KR101151795B1 - Biomarkers Indicative of Arthritis and Diagnosis Using the Same - Google Patents

Abstract

The present invention relates to a composition for diagnosing or prognostic arthritis and a kit comprising the same. ECRG 4 in the present invention is a marker with greatly improved accuracy and reliability as a marker for arthritis. In addition, the present invention can determine the early diagnosis and prognosis of arthritis very specifically from biological samples (eg, chondrocytes or tissues) using ECRG 4, which specifically reduces expression only in tissues or cells of arthritis patients. .

Description

Biomarkers for Arthritis and Diagnosis of Arthritis Using Them {Biomarkers Indicative of Arthritis and Diagnosis Using the Same}

The present invention relates to biomarkers specific for arthritis and uses thereof.

The number of arthritis patients exceeds 43 million in the United States, and degenerative arthritis is the leading cause of disability worldwide, ranking fourth in women and eighth in men. In Korea, knee joint disease is the single disease of orthopedic disease, which is the number one claim for outpatient insurance claims, accounting for 1.3% of total outpatient medical care benefits. Each year, more than $ 64 billion is spent on joint cartilage damage or disease when estimating the cost of treatment and the loss caused by illness.

In Korea, 80% of patients over 55 years of age and almost 70% of people over 70 years of age are degenerative arthritis. The market size of arthritis, which is continuously increasing in frequency, is estimated to be about US $ 9.5 billion in 2000, an average annual increase of 14%, US $ 20 billion in 2005 and US $ 35 billion in 2010. In 2009, the market was estimated at about 400 billion won. This trend is expected to accelerate in the future due to the increase in cartilage damage due to degenerative arthritis and trauma caused by the increase of elderly population and sports activities. In the next 10 years, the elderly population aged 65 or over is expected to reach 16.3% of the world, and new pharmaceuticals, cells, tissues and organs will continue to be required due to diseases and industrial and natural disasters caused by this aging. In particular, dysfunction of the musculoskeletal system not only seriously degrades the quality of life, but also leads to the loss of the individual and national economy.

Cartilage breakdown is a direct cause of arthritis, a refractory disease that occurs gradually and irreversibly and degrades quality of life. Increasing leisure activities and increasing elderly populations have highlighted the importance of treating arthritis, but the pathological causes and underlying treatments have not yet been developed. In addition to surgical methods, it is only used to relieve pain through anti-inflammatory drugs, and cartilage protective agents (hyaluronic acid, glucosamine, chondroitin) have been developed, but these effects have not been established for cartilage degeneration, promotion of cartilage production, and induction of regeneration. Therefore, prevention is the best way to prevent deterioration with early diagnosis of arthritis.

 Human knee joint cartilage does not regenerate itself once damaged, and the response to damage of articular cartilage is different from other tissues because the articular cartilage lacks blood vessels, nerves and lymphoid tissues. Damaged articular cartilage is difficult to recover because of the inability to transfer inflammatory cells, and regeneration is extremely limited.

Trauma, ligament injuries, meniscus injuries, osteochondritis dissecans, degenerative joint disease, and misalignment of joints malalignment of joint, osteonecrosis, inflammatory arthritis, and infection. Articular cartilage injuries are accompanied by pain and limitations of movement, eventually leading to typical degenerative arthritis. Causes of cartilage tissue degeneration include cartilage tissue degradation by extracellular matrix enzymes, dedifferentiation to read the characteristics as chondrocytes, inflammatory reactions, and cell death. The matrix metalloproteinase (MMP) produced by chondrocytes is MMP-1, -2, -3, -8, -9, -13, aggrecanase, MT1-MMP (MMP- 14), TIMP-1, -2, -3, and degeneration of cartilage tissue is an irreversible process of destruction of cartilage tissue caused by excessive expression (activity) of MMP due to metabolic imbalance and insufficient synthesis (inactivation) of TIMP. . Various MMPs produced from chondrocytes decompose various ECM molecules synthesized from chondrocytes, such as collagen, proteoglycan, and link protein, resulting in destruction of cartilage tissue and cartilage tissue ECM (Extracellular). matrix) changes the molecular composition.

Chondrocytes in cartilage tissues lose their differentiation characteristics by inflammatory cytokines such as IL-1β or NO, that is, dedifferentiation.The same phenomenon is induced during the in vitro passage of chondrocytes to treat chondrocytes. Is a major deterrent to mass production. The dedifferentiation of chondrocytes decreases the synthesis of cartilage specific ECM molecules such as type II collagen and proteoglycans, stops supplementation of new ECM molecules, and destroys homeostasis of cartilage tissues.

Inflammation of the cartilage tissue is accompanied by severe pain, which causes the joints to not move, and at the same time, the joint fluid cannot reach the deep chondrocytes, which eventually leads to cartilage cell death. Inflammatory cytokines such as IL-1β increase the expression of chondrocyte cytokine receptors, thereby amplifying the sensitivity of chondrocytes to cytokines, thereby activating phospholipase A2 and COX-2 expression, thereby producing PGE2. Induces inflammatory reactions, such as synoviitis.

Apoptosis of chondrocytes in cartilage degeneration is also an important factor in inducing cartilage degeneration. Cartilage cell death ultimately reduces ECM synthesis of cartilage tissue, destroying the homeostasis of cartilage ECM, leading to degeneration.

EST (expressed sequence tags) database and new gene identification using it is an effective identification tool for gene identification that induces or inhibits cartilage degeneration by obtaining new gene identification and information based on information from the EST database, which is a fragment of a transcript. to be.

The discovery and functioning of new genes that regulate cartilage degeneration can be used as direct biomarkers to control cartilage degeneration, and the development of new drugs to control these markers has led to the development of gene therapy for cartilage degeneration and cartilage regeneration. In addition to the development, it is expected to contribute greatly to the improvement of public health by overcoming the limitations of the treatment methods for cartilage degeneration up to now and the practical use of the development of new molecular medicine treatment for arthritis.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors have made diligent research efforts to discover novel biomarkers that can rapidly and accurately molecularly diagnose arthritis. As a result, the present inventors have completed the present invention by identifying that the biomarkers that are discovered can diagnose arthritis and determine the prognosis.

Accordingly, it is an object of the present invention to provide a kit for diagnosing or prognosticting arthritis.

Another object of the present invention is to provide a method for detecting arthritis markers in order to provide information necessary for the diagnosis or prognosis of arthritis.

Still another object of the present invention is to provide a method for screening a material for preventing or treating arthritis.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to an aspect of the present invention, the present invention provides a composition for diagnosing or prognostic arthritis comprising a binding agent specific for ECRG 4 protein, a nucleotide sequence encoding ECRG 4 protein, a sequence complementary to the nucleotide sequence, or a fragment of the nucleotide. To provide.

According to another aspect of the present invention, the present invention provides a kit for diagnosing or prognostic arthritis, comprising a binding agent specific for ECRG 4 protein, a nucleotide sequence encoding ECRG 4 protein, a sequence complementary to the nucleotide sequence, or a fragment of the nucleotide. To provide.

According to another aspect of the present invention, the present invention provides a method of detecting arthritis through a method of detecting the expression of the nucleotide sequence of ECRG 4 in a biological sample of human in order to provide information necessary for diagnosing or prognosis of arthritis.

The present inventors made extensive efforts to discover novel molecular markers capable of molecular diagnosis of arthritis quickly and accurately. As a result, the molecular markers described were able to diagnose arthritis early and determine the prognosis. It was. In particular, the marker of the present invention is a marker with greatly improved accuracy and reliability as a marker for arthritis.

ECRG 4 (esophageal cancer related gene 4) is a protein with a molecular weight of 17 kDa consisting of 148 amino acids. The nucleic acid sequence of the ECRG 4 gene of bos taurus is known under accession number NM_001038113.1, and information on proteins is known under accession number NP_001033202.1 respectively. The human ECRG 4 gene is present on chromosome 2 (2q14.1-q14.3) and information about the mRNA sequence is known from accession number AF325503. Information on proteins is known from Accession Number AAG42321.1, respectively. MRNA sequence information for mice is known from Unigene Mm. 50109 and information on glycoproteins is known from accession NP_077245.1.

As used herein, the term “biological sample” refers to any sample obtained from the human body or mammal, such as cells or tissues of cartilage (particularly articular cartilage), urine, saliva, blood, plasma or serum samples, but is not limited thereto. .

According to a preferred embodiment of the invention, the molecular marker ECRG 4 of the invention is included in cartilage, in particular articular cartilage samples.

In the context of the present invention, "arthritis" refers to a disease in which joints are inflamed by various causes.

The molecular marker of the present invention may be an indicator for the development and development of arthritis, and may be used to diagnose the development and development of arthritis.

According to a preferred embodiment of the present invention, the molecular markers of the present invention are trauma, ligament injuries, meniscus injuries, osteochondritis dissecans, degenerative arthritis. used to predict or diagnose arthritis due to joint disease, malalignment of joint, osteonecrosis, inflammatory arthritis or infection, more preferably inflammatory arthritis or degenerative It is used to predict or diagnose arthritis, and most preferably to predict or diagnose degenerative arthritis very accurately.

As used herein, the term “diagnosis” refers to determining the susceptibility of an object to a particular disease or condition, determining whether an object currently has a particular disease or condition, a particular disease or condition Determining the prognosis (eg, identifying arthritis status, determining the stage of arthritis, or determining the responsiveness of arthritis to treatment), or therametrics (eg, information on treatment efficacy). Monitoring the state of an object to provide it).

The term "prognosis" in the context of the present invention encompasses the predictive process of disease progression, in particular, the degree of disease remission, disease regeneration, arthritis recurrence. Preferably, the prognosis in the present invention means the possibility that the disease of the arthritis patient is cured.

According to a preferred embodiment of the present invention, the present invention can be carried out by immunoassay (ie, antigen-antibody reaction). In this case, the antibody or aptamer specifically binds to the arthritis marker of the present invention described above.

The antibody used in the present invention is a polyclonal or monoclonal antibody, preferably a monoclonal antibody. Antibodies may be commonly used in the art, such as fusion methods (Kohler and Milstein, European Journal of Immunology, 6: 511-519 (1976)), recombinant DNA methods (US Pat. No. 4,816,56) Or phage antibody library methods (Clackson et al, Nature , 352: 624-628 (1991) and Marks et al, J. Mol. Biol. , 222: 58, 1-597 (1991)). General procedures for antibody preparation are described in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley / Greene, NY, 1991, which are incorporated herein by reference. For example, the preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing an immortalized cell line with an antibody-producing lymphocyte, and the techniques necessary for this process are well known and readily practicable by those skilled in the art. Polyclonal antibodies can be obtained by injecting a protein antigen into a suitable animal, collecting the antiserum from the animal, and then separating the antibody from the antiserum using a known affinity technique.

When the method of the present invention is carried out using antibodies or aptamers, the present invention can be used to diagnose arthritis according to conventional immunoassay methods.

Such immunoassays can be performed according to various quantitative or qualitative immunoassay protocols developed in the prior art. The immunoassay format may include radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, enzyme-linked immunosorbant assay (ELISA), capture-ELISA, inhibition or hardwood analysis, sandwich analysis, flow cytometry, immunoassay. Including but not limited to fluorescent staining and immunoaffinity purification. The immunoassay or method of immunostaining is described in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J.M. ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, which is incorporated herein by reference.

For example, if the method of the present invention is carried out according to the method radioactive immunoassay, radioactive isotope is an antibody labeled (e.g., C _14, I _125, P ₃₂ and S ₃₅₎ of detecting the marker molecules of the present invention .

When the method of the present invention is carried out in an ELISA method, certain embodiments of the present invention comprise the steps of: (i) coating an unknown cell sample lysate to be analyzed on the surface of a solid substrate; (Ii) reacting said cell lysate with an antibody against a marker as a primary antibody; (Iii) reacting the result of step (ii) with an enzyme-conjugated secondary antibody; And (iii) measuring the activity of the enzyme.

Suitable as the solid substrate are hydrocarbon polymers (eg polystyrene and polypropylene), glass, metal or gel, most preferably microtiter plates.

The enzyme bound to the secondary antibody may include an enzyme catalyzing a chromogenic reaction, a fluorescence reaction, a luminescent reaction, or an infrared reaction, but is not limited thereto. For example, an alkaline phosphatase,? -Galactosidase, Radish peroxidase, luciferase, and cytochrome P450. When alkaline phosphatase is used as an enzyme binding to the secondary antibody, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate (naphthol-AS) Chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis) when chromogenic reaction substrates such as -B1-phosphate) and enhanced chemifluorescence (ECF) are used, and horse radish peroxidase is used -N-methylacridinium nitrate), lesoruppin benzyl ether, luminol, Amflex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), p-phenylenediamine-HCl and pyrocatechol (HYR), TMB (tetramethylbenzidine), ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol / pyronine, glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS a substrate like phenzaine methosulfate It can be.

When the method of the invention is carried out in a capture-ELISA mode, certain embodiments of the invention comprise (i) coating an antibody against a marker of the invention as a capturing antibody on the surface of a solid substrate; (Ii) reacting the capture antibody with the sample; (Iii) reacting the product of step (ii) with a detecting antibody having a label that generates a signal and which specifically reacts with ECRG 4 protein; And (iv) measuring a signal originating from said label.

The detection antibody carries a label which generates a detectable signal. The label may include chemicals (eg biotin), enzymes (alkaline phosphatase, β-galactosidase, horse radish peroxidase and cytochrome P450), radioactive substances (eg C14, I125, P32 and S35) , Fluorescent materials (eg, fluorescein), luminescent materials, chemiluminescent, and fluorescence resonance energy transfer (FRET). Various labels and labeling methods are described in Ed. Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999.

Measurement of the final enzyme activity or signal in the ELISA method and the capture-ELISA method can be carried out according to various methods known in the art. Detection of these signals allows for qualitative or quantitative analysis of the markers of the invention. If biotin is used as a label, the signal can be easily detected with streptavidin and luciferin if luciferase is used.

According to another variant of the present invention, an aptamer that specifically binds to the marker of the present invention may be used instead of the antibody. Aptamers are oligonucleic acid or peptide molecules, the general contents of which are described in Bock LC et al., Nature 355 (6360): 5646 (1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78 (8): 42630 (2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA . 95 (24): 142727 (1998).

Arthritis can be diagnosed by analyzing the final signal intensity by the above-described immunoassay process. That is, if the protein of the marker of the present invention is low in the biological sample and the signal is weaker than the normal biological sample (eg, articular cartilage cells or tissues, blood, plasma or serum), arthritis is diagnosed.

The composition of the present invention may further include other components in addition to the above components. For example, when the composition of the invention is subjected to a PCR amplification process, the composition of the invention may optionally contain reagents necessary for PCR amplification, such as buffers, DNA polymerases (eg, Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis or thermally stable DNA polymerase obtained from Pyrococcus furiosus (Pfu)), DNA polymerase cofactors and dNTPs. When preparing a kit containing the composition of the present invention can be prepared in a number of separate packaging or compartments containing the above reagent components.

According to a preferred embodiment of the present invention, the composition of the present invention may be a composition for a microarray.

According to a preferred embodiment of the present invention, the composition of the present invention is a composition for gene amplification.

When the composition of this invention is a composition for microarrays, the probe is immobilized on the solid surface of a microarray. When the composition of the present invention is a composition for gene amplification, it comprises a primer.

The probe or primer used in the diagnostic composition of the present invention has a sequence complementary to the ECRG 4 nucleotide sequence. As used herein, the term “complementary” means having complementarity enough to selectively hybridize to the above-described nucleotide sequence under certain specific hybridization or annealing conditions. Thus, the term “complementary” has a different meaning from the term perfectly complementary, and the primers or probes of the present invention may be capable of selectively hybridizing to the above-described nucleotide sequence so long as one or more mismatches ( mismatch) may have a nucleotide sequence.

As used herein, the term “primer” refers to a single that can serve as an initiation point for template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerases) at suitable temperatures. -Refers to stranded oligonucleotides. The suitable length of the primer is typically 15-30 nucleotides, although it varies with various factors such as temperature and use of the primer. Short primer molecules generally require lower temperatures to form hybrid complexes that are sufficiently stable with the template.

The sequence of the primer does not need to have a sequence completely complementary to a partial sequence of the template, and it is sufficient if the primer has sufficient complementarity within a range capable of hybridizing with the template and acting as a primer. Therefore, the primer in the present invention does not need to have a sequence that is perfectly complementary to the above-described nucleotide sequence as a template, and it is sufficient to have sufficient complementarity within a range capable of hybridizing to the gene sequence and acting as a primer. The design of such primers can be easily carried out by those skilled in the art with reference to the above-described nucleotide sequence, for example, by using a program for primer design (eg, PRIMER 3 program).

As used herein, the term “probe” refers to a linear oligomer of natural or modified monomers or linkages, includes deoxyribonucleotides and ribonucleotides, and can specifically hybridize to a target nucleotide sequence, naturally Present or artificially synthesized. The probe of the present invention is preferably a single strand, and is an oligodioxyribonucleotide.

The nucleotide sequence of the marker of the present invention, which should be referred to when constructing the primer or probe, can be identified in GenBank using the access number of ECRG 4 described above, and the primer or probe can be designed with reference to this sequence.

In the microarray containing the composition of the present invention, the probe is used as a hybridizable array element and is immobilized on a substrate. Preferred gases include, for example, membranes, filters, chips, slides, wafers, fibers, magnetic beads or non-magnetic beads, gels, tubing, plates, polymers, microparticles and capillaries, as suitable rigid or semi-rigid supports. The hybridization array elements are arranged and immobilized on the substrate. This immobilization is carried out by chemical bonding methods or by covalent binding methods such as UV. For example, the hybridization array element can be bonded to a glass surface modified to include an epoxy compound or an aldehyde group, and can also be bonded by UV at the polylysine coating surface. In addition, the hybridization array element may be coupled to the gas through a linker (e.g., ethylene glycol oligomer and diamine).

Meanwhile, the sample DNA applied to the microarray containing the composition of the present invention may be labeled and hybridized with the array elements on the microarray. Hybridization conditions can vary. The detection and analysis of the hybridization degree can be variously carried out according to the labeling substance.

Kits containing the composition for diagnosing arthritis of the present invention can be carried out based on hybridization. In this case, a probe having a sequence complementary to the nucleotide sequence of the marker of the present invention described above is used.

Arthritis may be determined by hybridization-based analysis using a probe hybridized to the nucleotide sequence of the marker of the present invention described above.

The label of the probe can provide a signal that allows detection of hybridization, which can be linked to oligonucleotides. Suitable labels include fluorophores (eg fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5 (Pharmacia), chromophores, chemilumines, magnetic particles, radioisotopes Elements (P32 and S35), mass labels, electron dense particles, enzymes (alkaline phosphatase or horseradish peroxidase), cofactors, substrates for enzymes, heavy metals (eg gold) and antibodies, streptavidin, biotin And hapten with specific binding partners such as digoxigenin and chelating groups. Labeling is carried out in a variety of ways conventionally practiced in the art, such as nick translation methods, random priming methods (Multiprime DNA labeling systems booklet, "Amersham" (1989)) and chination methods (Maxam & Gilbert, Methods). in Enzymology , 65: 499 (1986)). The label provides signals that can be detected by fluorescence, radioactivity, colorimetry, weighing, X-ray diffraction or absorption, magnetism, enzymatic activity, mass analysis, binding affinity, hybridization high frequency, and nanocrystals.

Nucleic acid samples to be analyzed can be prepared using mRNA obtained from various biological samples (biosamples), preferably can be prepared using mRNA obtained from articular cartilage tissue cells. The hybridization reaction-based assay may be performed by labeling the cDNA to be analyzed instead of the probe.

If a probe is used, the probe is hybridized with the cDNA molecule. In the present invention, suitable hybridization conditions can be determined in a series of procedures by an optimization procedure. This procedure is carried out by a person skilled in the art in order to establish a protocol for use in the laboratory. For example, conditions such as temperature, concentration of components, hybridization and wash times, buffer components and their pH and ionic strength depend on various factors such as probe length and GC amount and target nucleotide sequence. Detailed conditions for hybridization are described by Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); And MLM Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc .; NY (1999). For example, high stringency conditions were hybridized at 65 ° C in 0.5 M NaHPO ₄ , 7% SDS (sodium dodecyl sulfate) and 1 mM EDTA, followed by addition of 0.1 x SSC / 0.1% SDS Lt; RTI ID = 0.0 > 68 C < / RTI > Alternatively, high stringency conditions means washing at < RTI ID = 0.0 > 48 C < / RTI > in 6 x SSC / 0.05% sodium pyrophosphate. Low stringency conditions mean, for example, washing in 0.2 x SSC / 0.1% SDS at 42 ° C.

After the hybridization reaction, the hybridization signal coming out of the hybridization reaction is detected. The hybridization signal can be performed by various methods, for example, depending on the type of label bound to the probe. For example, if the probe is labeled by an enzyme, the substrate of the enzyme can be reacted with the hybridization product to confirm hybridization. Combinations of enzymes / substrates that can be used include peroxidase (eg horseradish peroxidase) and chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium Nitrate), resoruppin benzyl ether, luminol, amplex red reagent (10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2 , 2'-Azine-di [3-ethyl benzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol / pyronine; Alkaline phosphatase with bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate and ECF substrates; Glucose oxidase, t-NBT (nitroblue tetrazolium) and m-PMS (phenzaine methosulfate). When the probe is labeled with gold particles, it can be detected by silver dyeing using silver nitrate. Therefore, when the method for detecting the arthritis marker of the present invention is performed based on hybridization, specifically (i) hybridizing a probe having a sequence complementary to the nucleotide sequence of the marker of the present invention to a nucleic acid sample; (ii) detecting whether the hybridization reaction occurs. By analyzing the strength of the hybridization signal by the hybridization process, it is possible to determine whether arthritis. That is, if the hybridization signal for the nucleotide sequence of the marker of the present invention in the sample is weaker than the normal sample (eg, tissue or cells of articular cartilage), it is diagnosed as arthritis.

According to a preferred embodiment of the present invention, the human arthritis diagnostic kit of the present invention may be a gene amplification kit.

As used herein, the term "amplification" refers to a reaction that amplifies a nucleic acid molecule. Various amplification reactions have been reported in the art, which include polymerase chain reaction (PCR) (US Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), reverse transcriptase-polymerase chain reaction (RT-PCR) (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed.Cold Spring Harbor Press (2001)), Miller, HI (WO 89/06700) and Davey, C. et al. (EP 329,822), ligase chain reaction (LCR) ( 17, 18), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA, WO 88/10315), self-maintaining sequence replication (self sustained sequence replication, WO 90/06995), selective amplification of target polynucleotide sequences (US Pat. No. 6,410,276), consensus sequence primed polymerase chain reaction ( CP-PCR), US Pat. No. 4,437,975), optional print Arbitrarily primed polymerase chain reaction (AP-PCR), US Pat. Nos. 5,413,909 and 5,861,245, Nucleic acid sequence based amplification (NASBA), US Pat. No. 5,130,238, 5,409,818, 5,554,517, and 6,063,603), strand displacement amplification (21, 22) and loop-mediated isothermal amplification; LAMP) 23, but is not limited thereto. Other amplification methods that may be used are described in U.S. Patent Nos. 5,242,794, 5,494,810, 4,988,617 and U.S. Patent No. 09 / 854,317.

PCR is the most well-known nucleic acid amplification method, and many variations and applications thereof have been developed. For example, touchdown PCR, hot start PCR, nested PCR and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR. In addition, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), multiplex PCR, inverse polymerase chain reaction (inverse polymerase) chain reaction (IPCR), vectorette PCR and thermal asymmetric interlaced PCR (TAIL-PCR) have been developed for specific applications. For more information on PCR see McPherson, M.J., and Moller, S.G. PCR. BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, N.Y. (2000), the teachings of which are incorporated herein by reference.

When the diagnostic kit of the present invention is carried out using a primer, a gene amplification reaction is performed to examine the expression level of the nucleotide sequence of the marker of the present invention. Since the present invention analyzes the expression level of the nucleotide sequence of the marker of the present invention, the mRNA of the nucleotide sequence of the marker of the present invention in a sample (eg, articular cartilage tissue or cell, blood, plasma, serum or urine) to be analyzed. The amount is investigated to determine the expression level of the nucleotide sequence of the marker of the present invention.

Therefore, in principle, the present invention uses a mRNA in a sample as a template and performs a gene amplification reaction using a primer that binds to mRNA or cDNA.

To obtain mRNA, total RNA is isolated from the sample. Isolation of total RNA can be carried out according to conventional methods known in the art. See Sambrook, J. et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001); Tesniere. , C. et al., Plant Mol. Biol. Rep. , 9: 242 (1991); Ausubel, FM et al., Current Protocols in Molecular Biology , John Willey & Sons (1987); and Chomczynski, P. et al , Anal.Biochem. 162: 156 (1987)). For example, TRIzol can be used to easily isolate total RNA in a cell. Next, cDNA is synthesized from the separated mRNA, and this cDNA is amplified. Since the total RNA of the present invention is isolated from human samples, the end of the mRNA has a poly-A tail, and cDNA can be easily synthesized using oligo dT primers and reverse transcriptases using these sequence characteristics. PNAS USA, 85: 8998 (1988); Libert F, et al., Science , 244: 569 (1989); and Sambrook, J. et al., Molecular Cloning.A Laboratory Manual, 3rd ed.Cold Spring Harbor Press (2001). Next, the synthesized cDNA is amplified through gene amplification reaction.

The primer used in the present invention is hybridized or annealed at one site of the template to form a double-stranded structure. Suitable nucleic acid hybridization conditions for forming such a double-chain structure include Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization , A Practical Approach, IRL Press, Washington, DC (1985).

Various DNA polymerases can be used for amplification of the present invention and include “Clenow” fragments of E. coli DNA polymerase I, thermostable DNA polymerase and bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtained from various bacterial species, which include Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu). Include.

When performing the polymerization reaction, it is preferable to provide the reaction vessel with an excessive amount of the components necessary for the reaction. The excess amount of the components required for the amplification reaction means an amount such that the amplification reaction is not substantially restricted to the concentration of the component. It is desired to provide cofactors such as Mg ²⁺ , dATP, dCTP, dGTP and dTTP to the reaction mixture such that the desired degree of amplification can be achieved. All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, the buffer ensures that all enzymes are close to optimal reaction conditions. Thus, the amplification process of the present invention can be carried out in a single reactant without changing conditions such as addition of reactants.

Annealing in the present invention is carried out under stringent conditions allowing specific binding between the target nucleotide sequence and the primer. Stringent conditions for annealing are sequence-dependent and vary depending on the surrounding environmental variables.

The cDNA of the nucleotide sequence of the marker of the present invention thus amplified is analyzed by a suitable method to investigate the expression level of the nucleotide sequence of the marker of the present invention. For example, the degree of expression of the nucleotide sequence of the marker of the present invention is examined by gel electrophoresis of the amplification reaction product described above, and by observing and analyzing the resulting band. Through this amplification reaction, arthritis is diagnosed when the expression of the nucleotide sequence of the marker of the present invention in a biological sample is lower than that of a normal sample (eg, normal articular cartilage cells or tissues, blood, plasma or serum).

Therefore, when performing the detection method of the arthritis marker of the present invention based on the amplification reaction using cDNA, specifically (i) performing an amplification reaction using a primer annealed to the nucleotide sequence of the marker of the present invention; And (ii) analyzing the product of the amplification reaction to determine the expression level of the nucleotide sequence of the marker of the present invention.

The marker of the present invention is a biomolecule that is low in arthritis. Low expression of such markers can be measured at the mRNA or protein level. The term "low expression" as used herein refers to a case where the expression level of the nucleotide sequence of interest in the sample to be investigated is low compared to the normal sample. For example, expression assay methods commonly used in the art, such as RT-PCR methods or ELISA methods (Sambrook, J. et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)) In the case of expression analysis according to, it means a case where the expression is analyzed to be low. For example, as a result of analysis according to the above-described analytical method, when the marker of the present invention is 2-10 times lower than normal cells or tissues, it is determined as “low expression” in the present invention and is determined as arthritis. do.

According to another aspect of the present invention, the present invention provides a method for screening a material for preventing or treating arthritis, comprising the following steps:

(a) contacting a sample to be analyzed with a cell comprising the nucleotide sequence of ECRG 4; And

(b) measuring the expression level of the nucleotide sequence, and when the low expression of the nucleotide sequence is suppressed, the sample is determined as a substance for preventing or treating arthritis.

According to the method of the present invention, first, a sample to be analyzed is contacted with a cell containing the nucleotide sequence of the marker of the present invention. Preferably, the cell comprising the nucleotide sequence of the marker of the present invention is a human articular cartilage cell. As used to refer to the screening method of the present invention, the term "sample" refers to an unknown substance used in screening to examine whether it affects the expression level of the marker of the present invention. The sample includes, but is not limited to, chemicals, nucleotides, antisense-RNAs, small interference RNAs (siRNAs), and natural extracts.

Next, the expression level of the marker of the present invention is measured in the cells treated with the sample. The amount of expression can be measured as described above, and as a result of the measurement, when the low expression of the nucleotide sequence of the marker of the present invention is suppressed, the sample can be determined as a substance for preventing or treating arthritis.

The features and advantages of the present invention are summarized as follows:

(Iii) The present invention provides a composition for diagnosing or prognostic arthritis and a kit comprising the same.

(Ii) ECRG 4 in the present invention is a marker with greatly improved accuracy and reliability as a marker for arthritis.

(Iii) In addition, the present invention uses ECRG 4, which specifically reduces expression in tissues or cells of arthritis patients, to determine early diagnosis and prognosis of arthritis very specifically from biological samples (eg, chondrocytes or tissues). can do.

Figure 1a is a result of comparing the mRNA and the expression of ECRG4 in the cartilage tissue through the reverse transcriptase chain polymerization and real-time reverse transcriptase chain polymerization reaction in each tissue compared with other tissues. Figure 1b is the result confirmed by Northern blotting to confirm the mRNA amount of ECRG4. Figure 1c is the result of overexpressing in chondrocytes using the myc reporter gene to confirm the expression of the expressed protein of ECRG4 by Western blotting in the cytoplasm and cell culture, and confirmed that ECRG4 is a protein secreted out of the cell. Cartilage: Cartilage, Brain: Brain, Heart: Heart, Liver: Liver, Lung: Lung, Kidney: Kidney, Muscle Muscle, Skin: Skin, cellular ECRG4: Intracellular ECRG4, secreted ECRG4: Secreted ECRG4.
Figure 2a confirms the expression of ECRG4 during the process of differentiation into chondrocytes from undifferentiated stem cells. When micronized mesenchymal stem cells of Mautm were isolated and microcultured to induce differentiation into chondrocytes, ECRG4 showed expression patterns such as type II collagen and aggrecan indicating the degree of differentiation of chondrocytes. Figure 2b is the result of quantifying the same result through real-time reverse transcriptase chain polymerization. Figure 2c is the result of differentiating undifferentiated cells into chondrocytes by the microculture method and then cutting the ECRG4 transcript by in situ hybridization method. chondrogenesis: chondrocyte differentiation, hypertrophic maturation: chondrocyte enlargement, Coll: collagen, Aggrecan: aggrecan, MMP (matrix metallopreoteinase) extracellular matrixase, GAPHD (Glyceraldehye-3-phosphate dehydrogenase) 3-phosphate glyceraldehyde dehydrogenase Enzyme, MMP-13: extracellular matrix enzyme-3.
3 shows the results of confirming the expression of ECRG4 from the limbs of mouse embryos. Figure 3a and 3b shows the results of the ECRG4 expression through reverse transcriptase chain polymerization and real-time reverse transcriptase chain polymerization reaction by extracting mRNA from mouse embryos from E11.5 to E15.5 before chondrocyte differentiation occurs. It was expressed in the same pattern as the expression of type II collagen, a marker molecule of one chondrocyte. Figure 3c is a result of detecting the ECRG4 by in situ hybridization method by cutting the E14.5 rim to confirm the rim tissue, it was confirmed that the expression of ECRG4 in the type II collagen expression. Figure 3d is a 200-fold, 400-fold magnification observed to confirm that the expression of a lot of ECRG4 in the chondrocytes expressing type II collagen. PC (proliferatin chondrocyte) dividing chondrocytes, hypertrophic chondrocyte (HC) hyperchondral cells.
4 is an experimental result confirming the expression pattern of ECRG4 in the degenerative conditions of chondrocytes. Figure 4a is a result of treating the inflammatory cytokine IL-1β that induces the differentiation of chondrocytes, Figure 4b is a result of the reduction of type II collagen, a differentiation marker of chondrocytes when passaged. At this time, the expression of ECRG4 was also decreased by reverse transcriptase chain polymerization. 4c is an experimental result confirming that ECGR4 can regulate the expression of chondrocyte differentiation markers or degeneration-related extracellular matrixases. These results show that ECRG4 is overexpressed with lentiviral to normal chondrocytes or that ECRG4 is overexpressed with lentiviral under chondrocyte degeneration conditions. Figure 4d shows the decreased results when chondrocyte division overexpressed ECRG4.
5 shows normal cartilage tissue without arthritis and cartilage tissue with high arthritis in arthritis patients cartilage tissue, and reverse transcriptase chain polymerization reaction and real-time reverse transcriptase chain polymerization reaction to confirm the expression level of ECRG4 in the tissue. Is the result of The white graph shows normal cartilage tissue and the black graph shows arthritis cartilage tissue. Figure 5a is a result showing that the expression of CERG4 decreased with type II collagen in the area where cartilage degeneration progressed. Figure 5b is a result of inducing degenerative arthritis with collagenase (collagense) in mice and cutting the degeneration of cartilage tissue by saffron O staining, reverse transcriptase chain after separation of mRNA from the cartilage tissue in which cartilage degeneration occurs The expression of CERG4 was decreased by polymerization and real-time reverse transcriptase chain polymerization. N (normal) normal, osteoarthritis degenerative arthritis (OA), [FIG. 5b] phosphate buffered saline (PBS).

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Unless stated otherwise, solids / solids are (weight / weight) parts or%, solids / liquids are (weight / volume) parts or%, and liquids / liquids are (volume / volume) parts or%.

Materials and methods

In silico analysis

CDNA sequences for novel genes were retrieved from the Unigene transcript library (www.ncbi.nlm.nih.gov/Unigene). Goblet (www.goblet.molgen.mpg.de) and AmiGO (www.godatabase.org/cgi-bin/go.cgi) programs were used to predict gene ontology, and SMART was used to predict various protein domain regions. The program (www.smart.embl-heidelberg.de) was used. SIgnalP (www.cbs.dtu.dk/services/SignalP) was applied to confirm the presence of the signal peptide at the N-terminal site.

Secure tissue from humans and mice

Limbuds were isolated from the limb buds of the front and hind limbs of the mouse embryos at the indicated developmental stages, respectively. RNA was extracted from the isolated lymbud and reverse transcriptase chain polymerization was used to confirm the expression of type IIb collagen and ECRG4. To perform in situ hybridization for the identification of type IIb collagen and ECRG4 transcripts, tissue isolated from the forelimb limbs of mouse embryos 14.5 days after fertilization were used (Kim et al. 2008). Cartilage and all other tissues were obtained from newborn mice, and expression of type IIb collagen and ECRG4 was confirmed by reverse transcriptase chain polymerization. Human degenerative arthritis cartilage tissue was obtained from patients (51-72 years) who underwent arthroplasty for the degenerative arthritis knee joint. The Human Subjects Committees have been approved for the transfer of human tissue. All patient cases met the American College of Rheumatology classification criteria for knee degenerative arthritis (Altman et al. 1986). Within 60 minutes after the operation, cartilage tissue was collected from the tibial plateau of each sample to the subchondral bone area. Intact cartilage tissue was used as a control and severely damaged cartilage tissue was used as degenerative arthritis cartilage tissue. RNA was extracted from normal, degenerative arthritis cartilage tissue, and the expression of type IIb collagen and ECRG4 was confirmed by reverse transcriptase chain polymerization.

Production and Embryo Analysis of ECRG4 Recombinant Mice-No Relevant Contents in Drawings

For the production of chondrocyte-specific ECRG4 recombinant mice, Ueta et al. As described in (2001), the ECRG4 gene was inserted into the NotI site of the expression vector containing the promoter and enhancer of the mouse Col2a1 gene. ECRG4 recombinant mice were purchased from Macrogen (Seoul, South Korea). Hybridization of founder recombinant mice and wild type C57BL / 6 mice was performed to produce F1 generation hybrids. Primers of 5'-TGGGTCCAGATGGCATAAGTGG-3 'and 5'-ATAGTTGACACTGGCCTCCATGCC-3' were used for genotyping of 3-4 week-old F1-F4 generations. To characterize ECRG4 in developmental stages, 14.5 and 16.5 days of embryonic skin and intestine were removed before staining, fixed for 4 days with 95% ethanol and then soaked in acetone for 3 days, followed by Alcian blue. Skeleton was stained for 10 days with) / Alizarin red staining reagent (0.015% Alcyan blue in 70% ethanol, 0.05% Alizarin red in 95% ethanol, 5% acetic acid in 70% ethanol). After staining the embryos were washed for 2 days with 1% KOH and stored in 100% glycerol.

Collagen Degrading Enzyme-induced Cartilage Damage

Experimental induction of knee degenerative arthritis by injecting 4 units of collagen degrading enzyme (Sigma, st.Louis, MO) into the joints of 10-week-old C57BL / 6 male mice (multiple sciences) on day 0 and day 2, respectively It was. Four weeks later, the knee joints were separated and subjected to histological evaluation as described below.

After fixation of 5% paraformaldehyde knee joint, decalcification solution (Merk, Darmstadt, Germany) was treated for 14 days. Paraffin-penetrated tissues were cut into slices of 8 μm thickness and the slices were fixed on Superfrost slides (Menzel-Glaser, Braunschweig, Germany). Moisture and paraffin were removed from the flakes and stained with hematoxylin for 10 minutes. After washing for 10 minutes in running water, and then dyed for 3 minutes with a Fast Green solution, and then quickly washed with a 1% acetic acid solution in a range not exceeding 10 seconds. Finally the samples on the slides were stained with 0.1% Safranin-O solution for 5 minutes and then washed in running water for 3 minutes. The degree of cartilage damage is reported by Mankin et al. It was measured according to the method of (1971). For biochemical analysis, Blom et al. Normal and degenerative arthritis cartilage tissues were collected by the method described in (2007), and the expression levels of various molecules were measured.

Cell culture

As described in Kim et al. (2008), undifferentiated mesenchymal cells derived from the end of the rim bud of mouse embryo 11.5 days after fertilization to induce chondrocyte differentiation using micromass culture method Incubated together.

Suspension of 4.0 x 10 ⁷ / ml cells in DMEM (Dulbecco's modified Eagle's medium; Gibco-BRL, Gaithersburg, MD) containing 10% (v / v) Fetal bovine serum It was incubated in the culture dish in the form. Chondrocyte differentiation was confirmed by reverse transcriptase chain polymerization and Alcian blue staining for the expression of type II collagen and the accumulation of sulfided glycosaminoglycans, respectively. On day 6 of micromass culture, DMEM was exchanged with DMEM / F-12 (2: 3) medium containing 50 μg / ml of ascorbic acid and 5 mM of β-glycerol phosphate. Hypertrophic maturation of chondrocytes was induced by culturing up to 1 day (Mello and Tuan, 1999; Kim et al. 2008). As described in Kim et al. (2008), hypertrophic maturation of chondrocytes was confirmed by the expression of type X collagen and MMP-13 through reverse transcriptase chain polymerization, or the degree of mineralization of chondrocytes through alizarin red staining. It was confirmed by measuring. On day 5 of culture, the micromass culture drops were removed, fixed in paraffin wax, and subjected to in situ hybridization to type II collagen and ECRG4. According to the method described by Yoon et al. (2002), cartilage sections of two-week-old New Zealand white rabbits (Fine test animals) were digested with enzymes to extract cartilage cells of rabbit joints. Cartilage sections were digested for 4 hours with 0.2% type II collagen degrading enzyme (381 units / mg; Sigma) contained in DMEM. As described in Kim et al. (2008), rib chondrocytes were harvested from 3 days old mice. Cartilaginous rib cages were digested for 45 minutes with 0.2% collagenase type II. Cells isolated from joints and ribs were resuspended in DMEM containing 10% (v / v) fetal bovine serum and 50 μg / ml streptomycin and 50 units / ml penicillin. Incubations were made at a concentration of 5 × 10 ⁴ cells / cm ² . The medium was changed every 1.5 days and the concentration of cells peaked between 4-5 days. This was expressed as passage 0 (P0) and passaged at a concentration of 5 × 10 ⁴ cells / cm ² up to P3 (Yoon et al. 2002). Chondrocyte differentiation was measured through the expression of type II collagen. Proliferation of primary cultured rabbit chondrocytes was measured using Ez-Cytox enhanced cell viability assay kit (Itsbio, Seoul, Korea).

Structure of ECRG4 Expression Vectors and Lentiviruses

Mouse ECRG4 (Mm.50109) cDNA was obtained by performing reverse transcriptase chain polymerization using ECRG4 primers designed to add Bam HI and Xba I recognition sequences to 5 'and 3' ends, respectively. The obtained cDNA was inserted into the pcDNA3.1 / myc-His vector (Invitrogen, Carlsbad, CA). pCDNA-ECRG4-myc / his vector was introduced into primary chondrocytes using Metafectene (Biotex, Martinsried, Germany) according to the manufacturer's protocol. Introduced chondrocytes were incubated for 36 hours and used in subsequent experiments. Lentiviruses expressing ECRG4 were synthesized by the method described below (Macrogen Lentivector Laboratories, Korea).

The pCDNA-ECRG4-myc / his vector was cut with Bam HI and Xba I restriction enzymes and inserted into the lentiviral vector (Lenti-mCMV-IRES-puro), which was then used with Lipofectamine Plus (Invitrogen). Was introduced into 293T cells. Cultures containing viral particles were harvested 48 hours after introduction. Virus titers were measured using type I human immunodeficiency virus p24 enzyme immunoassay (Enzyme-linked Immunosorbent Assay, ELISA). Mock lentivirus and ECRG4 lentivirus were introduced into undifferentiated mesenchymal cells and rabbit primary chondrocytes and cultured for 48 hours.

Securing ECRG4-Conditioned media

PCDNA-ECRG4 vector was introduced into rabbit primary chondrocytes, and when control and ECRG4 expressing chondrocytes reached 80% confluency, each was washed and incubated in serum-free DMEM (Serum-free DMEM) for 48 hours. After filtration of the conditioned media through a 0.2 μm filter, ultrafiltration using YM membrane and Amicon stirred cells (Millipoer, Billerica, MA) to filter proteins of 5 kDa molecular weight ( Concentrated 30 times using Ultrafiltration). After the mesenchymal stem cells were cultured through a micromass culture method, the cultured mesenchymal stem cells were treated with a control medium and a culture solution containing ECRG4, respectively, and cultured for 5 to 13 days.

Analysis of reverse transcriptase chain polymerization group and real-time reverse transcriptase chain polymerization reaction

RNA was extracted using TRI-reagent (MRC, Inc., Cincinati, OH) according to the manufacturer's protocol and subjected to reverse transcription using Improm-II ^TM reverse transcriptase (Promega) to obtain cDNA. cDNA was amplified by polymerase chain reaction using Taq polymerase (Intron, Gyeonggido, Korea). The primers used are summarized in Table 1. Real time reverse transcriptase chain polymerization was performed using a chromo 4 cycler (Bio-Rad, Hercules, CA) and SYBR Premix Ex Taq ^™ (TaKaRa bio, Inc., Shiga, Japan). All real-time reverse transcriptase chain polymerization reactions were performed twice and the amplified signal of the target gene was corrected by the signal of glyceraldehyde-3-phosphate dehydrogrenase (GAPDH) in the same reaction.

Table 1

Northern blot and western blot

Northern and western blotting methods were performed in the same manner as in the previous paper (Huh et al., 2007; Kim et al., 2008). RNA was sorted on formaldehyde / agarose gels and transferred to S and S Nytran N nylon membranes. Prehybridization and hybridization were carried out, and the ECRG4 transcript was probed with a partial cDNA prepared by reverse transcriptase chain polymerization. Random probes showing high specific activity were prepared using the T7 Quick Primer Kit (Amersham Bioscience, Inc., Piscataway). The filter was washed three times with 0.2 X SSC / 0.1% SDS and developed by increasing and decreasing the temperature at -70 ° C with Kodac X OMAT film. For Western blot analysis, cell lysates were sorted according to protein size by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel and transferred to nitrocellulose membrane. . ECGR4 protein was detected using an anti-myc antibody of Cell signaling Technology (Beberly, MA). Protein signals (Blots) were developed using a peroxidase-conjugated secondary antibody (SIGMA) and a chemilumines-cence system.

In situ hybridization

To identify type IIb collagen and ECGR4, a method described below was performed using a Digoxigenin-labeled ribonucleic acid probe (Riboprobes) using a Digoxigenin RNA labeling mix (Roche Diagonestics Corp., Indianapolice, IN). It was synthesized using (Ryu and Chun, 2006).

Collagen type II and ECGR4 cDNA fragments of mice were amplified by polymerase chain reaction using primers containing Hind III (sence) and Eco RI (antisence) recognition sequences at the 5 'end. Type II collagen and ECGR4 cDNA were inserted into the pSPT-18 vector and linearized and transcribed using either SP6 or T7 RNA polymerase to form antisence probes and sense probes. In order to hybridize, the lymph buds and micromass culture spots of the forelimbs of the mouse embryo, which were 14.5 days after fertilization, were fixed in 4% paraformaldehyde at 4 ° C. for 18 hours, dehydrated with ethanol, and then infiltrated with paraffin to 4 μm. The flakes were cut to thickness. 10 minutes of 0.2 N HCl was used to remove paraffin from paraffin infiltrated flakes, and then 20 μg proteinase K was treated at 37 ° C. for 10 minutes to impart permeability. Hybridization buffer (40% formaldehyde) containing riboprobe labeled with denatured sense or antisense digoxigenin after acetylation with 0.25% acetic anhydride for 10 minutes , 10% dextran sulfate, 1 X Denhardt's solution, 4 X SSC, 10 μM DTT, 1 mgg / ml yeast tRNA, 2 mg / ml salmon sperm DNA). The flakes were treated with 10 mg / ml RNase A at 37 ° C. for 30 minutes and then detected using an anti-digoxigenin detection kit (Roche Diagonetics Corp.). Hybridization with 4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-iodolyl-phosphate The signal was visualized.

Experiment result

ECRG4 Expression in Chondrocytes

To identify genes that are highly expressed in cartilage tissue, we analyzed human normal cartilage library, Lib.8940, using the Unigene database provided by NCBI. Hs.43125 was isolated into cartilage specific and unknown genes through SAGE (serial analysis of gene expression) / cDNA virtual Northern. The overall gene sequence of the human gene Hs.43125 was consistent with ECRG4 (Mm. 50109) in mice. Initial in silico approach confirmed that ECRG4 was cartilage specific in various tissues of mice. Reverse transcriptase chain polymerization (RT-PCR) and real-time reverse transcriptase chain polymerization (qRT-PCR) confirmed that ECRG4 was expressed more in cartilage than in other tissues (FIG. 1A). Transcripts of ECRG4 were identified by Northern blot in mouse rib chondrocytes (FIG. 1B). The gene sequence of ECRG4 consists of four exons, three introns, and contains a signal peptide at the N-terminus. It was found that 148 amino acids constitute an open reading frame (ORF). An artificial overexpression of ECRG4 in chondrocytes was detected at a size of approximately 17 kDa, and a smaller 14 kDa ECRG4 was detected when chondrocytes were detected in cultured media (FIG. 1C). These results indicate that there is 'post-translation break' after extracellular secretion.

ECRG4 expression in dividing chondrocytes or hypertrophic chondrocytes

The microculture method of differentiating undifferentiated mesenchymal stem cells into chondrocytes further confirmed the expression of chondrocyte-specific ECRG4 during chondrocyte differentiation and chondrocyte enlargement of chondrocytes. When the mesenchymal stem cells were cultured by microculture, they were differentiated into chondrocytes up to 6 days. After that, the culture medium was changed and the mesenchymal stem cells were differentiated into hypertrophic chondrocytes 15 days after the culture. Type IIb collagen and aggrecan, markers of chondrocytes, began to appear from day 3 and were most expressed on day 6 and decreased during cartilage hypertrophy (FIGS. 2A and 2B). ECRG4 is expressed very low in undifferentiated stem cells into chondrocytes very similarly to type IIb collagen, whereas ECRG4 is expressed during chondrocyte differentiation and decreased during chondrocyte enlargement (FIGS. 2A, B). Type X collagen and MMP-13, markers of hypertrophic chondrocytes, were detected from day 12 after culture (FIGS. 2A and 2B). In addition, microcultured chondrocyte spots cultured for 6 days were cut and confirmed by in situ hybridization. As a result, ECRG4 was expressed only in cartilage nodules formed by differentiated chondrocytes (FIG. 2C).

ECRG4 expression in developing rim buds

In order to confirm the expression of ECRG4 during cartilage development in vivo, rim buds were isolated from 11.5-15.5 day mouse embryos after cartilage development occurred and confirmed by RT-PCR and qRT-PCR. Type IIb collagen, the marker molecule of differentiated chondrocytes, was the highest expression in 14.5 and 15.5 dps rim buds, which are the cartilage-producing progressively carcinogenic parts (FIGS. 3A and 3B). Similar to type IIb collagen, the expression of ECRG4 was strong in the 14.5 and 15.5 rims (FIGS. 3A and 3B). In situ hybridization experiments showed that ECRG4 specifically expressed only at the site where the type IIb collagen, a chondrocyte marker molecule, expressed (FIG. 3D).

Decreased ECRG4 Expression in Dedifferentiated Chondrocytes

Differentiated chondrocytes lost their differentiated properties in IL-1β, tomographic culture. Loss of differentiated properties is called dedifferentiation and is an important cause of degenerative arthritis. As shown in Figs. 4A and B, chondrocytes were treated with IL-1β or passage culture to induce chondrocyte dedifferentiation in which type II collagen was reduced. Similar to type II collagen, ECRG4 was also reduced in chondrocytes treated with IL-1β or subjected to tomographic passage three times (FIGS. 4A and 4B). This clearly showed a decrease in ECRG4 during chondrocyte dedifferentiation during cartilage degeneration. In order to elucidate the function of ECRG4 in chondrocyte dedifferentiation, ECRG4 was overexpressed using lentivirus in chondrocytes treated with IL-1β. Overexpression of ECRG4 in IL-1β-treated chondrocytes did not play any role in the expression of type II collagen, COX-2, a typical inflammatory response molecule, and extracellular matrixases (MMPs), which play an important role in cartilage degeneration. 4c). In contrast to the lack of a role in chondrocyte dedifferentiation, overexpression of chondrocytes with lentiviral of ECRG4 reduced chondrocyte division (FIG. 4D).

Osteoarthritis (OA) Reduces ECRG 4 Expression in Cartilage Tissue and Chondrocytes

Finally, expression of CERG4 was observed in degenerative arthritis joint tissues. Human degenerative arthritis joint tissue was taken from a patient who underwent artificial joint surgery, and experimental degenerative arthritis joint samples were collected by injecting collagenase into the knee to induce cartilage degeneration in the mouse knee. Similar to the collagen type II collagen, which is a chondrocyte marker molecule, the expression of ECRG4 was reduced in comparison with the normal cartilage site at the degenerative site of human degenerative arthritis cartilage (FIG. 5A). Injection of collagenase induced cartilage degeneration, such as cutting mouse cartilage and staining with safranin O (FIG. 5B). The Mankin score, which quantifies the degree of degenerative arthritis, was 0.4 ± 0.9 in the PBS injection sample and 6.6 ± 1.3 in the collagenase injection sample. RT-PCR and qRT-PCR analysis showed a decrease in type II collagen, a chondrocyte marker molecule as well as ECRG4. These results clearly show that in vivo ECRG4 expression decreases in chondrocyte dedifferentiation and cartilage degeneration.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

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Claims (14)

delete delete delete delete delete delete delete delete How to detect arthritis diagnosis or prognostic markers by analyzing the reduction of expression of nucleotide sequences encoding ECRG 4 protein or ECRG 4 protein in a human biological sample to provide information necessary for diagnosing or prognostic arthritis .
10. The method of claim 9, wherein the arthritis is arthritis due to trauma, joint ligament injury, meniscus damage, exfoliative osteochondritis, degenerative arthritis, misalignment of the joint, avascular necrosis, inflammatory arthritis or infection.
10. The method of claim 9, wherein the method is carried out in an antigen-antibody reaction mode.
10. The method of claim 9, wherein the method is performed in a microarray manner.
10. The method of claim 9, wherein the method is performed by gene amplification.
Screening methods for preventing or treating arthritis, comprising the following steps:
(a) contacting a sample to be analyzed with a cell comprising the nucleotide sequence of ECRG 4; And
(b) measuring the expression level of the nucleotide sequence, and when the low expression of the nucleotide sequence is suppressed, the sample is determined as a substance for preventing or treating arthritis.

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