, and the expression of DICAM was confirmed by immunohistostaining. DICAM showed high expression in normal knee insulated bone, but expression was significantly decreased in knee articular cartilage of mice with osteoarthritis (FIG. 11).

In order to confirm the role of DICAM in the pathogenesis of osteoarthritis, osteoarthritis was injected 4 weeks after the injection of adenovirus (Ad-Dicam) and Ad-LacZ, a control group, overexpressing DICAM into the knee joint cavity of osteoarthritis-induced normal mice. The severity of was compared. It was confirmed that the osteoarthritis severity of the articular cartilage injected with Ad-DICAM was significantly reduced compared to the control Ad-LacZ (FIG. 12).

Also, the incidence of osteoarthritis in Col2-Dicam Tg was compared with that of wild type. When osteoarthritis was induced in the wild type and Col2-Dicam Tg mice by the above method and the severity was compared after 8 weeks, it was confirmed that the incidence of osteoarthritis was reduced in the Col2-Dicam Tg mice compared to the wild type (FIG. 13).

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims (11)

1. A pharmaceutical composition for the prevention or treatment of degenerative arthritis, comprising a DICAM (Dual Ig domain containing cell adhesion molecule) protein or a nucleic acid molecule encoding a DICAM protein as an active ingredient.
2. The method of claim 1,

   The DICAM protein, characterized in that consisting of the amino acid sequence represented by SEQ ID NO: 1, a pharmaceutical composition for the prevention or treatment of degenerative arthritis.
3. The method of claim 1,

   The nucleic acid molecule encoding the DICAM protein is characterized by consisting of a nucleotide sequence represented by SEQ ID NO: 2, pharmaceutical composition for the prevention or treatment of degenerative arthritis.
4. The method of claim 1,

   The nucleic acid molecule encoding the DICAM protein is contained in a vector, a pharmaceutical composition for preventing or treating degenerative arthritis.
5. The method of claim 4, wherein

   The vector is at least one member selected from the group consisting of linear DNA, plasmid DNA and recombinant viral vectors, pharmaceutical composition for the prevention or treatment of degenerative arthritis.
6. The method of claim 5,

   The recombinant viral vector is AAV (Adeno-associated virus), characterized in that the prevention or treatment of degenerative arthritis pharmaceutical composition.
7. The method of claim 1,

   The composition is characterized in that the proliferation of chondrocytes or to differentiate into chondrocytes, pharmaceutical composition for the prevention or treatment of degenerative arthritis.
8. Composition for proliferation or differentiation of chondrocytes comprising a dual lg domain containing cell adhesion molecule (DICAM) protein or a nucleic acid molecule encoding a DICAM protein as an active ingredient.
9. Treating the test substance with a dual lg domain containing cell adhesion molecule (DICAM) expressing cell line (S1);

   Measuring the expression or activity level of DICAM in the cell line of step S1 (S2); And

   A screening method for a degenerative arthritis therapeutic agent comprising a step (S3) of selecting a test substance whose level of DICAM expression or activity of step S2 is increased compared to a control group not treated with the test substance.
10. The method of claim 9,

    Methods for measuring the expression level are reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (competitive RT-PCR), real time quantitative RT-PCR, RNase protection assay (RNase protection method), Northern blotting (Northern blotting) and DNA chip technology (DNA chip technology), characterized in that at least one method selected from the group consisting of, the method for screening candidate drugs for the treatment of degenerative arthritis.
11. The method of claim 9,

    The method of measuring the activity level is Western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunoasiffusion (radial immunodiffusion), Ouchterlony immunodiffusion (Ouchterlony immunodiffusion) Rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography, immunochromatography, immunochromatography, A method for screening a candidate for treating degenerative arthritis, characterized in that it is at least one method selected from the group consisting of FACS analysis (fluorescenceactivated cell sorter analysis) and protein chip technology.

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