WO2003001906A1 - Method of constructing joint disease model animal - Google Patents

Abstract

A method of constructing a model nonhuman mammal of meniscetomy as a model animal of a joint disease such as arthritis deformans characterized by comprising cutting the tibial collateral ligament, anterior cruciate ligament and posterior cruciate ligament of a nonhuman mammal (in particular, a rat), and then further cutting its inner meniscens of the same leg to thereby substantially induce arthritis mutilans.

Description

 Specification

Method for producing animal model of joint disease

Technical field

 The present invention relates to functional analysis of a disease-specific variable gene. More specifically, drugs and genes used to analyze the effects of joint diseases and joint disease-specific variable genes on joints, joint tissues and cells constituting joints involving osteoarthritis, rheumatoid arthritis or osteoporosis, etc. Or a model animal to which the gene product is administered. Background art

Joint diseases are mainly classified into bone diseases, cartilage diseases and arthritis. Bone diseases include non-metabolic bone diseases such as fractures, bone deformation, and osteogenesis imperfecta, and metabolic bone diseases such as osteoporosis and osteomalacia. Cartilage diseases include chronic diseases such as osteoarthritis and rheumatoid arthritis. Arthritis includes inflammatory diseases such as periarthritis of the shoulder, tendon, tendon sheath and peritenitis. The joint disease that often affects patients refers to a disease in which degeneration of articular cartilage is the main lesion (eg, rheumatoid arthritis or osteoarthritis). For example, in the case of cartilage disease represented by osteoarthritis, which has the largest number of patients, the extracellular matrix composed of collagen and proteodalican depends on various factors for proteodalican, then collagenase-3 in the order of collagen. Is degraded by an extracellular streptolytic enzyme called matrix metaoral protease. This causes the cartilage that covers the surfaces of the joints such as knees, jaws, elbows, hips, fingers and jaws to harden and break down, deforming the joints and leading to dysfunction in severe cases. It is known that the incidence of osteoarthritis increases with age, and it is expected that the number of patients will increase in aging societies including Europe and the United States. The main clinical symptoms are inadequate knee expansion and contraction of the jaw due to joint pain, and when the knee joint or hip joint develops, walking may be difficult. As a treatment method, cartilage tissue has poor regeneration ability, so once cartilage destruction progresses, it takes time to regenerate cartilage.If severe pain is involved, total joint replacement or osteotomy is performed. A method of preserving the knot function is taken. Conventionally, palliative treatments such as hyaluronic acid preparations and anti-inflammatory drugs have been used to prevent and treat osteoarthritis for the purpose of reducing pain. It has not been developed and there is no fundamental treatment. Disclosure of the invention

 The present invention is intended for the manufacture of a model animal to which a drug, a gene or a gene product thereof is administered in relation to the development of a therapeutic agent from the viewpoint of creating a joint destruction inhibitor and a therapeutic agent for joint repair, and to analyze the function of the search target. In addition, the present invention provides a method for producing a model animal to which a drug, a gene or a gene product thereof is efficiently and specifically administered to a joint disease for the purpose of evaluating the efficacy of the drug.

 The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, have found that a conventional method of cutting a ligament of a knee joint to artificially induce a joint disease (osteolite squirt cartage, ninth ed. Volume, p. 308, 2001), the method of artificially inducing joint disease by adding meniscal resection of the knee joint to the medial collateral ligament, anterior cruciate ligament and posterior cruciate ligament amputation is unexpected. It was found that even more severe cartilage destruction could be induced. The present inventors have conducted further studies based on these findings, and as a result, have completed the present invention.

 That is, the present invention

 (1) Cut off the medial collateral ligament, anterior cruciate ligament and posterior cruciate ligament of a non-human mammal (preferably a rat), and then resect the medial meniscus of the same limb to substantially induce joint destruction A method for producing a non-human mammal having a meniscal ligament resection model, a joint thereof, a joint tissue thereof, or a cell constituting the joint,

 (2) A meniscal ligament resection model non-human mammal containing the gene, characterized in that the gene is administered to the non-human mammal obtained by the production method according to (1), a joint thereof, and a joint thereof. A method for producing cells constituting a tissue or its joints,

(3) A non-human mammal capable of producing the gene product, characterized in that the gene is administered to the non-human mammal obtained by the production method according to (1), a joint thereof, and a joint tissue thereof. Or a method for producing cells constituting the joint, (4) The method according to any of (1) to (3) above, wherein the non-human mammal is an animal from which the drug effect or gene function of a drug, gene or gene product can be inferred.

 (5) The method according to (2) or (3) above, wherein the gene is (1) sense DNA, (2) antisense DNA, (3) a viral vector or (2) a plasmid vector, (6) the gene is a joint disease-specific variable gene. The production method according to (2) or (3) above,

 (7) A method for elucidating the function of the gene or its gene product, which comprises administering a gene of unknown function to a non-human mammal obtained by the production method according to (1),

 (8) The method according to the above (7), wherein changes in the non-human mammal, its joints, joint tissues or cells constituting the joints are searched for when a gene of unknown function is administered and when the gene is not administered. Method,

 (9) The function of (7) above, wherein the function of the non-human mammal, its joint, the joint tissue or the cell constituting the joint is searched for when the gene whose function is unknown is administered and when it is not administered. Method,

 (10) A kit for elucidating the function of a gene or a gene product thereof comprising a non-human mammal, its joint, its joint tissue or cells constituting the joint obtained by the production method according to (1) above ,

 (1 1) A non-human mammal containing the gene obtained by the production method described in (2) above, a joint thereof, a joint tissue thereof, or a cell constituting the joint; Function elucidation kit,

 (12) A non-human mammal having the ability to produce a gene product obtained by the production method described in (3), a joint thereof, a joint tissue thereof, or a cell constituting the joint, wherein the gene or A kit for elucidating the function of the gene product, (13) The function is elucidated using the method according to any one of (7) to (9) or the kit according to any one of (10) to (12). A medicament comprising the genetically modified gene,

(14) A gene product of a gene whose function has been elucidated using the method according to any one of the above (7) to (9) or the kit according to any one of the above (10) to (12). A pharmaceutical comprising

 (15) the medicament according to the above (13) or (14), which is a joint disease regulator (preferably a therapeutic agent for joint disease);

 (16) Fracture, refracture, bone deformity / osteomyelosis, osteosarcoma, myeloma, osteogenesis imperfecta, scoliosis, bone loss, osteoporosis, osteomalacia, rickets, fibrous osteomyelitis, renal osteodystrophy The medicine according to (15), which is a prophylactic or therapeutic agent for osteoarthritis, bone Behcet's disease, ankylosing myelitis, osteoarthritis, or rheumatoid arthritis (preferably, the above-mentioned diseases in the field of orthopedic surgery).

 (17) A non-human mammal, a joint thereof, a joint tissue thereof, or a cell constituting the joint, which contains a joint disease-specific variable gene obtained by the production method described in (2) above. A screening method for a joint disease regulator,

 (18) Use of a non-human mammal, a joint thereof, a joint tissue thereof, or a cell constituting the joint capable of producing a joint disease-specific variable gene product obtained by the production method described in (3) above. A screening method for a joint disease modulator characterized by the following: (19) the joint disease in the case where the test compound is added to or not to the non-human mammal, the joint, the joint tissue, or the cells constituting the joint; The screening method according to the above (17) or (18), wherein the amount of mRNA corresponding to the specific variable gene is measured.

 (20) measuring the production amount of the joint disease-specific fluctuating gene product when the test compound is added to the non-human mammal, the joint, the joint tissue, or the cells constituting the joint, with or without the test compound; The screening method described in (17) or (18) above,

 (21) When the test compound is added to the non-human mammal, its joint, its joint tissue or cells constituting the joint, and when the test compound is not added, the non-human mammal, its joint, its joint tissue or The screening method according to the above (17) or (18), wherein the function of a cell constituting the joint is searched for,

(22) A non-human mammal containing a joint disease-specific variable gene obtained by the production method according to (2), a joint thereof, a joint tissue thereof, or a cell constituting the joint. A kit for screening for a joint disease modulator, (23) A non-human mammal having the ability to produce a joint-related disease gene product obtained by the production method according to (3), a joint thereof, a joint tissue thereof, or a cell constituting the joint. Screening kit for a joint disease modulator

(24) A joint disease modulator obtained by using the method according to any of (17) to (21) or the screening kit according to (22) or (23),

(25) a medicament comprising a joint disease regulator obtained by using the method according to any of (17) to (21) or the screening kit according to (22) or (23);

 (26) Fracture, refracture, bone deformity / osteomyelosis, osteosarcoma, myeloma, osteogenesis imperfecta, scoliosis, bone loss, osteoporosis, osteomalacia, rickets, fibrous osteomyelitis, renal osteodystrophy A medicament according to the above (25), which is a therapeutic agent for the prevention or treatment of bone disease, bone Petiet's disease, ankylosing myelitis, osteoarthritis or rheumatoid arthritis (preferably, the above diseases in the field of orthopedic surgery);

 (27) Effectiveness of a joint disease modulator obtained by using the method according to any one of (17) to (21) or the screening kit according to (22) or (23) for a mammal. A method for preventing and treating joint diseases, characterized in that

 (28) A method for evaluating the efficacy of a compound, comprising administering a test compound to a non-human mammal, a joint thereof, a joint tissue thereof, or a cell constituting the joint obtained by the production method described in (1) above. ,

 (29) The method according to (28), wherein a change in a non-human mammal, its joint, joint tissue or a cell constituting the joint is searched for in a case where the test compound is administered and in a case where the test compound is not administered,

 (30) The method according to the above (28), wherein the function of the non-human mammal, its joint, joint tissue or the cell constituting the joint is searched with and without the administration of the test compound.

 (31) The method according to the above (28) to (30), wherein the test compound is a gene product,

(32) a medicament comprising a compound whose efficacy has been evaluated using the method according to any of (28) to (31) above, (33) The medicament according to the above (32), which is a joint disease modulator (preferably a therapeutic agent for joint disease).

 (34) Fractures, refractures, bone deformities, osteosarcoma, osteomyeloma, bone dysplasia, scoliosis, bone loss, osteoporosis, osteomalacia, rickets, fibrous ostitis, The medicament according to the above (33), which is a prophylactic or therapeutic agent for renal osteodystrophy, bone Behcet's disease, ankylosing myelitis, osteoarthritis, or rheumatoid arthritis (preferably, the above-mentioned diseases in the field of orthopedic surgery). ,

 (35) a gene product whose function or efficacy has been elucidated by the method according to (7) or (28) above, an antibody against a partial peptide thereof or a salt thereof,

 (36) an antisense nucleic acid against a gene whose function has been elucidated by the method described in (7),

 (37) a medicament comprising the antibody according to the above (35),

 (38) a medicament comprising the antisense nucleic acid according to the above (36),

 (39) the medicament according to the above (37) or (38), which is a joint disease regulator (preferably a therapeutic agent for joint disease);

 (40) Fracture, refracture, bone deformity / osteomyelosis, osteosarcoma, myeloma, osteogenesis imperfecta, scoliosis, bone loss, osteoporosis, osteomalacia, rickets, fibrous osteomyelitis, renal osteodystrophy The medicament according to the above (39), which is a prophylactic / therapeutic agent for osteoarthritis, bone Behcet's disease, ankylosing myelitis, osteoarthritis or rheumatoid arthritis (preferably, the above-mentioned diseases in the field of orthopedic surgery).

 (41) a diagnostic agent for joint disease comprising the antibody according to (35),

 (42) a diagnostic agent for joint disease comprising the antisense nucleic acid according to (36), and

(43) Fracture, refracture, bone deformity / osteomyelosis, osteosarcoma, myeloma, osteogenesis imperfecta, scoliosis, bone defect, osteoporosis, osteomalacia, rickets, fibrous osteomyelitis, renal osteodystrophy The diagnostic agent according to (41) or (42), which is a diagnostic agent for osteoarthritis, bone-peetet's disease, ankylosing myelitis, osteoarthritis or rheumatoid arthritis (preferably, the above-mentioned diseases in the field of orthopedic surgery). I will provide a. BEST MODE FOR CARRYING OUT THE INVENTION

 (Method for manufacturing a meniscal ligament resection model non-human mammal)

 The method for producing a meniscal ligament resection model non-human mammal (hereinafter abbreviated as a model animal) of the present invention substantially comprises the medial collateral ligament, anterior cruciate ligament and posterior cruciate ligament of the knee joint of a non-human mammal. It is characterized by artificially inducing joint disease by applying medial meniscectomy to the cutting of the three ligaments of the ligament, thereby inducing significant cartilage destruction. The model animals obtained by this production method are targeted for joint drug-specific and efficient drug, gene or gene product administration for the purpose of functional analysis of drug discovery and drug efficacy evaluation.

 Examples of non-human mammals include, for example, guinea pigs, rats, mice, egrets, bushes, higgins, magpies, monkeys, and the like.Small animals are preferred, and rodents such as rats and mice are particularly preferred. Rats are particularly preferred.

 Meniscectomy of the knee joint refers to opening the knee joint and removing part or all of the cartilage meniscus intervening between the bones that form the upper and lower joint cavities. As a result, the mobility of the joint is impaired, and the upper and lower bones come into contact with each other, thereby destroying the cartilage existing at the epiphysis. Meniscectomy can be performed by a method known per se, for example, a method for producing an arthritis model animal (Toxicolological Pathology 1, Vol. 27, p. 134, 1999) or a method analogous thereto.

 Drugs, genes, gene products (eg, proteins, peptides, etc.) are used as substances to be administered to meniscal ligament resection model animals.

 The drug is not particularly limited as long as it has a therapeutic / preventive effect on various diseases such as inflammation as well as joint diseases. Specifically, drugs listed in the Japanese Pharmacopoeia are used.

 Further, as the drug, a test compound whose pharmacological action is unknown, a drug whose second pharmaceutical use is unknown, and the like can also be used. As test compounds, for example, non-peptidic compounds derived from living organisms (such as carbohydrates and lipids), synthetic compounds (including peptides), microbial cultures, cell extracts, plant extracts, and animal tissue extracts are used. These compounds may be new compounds or known compounds.

Genes can be either genes whose function is unknown or genes whose function is elucidated. And it may be either genomic DNA or cDNA. Further, the gene may be either sense DNA or antisense DNA. The DNA can be administered alone, but it may be administered in the form of ribosome or a viral vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector; It can be used by inserting it into a vector.

 Further, these genes may encode a full-length protein or peptide, or may be gene fragments encoding a part of the protein or peptide.

 The gene product may be any of a protein and a peptide. In addition, either a gene product whose function is unknown or a gene product whose function is elucidated can be used.

 In the method for producing the model animal of the present invention, the order of the surgical procedures is not particularly limited, and specifically, usually, incision of the skin, cutting of the medial collateral ligament for exposing the joint cavity, cutting of the anterior cruciate ligament, A method of cutting the posterior cruciate ligament, followed by medial meniscectomy, and suturing to close the joint cavity is used.

 In the production method of the present invention, as long as substantially the same method as the specific method is used, the method of ablation and the like can be appropriately changed. That is, surgery on a non-human mammal is usually composed of about 1 to 4, preferably about 15 to 30, individual procedures. Can be replaced with the acquired technique. These procedures can be obtained from known books or literatures. Therefore, the method for producing a model animal of the present invention can be performed using substantially the same method as the above-described specific method, and performing the three ligament cuts and medial meniscectomy shown above for the target non-human mammal. It can be modified as appropriate.

The degree of modification is, for example, about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more identical to the above-mentioned production method. Range. In particular, the method includes the step of resecting the inner meniscus with the three ligaments of the knee joint of a non-human mammal cut, and as a whole about 70% or more, preferably about 80% or more, more preferably about 9% or more. 0% or more, most preferably about 95% or more identity Is preferred.

 By using such a production method of the present invention, a joint disease model animal can be produced specifically and efficiently.

 Usually, in small animals (especially rats), treatment such as damage to articular cartilage does not result in the condition of osteoarthritis (OA) and often resolves quickly. However, according to the production method of the present invention, an OA model animal can be efficiently produced even using a small animal such as a rat. Further, since small animals can be used, for example, there is an advantage that the amount of a compound or gene to be administered per body weight can be saved.

 From the model animal produced by the production method of the present invention, joints, joint tissues or cells constituting the joints can be extracted by conventional means.

 The cells constituting the joint include bone cells, osteoblasts, osteoclasts, chondrocytes, joint synovial cells, fibroblasts and the like.

 [Method for producing model animal containing gene]

 In the method for producing a model animal containing the gene of the present invention, the above-described gene may be contained in the above-described model animal of the present invention using a virus vector or the like.

 By using this production method, a model animal containing the gene or a model animal capable of producing the gene product can be produced. In addition, a joint containing the gene, a cell constituting the joint tissue or a cell constituting the joint, or a joint capable of producing the gene product, a joint tissue or a cell constituting the joint is isolated from these model animals by a conventional means. be able to.

 [Method for producing cells constituting joints containing genes]

 The method for producing the cells constituting the joint containing the above-mentioned gene is substantially performed by cutting three ligaments of the medial collateral ligament, the anterior cruciate ligament and the posterior cruciate ligament in a non-human mammal knee joint. The method is characterized by removing the inner meniscus. The specific operation may be such that the above-described gene is contained in the above-described model animal of the present invention using a viral vector or the like.

By using this production method, it is possible to produce cells forming a joint containing the gene or cells forming a joint capable of producing the gene product. Can be.

 The method for isolating the cells constituting the joint from the joint is known per se, for example, the method described in Osteolites cultivation (Vol. 9, pp. 73-84, 2001) or a method similar thereto. A method is used.

 [Method of elucidating the function of a gene whose function is unknown]

 The method for elucidating the function of a gene of the present invention is to elucidate the function of the gene by administering a gene whose function is unknown to the model animal of the present invention.

 As described above, the gene whose function is unknown may be genomic DNA or cDNA. Further, the gene may encode a full-length protein or peptide, or may be a gene fragment encoding a part of the protein or peptide.

 The function includes, for example, a function relating to the mobility of a joint, and more specifically, a joint destruction inhibitory action, a joint repair action, and the like.

 The elucidation of the function, for example,

 (1) Search for changes in the function of model animals, their joints, joint tissues or cells constituting joints when a gene whose function is unknown or not is administered (any of assay, detection, or measurement is preferable. Is to measure)

 (2) Search for the function of model animals, their joints, joint tissues, or cells constituting joints when a gene whose function is unknown is administered or not administered (either assay, detection or measurement, but preferably measurement ), Etc.

 In the method (1), the search for the function of the joint (which may be any of assay, detection, or measurement, but preferably measurement) is performed by, for example, Arthritis Rheum, Vol. 44, 107, 1081 , 2001).

 In the method (2), the search for the function of the cells constituting the joint (either assay, detection or measurement, but preferably measurement) is performed, for example, by the method of Arthritis Rheum, Vol. 1071-1081, 2001).

Culture of the cells constituting the joint is performed by a method known per se, for example, The method can be carried out using the method described in Arthritis Rheum, Vol. 44, pp. 1071-11081, 2001.

 Furthermore, by culturing the cells constituting the joint under pressurized conditions or culturing them under lethal conditions, the functions of the cells constituting the joint can be elucidated more efficiently.

 The pressurized condition refers to a condition as if the bone were pressurized by applying a centrifugal operation or the like in the process of culturing the cells constituting the joint, for example, osteoblasts. Under pressurized conditions, osteoblasts are subjected to pressurized stress, and the cells organize, transmit, and provide a means for stress.

 Fatal conditions include serum removal and administration of highly toxic anticancer drugs (eg, adriamycin) to the cells that make up the joint.

 The kit for elucidating the function of the present invention comprises a model animal containing the gene, a joint thereof, a joint tissue thereof, or a cell constituting the joint, or a model animal capable of producing the gene product, a joint thereof, a joint tissue thereof. Or it contains the cells that make up the joint.

 Examples of the function elucidating kit of the present invention include the following.

①Measurement buffer and washing buffer

 Hanks' Balanced Salt Solution (manufactured by Gibco) plus 0.05% serum albumin (manufactured by Sigma).

 The solution may be sterilized by filtration through a 0.45 m filter, and stored in, or may be prepared at use.

 (2) Cells forming a joint containing the gene or cells forming a joint capable of producing the gene product

The cells constituting these joints, 1 2-well plates and passaged 5 X 1 0 ⁵ or holes, 3 7, 5% C 0 2, 9 with 5% air 2 days followed by culturing.

By using the method or kit for elucidating the function described above, for example, the function of a joint containing a gene whose function is unknown is compared with that of a joint that does not contain a gene whose function is unknown. About 20% or more, preferably about 30% or more Preferably, when it is increased by about 50% or more, it can be determined that the gene has a joint function promoting (or improving or enhancing) effect.

 On the other hand, compared to a joint that does not contain a gene whose function is unknown, the function of the joint when it does, and the function of the cells that make up the joint are about 20% or more, preferably about 30% or more. Preferably, when the gene is reduced by about 50% or more, it can be determined that the gene has a joint function lowering action.

 Genes elucidated to have a joint function promoting effect or genes elucidated to have a joint function lowering effect are referred to as joint function regulating genes, respectively, as pharmaceuticals such as joint function regulators (preferably drugs for treating joint diseases). Useful as a composition. In particular, genes that have been found to have joint function-promoting effects are useful, for example, as agents for preventing and treating bone or joint diseases. Specific examples of bone or joint diseases include non-metabolic bone diseases such as fractures, refractures, bone deformities and osteoporosis, osteosarcoma, myeloma, osteogenesis imperfecta, and scoliosis in the field of orthopedic surgery; bone defects, osteoporosis Metabolic bone diseases such as osteomalacia, rickets, fibrous osteomyelitis, renal osteodystrophy, bone petiet's disease, ankylosing myelitis; representatives of cartilage diseases such as osteoarthritis and rheumatoid arthritis Joint disease.

 In addition, the genes that have been shown to have joint function-promoting effects can be used as bone tissue repair agents after surgery for multiple myeloma, lung cancer, breast cancer, etc. In the dental field, treatment of periodontal disease, periodontal disease It can be used to repair periodontal tissue defects in disease, stabilize artificial dental roots, repair ridge formation and cleft palate.

When the gene whose function has been elucidated is used as the above-mentioned pharmaceutical composition, the gene may be used alone or after being inserted into an appropriate vector such as a retrovirus vector, an adenovirus vector, or an adenovirus associated virus vector. It can be carried out according to conventional means. The gene can be administered as is or with an auxiliary to promote uptake, such as with a gene gun or a catheter such as Hyde-mouth gel catheter. For example, the gene can be used as a sugar-coated tablet, capsule, elixir, microcapsule or the like as needed, orally, or aseptic solution with water or other pharmaceutically acceptable liquid, Alternatively, they can be used parenterally in the form of injections such as suspensions. For example, the gene It can be manufactured by admixing it with known approved carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc. in the unit dosage form generally required for the practice of formulations. it can. The amount of active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.

 Additives that can be incorporated into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid, etc. Swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry. When the unit dosage form is a capsule, the above type of material can further contain a liquid carrier such as an oil or fat. Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. it can. Examples of the aqueous liquid for injection include physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like. Agents, for example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene daricol), non-ionic surfactant (eg, Polysorbate 80 ™, HCO-50) May be. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. For example, phosphate buffer, sodium acetate buffer), soothing agent (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizer (eg, human serum albumin, polyethylene dalicol, etc.), preservative (eg, benzyl) Alcohol, phenol, etc.) and antioxidants. The prepared injection solution is usually filled in a suitable ampoule.

 Since the pharmaceutical composition thus obtained is safe and has low toxicity, it can be used, for example, in mammals (eg, humans, rats, mice, rabbits, sheep, pigs, horses, cats, dogs, monkeys, etc.). Can be administered.

The dosage of the gene varies depending on the administration target, target organ, symptoms, administration method, etc. However, in the case of oral administration, in general, for example, in patients with osteoarthritis (60 kg), about 0.1 to 100 mg, preferably about 1.0 to 5 Omg, more preferably Is about 1.0 to 2 Omg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc. For example, in the case of an injection, it is usually used, for example, in patients with osteoarthritis (6 O kg ), About 0.01 to 3 Omg, preferably about 0 .: to about 2 Omg, and more preferably about 0.1 to 1 Omg by intravenous injection. It is convenient. For other animals, the dose can be administered in terms of 6 O kg.

 (Screening method of drug candidate compound)

 When the function of the gene administered to the model animal of the present invention has been elucidated, the drug candidate compound can be screened using the following method.

 (Screening method for drug candidate compounds using cells constituting joints)

 The present invention

 (1) a method for screening for a drug for regulating a joint function, which comprises using cells constituting a joint of a model animal of the present invention containing a gene, and

 (2) To provide a method for screening a joint function-regulating agent, which comprises using cells constituting a joint of a model animal of the present invention having the ability to produce a gene product. The same cells as those described above are used as the cells constituting the joint. The cells constituting the joint can be cultured in the same manner as described above.

 Examples of the gene include a gene involved in joint function, a joint disease-specific variable gene such as a joint function regulating gene, and the like. More specifically, Connective Tissue Res. Research (Connect Tissue Res 41, 175-184, 2000) and the like are used.

 This screening method is, for example, as described above,

 (1) To stimulate the cells constituting the joint under pressure conditions,

 (2) The cells constituting the joint can be cultured under lethal conditions.

Fatal conditions include serum removal and high toxicity to joint cells. Administration of anticancer drugs (eg, adriamycin).

 More specifically, this screening method includes, for example,

 (1) measuring the amount of mRNA corresponding to the gene when the test compound is added to the cells constituting the joint and when the test compound is not added;

 (2) It can be carried out by, for example, measuring the production amount of the gene product when the test compound is added to the cells constituting the joint and when it is not added. As the test compound, for example, a non-peptide compound derived from a living body (such as a carbohydrate or a lipid), a synthetic compound (including a peptide), a microorganism culture, a cell extract, a plant extract, or an animal tissue extract can be used. These compounds may be new compounds or known compounds.

 The expression level of mRNA can be determined by a method known per se, for example, Northern blotting or reverse transcription-polymerase chain reaction (RT-PCR)-^ TaqMan polymerase chain reaction or the like. It can be measured according to the method.

 The production amount of the gene product can be measured by a method known per se, for example, a method using an antibody, a method using chromatography, and the like.

 This screening kit contains cells constituting the joint, and the same kit as the above-described kit for elucidating the function of a gene is used.

 By using the screening method or the screening kit described above, the amount of mRNA and gene product when administered is about 20% or more, preferably about 30% or more, compared to the case where no test compound is administered. Preferably, when it increases by about 50% or more, it can be determined that the test compound has a joint function promoting action.

On the other hand, the amount of mRNA and the amount of gene product when administered are reduced by about 20% or more, preferably about 30% or more, more preferably about 50% or more, compared to the case where no test compound is administered. In this case, it can be determined that the test compound has a joint function lowering effect. Each of the test compounds having a joint function promoting action or a joint function reducing action is useful as a pharmaceutical composition such as a joint function regulator (preferably a therapeutic agent for joint disease). In particular, a test compound having a joint function promoting effect is useful, for example, as an agent for preventing or treating bone or joint diseases. Specific examples of bone or joint disease include Non-metabolic bone diseases such as bone fractures, refractures, bone deformities and osteoporosis, osteosarcoma, myeloma, dysplasia, and scoliosis in the field of plastic surgery; bone defects, osteoporosis, osteomalacia, rickets, and lines Metabolic bone diseases such as fibrous osteomyelitis, renal osteodystrophy, bone Petiet's disease, and stiff myelitis; and joint diseases represented by cartilage diseases such as osteoarthritis and rheumatoid arthritis.

 In addition, test compounds having a joint function-promoting effect can be used as a bone tissue repair agent after surgery for multiple myeloma, lung cancer, breast cancer, etc., and in the dental field, for treatment of periodontal disease and dental treatment for periodontal disease. It can be used for repair of peri-tissue defects, stabilization of artificial dental roots, ridge formation and repair of cleft palate.

 When the compound obtained by using the above-described screening method or screening kit is used as the above-mentioned pharmaceutical composition, it can be prepared into a pharmaceutical preparation by using a conventional method. For example, the compound can be administered as a sugar-coated tablet, capsule, elixir, microcapsule, orally, or aseptic solution with water or other pharmaceutically acceptable liquid, as appropriate, Or parenteral use in the form of injections such as suspensions. For example, the compound can be mixed with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc. in generally accepted unit dosage forms required for the formulation. By doing so, it can be manufactured. The amount of active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.

Additives that can be incorporated into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid, etc. Swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cellulose. When the unit dosage form is a capsule, the above type of material can further contain a liquid carrier such as an oil or fat. Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. it can. Aqueous liquids for injection include, for example, saline, dextrose and others An isotonic solution containing an auxiliary agent (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) is used, and a suitable solubilizing agent, for example, alcohol (eg, ethanol), polyalcohol (eg, propylene) Glycol, polyethylene glycol) and nonionic surfactants (eg, Polysorbate 80 ™, HCO-50). As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. In addition, the above-mentioned pharmaceutical composition includes, for example, a buffer (eg, phosphate buffer, sodium acetate buffer), a soothing agent (eg, benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (eg, human serum It may be combined with preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc. The prepared injection solution is usually filled in a suitable ampoule.

 Since the pharmaceutical composition thus obtained is safe and has low toxicity, it can be used, for example, in mammals (for example, humans, rats, mice, rabbits, sheep, pigs, rabbits, cats, dogs, monkeys, etc.). Can be administered.

 The dose of the compound varies depending on the administration target, target organ, symptoms, administration method, and the like. In the case of oral administration, in general, for example, in a patient with osteoarthritis (60 kg), one day About 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 2 Omg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc. For example, in the case of an injection, it is usually used, for example, in patients with osteoarthritis (6 O kg ), It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 Omg per day by intravenous injection. is there. In the case of other animals, the dose can be administered in terms of 60 kg.

 (Screening method of drug candidate compound using model animal or its joint) The present invention, for example,

 (1) a method for screening for a joint function-regulating agent, characterized by using the model animal of the present invention containing the gene or the joint thereof, and

(2) A model animal of the present invention having the ability to produce a gene product or a joint thereof Provided is a method for screening for a joint function modulator, which is characterized in that it is used. As the model or its joint, the same one as described above is used.

 As the test compound, for example, a non-peptide compound derived from a living body (such as a carbohydrate or a lipid), a synthetic compound (including a peptide), a microorganism culture, a cell extract, a plant extract, or an animal tissue extract can be used. These compounds may be new compounds or known compounds.

 Examples of the gene include a gene involved in joint function, a joint disease-specific variable gene such as a joint function regulating gene, and the like. More specifically, Connective Tissue Resume (Vol. 41, pp. 175-184, 2000) and the like are used.

 In this screening method, the joint function of the model animal or its joints is searched for (either assay, detection or measurement, preferably measurement), with or without the test compound added to the model animal. It can be implemented by doing The measurement of the joint function can be performed in the same manner as described above.

 By using this screening method, the joint function when administered was increased by about 20% or more, preferably about 30% or more, and more preferably about 50% or more, compared to the case where no test compound was administered. In this case, it can be determined that the test compound has a joint function promoting effect.

 On the other hand, the joint function and the function of the cells constituting the joint are reduced by about 20% or more, preferably about 30% or more, more preferably about 50% or more as compared with the case where the test compound is not administered. In this case, it can be determined that the test compound has a joint function lowering effect.

A test compound having a joint function promoting action or a joint function lowering action is useful as a pharmaceutical composition such as a joint function modulator (preferably a therapeutic agent for joint disease). In particular, a test compound having a joint function promoting action is useful, for example, as a prophylactic / therapeutic agent for bone or joint disease. Specific examples of bone or joint diseases include non-metabolic bone diseases such as bone fractures, refractures, bone deformities and osteoporosis, osteosarcoma, myeloma, osteogenesis imperfections, and scoliosis in the field of orthopedic surgery; Metabolic bone diseases such as osteoporosis, osteomalacia, rickets, fibrous osteomyelitis, renal osteodystrophy, bone petietosis, ankylosing myelitis; Joint diseases represented by cartilage diseases such as arthritis and rheumatoid arthritis. In addition, the test compound having a joint function-promoting effect is used as a bone tissue repair agent after surgery for multiple myeloma, lung cancer, breast cancer, etc., and in the dental field, treatment of periodontal disease, dental treatment for periodontal disease It can be used for repair of peri-tissue defects, stabilization of artificial dental roots, ridge formation and repair of cleft palate.

 When the compound obtained by using the above-described screening method or screening kit is used as the above-mentioned pharmaceutical composition, it can be prepared into a pharmaceutical preparation by using a conventional method. For example, the compound can be administered as a sugar-coated tablet, capsule, elixir, microcapsule, orally, or aseptic solution with water or other pharmaceutically acceptable liquid, as appropriate, Or parenteral use in the form of injections such as suspensions. For example, the compound can be mixed with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc. in the unit dosage form generally required for the practice of formulations. By doing so, it can be manufactured. The amount of active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.

Excipients that can be incorporated into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cell mouth, corn starch, gelatin, Swelling agents such as alginic acid, lubricating agents such as stear magnesium phosphate, sweetening agents such as sucrose, lactose or saccharine, and flavoring agents such as peppermint, cocoa oil or cherry. When the unit dosage form is a capsule, the above type of material can further contain a liquid carrier such as an oil or fat. Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. it can. Examples of the aqueous liquid for injection include physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like. Agents such as alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol), non-ionic surfactants (eg, polysorbate 80 ™, HCO-50) Any combination may be used. As the oily liquid, for example, sesame oil, soybean oil, and the like are used. The oily liquid may be used in combination with a solubilizing agent such as benzyl benzoate or benzyl alcohol. (For example, phosphate buffer, sodium acetate buffer), soothing agent (for example, benzalkonium chloride, pro-hydrochloride, etc.), stabilizer (for example, human serum albumin, polyethylene glycol, etc.), preservative (for example, For example, benzyl alcohol, phenol, etc.) and antioxidants may be blended. The prepared injection solution is usually filled in a suitable ampoule. Since the pharmaceutical composition thus obtained is safe and has low toxicity, it can be used, for example, in mammals (eg, humans, rats, mice, rabbits, sheep, pigs, horses, cats, dogs, monkeys, etc.). Can be administered.

 The dose of the compound varies depending on the administration subject, target organ, symptoms, administration method, and the like. In the case of oral administration, for example, in osteoarthritis patients (as 6 O kg), It is about 0.1 mg to 100 mg per day, preferably about 1.0 to 5 Omg, more preferably about 1.0 to 2 Omg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc. For example, in the case of an injection, it is usually used, for example, in patients with osteoarthritis (6 O kg ), About 0.01 to 3 Omg per day, preferably about 0 :! It is convenient to administer about 20 mg, more preferably about 0.1-1 Omg, by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.

 [Gene therapy],

 The joint function of a mammal can be regulated by administering to the mammal an effective amount of a joint function-regulating gene whose function has been clarified by using the method for elucidating the function of a gene of the present invention.

The dose of the gene to a mammal varies depending on the administration subject, symptoms, and the like. Usually, for example, in a patient with osteoarthritis (as 6 O kg), about 0.01 to 3 O mg, preferably about It is advantageous to administer about 0.1-20 mg, more preferably about 0.1-1 Omg, directly into the joint cavity. In the case of other animals, the dose can be administered in terms of 60 kg. (Compound evaluation method)

 The present invention

 (1) A method for evaluating the efficacy of a compound of the present invention, which comprises administering a test compound to a model animal, a joint thereof, a joint tissue thereof, or a cell constituting the joint, is provided.

 Test compounds other than genes, for example, gene products, non-peptide compounds derived from living organisms (such as carbohydrates and lipids), synthetic compounds (including peptides), microbial cultures, cell extracts, and plant extracts An animal tissue extract or the like is used, and these compounds may be novel compounds or known compounds. When a gene is used, it can be carried out in the same manner as the above-described method for elucidating the function of a gene.

 The administration method of the test compound is the same as the administration method of the present invention described above.

 In this drug efficacy evaluation method, for example,

 (1) Searching (or detecting or detecting or detecting or measuring) changes in the function of the model animal, its joints, its joint tissues or the cells constituting the joints with and without administration of the test compound;

 (2) Search for (or assay or detect or measure) the function of model animals, their joints, their joint tissues or the cells that make up their joints with and without the administration of the test compound. Evaluate the efficacy of the compound.

 In the above method (2), for example,

(1) To stimulate and culture the cells that make up the joints under pressurized conditions.

 (2) It is possible to evaluate the efficacy more effectively by culturing the cells constituting the joint under lethal conditions. Fatal conditions include serum removal and administration of highly toxic anticancer drugs (eg, adriamycin) to the cells that make up the joint.

 This drug efficacy evaluation method can be carried out in the same manner as the above-described method for elucidating the function of the gene of the present invention and the method for screening a drug candidate compound.

By using the above drug efficacy evaluation method, the joint function or the function of the cells constituting the joint when administered is about 20% or more, preferably about 30% or more as compared with the case where the test compound is not administered. , More preferably about 50% or more, It can be determined that the test compound has a joint function promoting action.

 On the other hand, the joint function or the function of the cells constituting the joint when administered is about 20% or more, preferably about 30% or more, and more preferably about 50% or more, compared to when the test compound is not administered. When it decreases, it can be determined that the test compound has a joint function lowering effect.

 A test compound having a joint function promoting action or a joint function lowering action is useful as a pharmaceutical composition such as a joint function modulator (preferably a therapeutic agent for joint disease). In particular, a test compound having a joint function promoting action is useful, for example, as a prophylactic / therapeutic agent for bone or joint disease. Specific examples of bone or joint diseases include non-metabolic bone diseases such as bone fractures, refractures, bone deformities and osteoporosis, osteosarcoma, myeloma, osteogenesis imperfections, and scoliosis in the field of orthopedic surgery; For metabolic bone diseases such as osteoporosis, osteomalacia, rickets, fibrous osteomyelitis, renal osteodystrophy, bone Behcet's disease, ankylosing myelitis; for cartilage diseases such as osteoarthritis and rheumatoid arthritis Representative joint diseases are mentioned. In addition, test compounds having a joint function-promoting effect can be used as a bone tissue repair agent after surgery for multiple myeloma, lung cancer, breast cancer, etc., and in the dental field, for treatment of periodontal disease and dental treatment for periodontal disease. It can be used for repair of peri-tissue defects, stabilization of artificial dental roots, ridge formation and repair of cleft palate.

 When the compound obtained by using the above-described screening method or screening kit is used as the above-mentioned pharmaceutical composition, it can be prepared into a pharmaceutical preparation by using a conventional method. For example, the compound can be administered as a sugar-coated tablet, capsule, elixir, microcapsule, orally, or aseptic solution with water or other pharmaceutically acceptable liquid, as appropriate, Or parenteral use in the form of injections such as suspensions. For example, the compound can be mixed with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc. in the unit dosage form generally required for the practice of formulations. By doing so, it can be manufactured. The amount of active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.

Examples of additives that can be mixed with tablets, capsules, etc. include binders such as gelatin, corn starch, tragacanth, gum arabic, and crystalline cells. Excipients such as loin, leavening agents such as corn starch, gelatin, alginic acid, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharine, peppermint, coconut oil or cherry. Such flavoring agents are used. When the unit dosage form is a capsule, the above type of material can further contain a liquid carrier such as an oil or fat. Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. it can. Examples of the aqueous liquid for injection include physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like. Agents, such as alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene daricol), nonionic surfactants (eg, Polysorbate 80 ™, HCO-50) Is also good. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. In addition, the above-mentioned pharmaceutical composition includes, for example, a buffer (eg, phosphate buffer, sodium acetate buffer), a soothing agent (eg, benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (eg, human serum It may be combined with preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc. The prepared injection solution is usually filled in a suitable ampoule.

 Since the pharmaceutical composition thus obtained is safe and has low toxicity, it can be used, for example, in mammals (eg, humans, rats, mice, rabbits, sheep, pigs, horses, cats, dogs, monkeys, etc.). Can be administered.

The dose of the compound varies depending on the administration target, target organ, symptoms, administration method, and the like. In the case of oral administration, in general, for example, in a patient with osteoarthritis (60 kg), one day About 0.1 mg to 100 mg, preferably about 1.0-5 mg, more preferably about 1.0-2 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc. For example, in the case of an injection, it is usually used, for example, in patients with osteoarthritis (6 O kg ), About 0.01 to 3 Omg per day, preferably about 0:! To 20 mg It is convenient to administer the drug by intravenous injection, for example, about 0.1 to 1 Omg. For other animals, the dose can be administered in terms of 6 O kg.

 (Antibody)

 The present invention provides an antibody against a gene product, a partial peptide or a salt thereof, whose function or efficacy has been clarified by using the method for elucidating the function or the efficacy evaluation method of the invention described above.

 The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody as long as it can recognize the gene product, its partial peptide, or a salt thereof.

 Antibodies against the gene product, a partial peptide thereof, or a salt thereof (hereinafter, abbreviated as the gene product) can be produced by using the gene product as an antigen in accordance with a known method for producing an antibody or antiserum. it can.

 [Preparation of monoclonal antibody]

 (a) Preparation of monoclonal antibody-producing cells

 The gene product is administered to a mammal at a site capable of producing an antibody by administration, itself or together with a carrier or diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. Administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times. Examples of mammals to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and mice and rats are preferably used.

When producing monoclonal antibody-producing cells, select a warm-blooded animal immunized with an antigen, for example, an individual with an antibody titer from a mouse, and collect the spleen or lymph node 2 to 5 days after the final immunization Then, a monoclonal antibody-producing hybridoma can be prepared by fusing the antibody-producing cells contained therein with myeloma cells. The antibody titer in the antiserum can be measured, for example, by reacting the labeled gene product described below with the antiserum, and then measuring the activity of a labeling agent bound to the antibody. The fusion operation is performed by known methods, for example, the method of Koehler and Milstein [Ne Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus. Preferably, PEG is used.

 Examples of myeloma cells include NS-1, P3U1, and 3 to 20, and P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably, PEG 1000 to PEG6000) is about 10 to 80%. The cell fusion can be carried out efficiently by incubating at about 20 to 40: preferably about 30 to 37 for about 1 to 10 minutes.

 Various methods can be used to screen the monoclonal antibody-producing hybridomas. For example, the hybridoma culture supernatant is applied to a solid phase (eg, a microplate) on which the antigen of the gene product is directly or adsorbed together with a carrier. Then, add an anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used if the cell used for cell fusion is a mouse) or protein A, labeled with a radioactive substance or an enzyme. A method for detecting monoclonal antibodies bound to, an immunoglobulin antibody or protein A-adsorbed solid phase, and adding the eight-hybridoma culture supernatant, adding the gene product labeled with radioactive substances, enzymes, etc. And a method of detecting a monoclonal antibody bound to the antibody.

The selection of the monoclonal antibody can be carried out according to a method known per se or a method analogous thereto. Usually, it can be carried out in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added. As a selection and breeding medium, any medium can be used as long as it can grow a hybridoma. For example, RPMI 1640 medium containing 1-20%, preferably 10-20% fetal calf serum, GIT medium containing 1-10% fetal calf serum (Wako Pure Chemical Industries, Ltd.) A serum-free culture medium for doma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) or the like can be used. The culturing temperature is usually 20 to 40, preferably about 37. The culture time is generally 5 days to 3 weeks, preferably 1 week to 2 weeks. The culture can be usually performed under 5% carbon dioxide. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above. (b) Purification of monoclonal antibodies

 Monoclonal antibodies can be separated and purified in the same manner as normal polyclonal antibodies. [Examples: salting out, alcohol precipitation, isoelectric focusing, electrophoresis, ion exchangers (ex. , DEAE), ultracentrifugation, gel filtration, antigen-binding solid phase or specific antibody that is collected by using an active adsorbent such as protein A or protein G to dissociate the bond and obtain the antibody. Purification method].

 (Preparation of polyclonal antibody)

 The polyclonal antibody of the present invention can be produced according to a method known per se or a method analogous thereto. For example, a complex of an immunizing antigen (the gene product) and a carrier protein is formed, and a mammal is immunized in the same manner as in the above-described method for producing a monoclonal antibody. The antibody can be produced by collecting the antibody and separating and purifying the antibody.

 Regarding a complex of an immunizing antigen and a carrier protein used for immunizing a mammal, the type of the carrier protein and the mixing ratio of the carrier and the hapten are determined by the antibody against the hapten immunized by cross-linking the carrier. As long as it can be efficiently carried out, any material can be cross-linked at any ratio.For example, 血清 serum albumin, ゥ thyroglobulin, keyhole 'limpet, hemocyanin, etc. are converted into hapten 1 by weight ratio. On the other hand, a method of coupling at a ratio of about 0.1 to 20, preferably about 1 to 5 is used.

 Further, various condensing agents can be used for force coupling between the hapten and the carrier. For example, an active ester reagent containing a daltaraldehyde, a carbodiimide, a maleimide active ester, a thiol group or a dithioviridyl group is used.

The condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site capable of producing antibodies. Complete Freund's adjuvant / incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration can usually be performed once every about 2 to 6 weeks, for a total of about 3 to 10 times. The polyclonal antibody can be collected from blood, ascites, etc., preferably from blood, of the mammal immunized by the above method. The measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the serum described above. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described separation and purification of the monoclonal antibody. Since the antibody of the present invention can specifically recognize the gene product, it can be used for quantification of the gene product in a test solution, particularly for quantification by sandwich immunoassay. That is, the present invention provides, for example,

 (i) reacting the antibody of the present invention with a test wave and a labeled gene product in a competitive manner, and measuring the ratio of the labeled gene product bound to the antibody; the gene product in the test wave; Quantification method,

 (ii) Simultaneously or continuously reacting the test solution with the antibody of the present invention and the labeled antibody of the present invention insolubilized on the carrier, and then measuring the activity of the labeling agent on the insolubilized carrier. Provided is a method for quantifying the gene product in a test wave, which is a characteristic feature.

In the above (ii), it is preferable that one antibody is an antibody that recognizes the N-terminal of the gene product and the other antibody is an antibody that reacts with the C-terminal of the gene product. The gene product can be measured using a monoclonal antibody against the gene product (hereinafter, sometimes referred to as the monoclonal antibody of the present invention), and can also be detected by tissue staining or the like. For these purposes, the antibody molecule itself may be used, or the F (ab ') ₂ , Fab', or Fab fraction of the antibody molecule may be used. The measurement method using an antibody against the gene product is not particularly limited, and the amount of the antibody, the antigen, or the antibody-antigen complex corresponding to the amount of the antigen in the test solution (for example, the amount of the gene product) is determined. Any measurement method may be used as long as it is detected by chemical or physical means and is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, a competitive method, an immunometric method, and a sandwich method are suitably used, but the sandwich method described later is particularly preferable in terms of sensitivity and specificity.

As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ^I4 C] and the like are used. As the above enzyme, a stable enzyme having a large specific activity is preferable. For example, / 3-galactosidase, β Dalcosidase, alkaline phosphatase, oxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiosinate and the like are used. As the luminescent substance, for example, luminol, luminol derivative, luciferin, lucigenin and the like are used. Further, a biotin-avidin system may be used for binding the antibody or antigen to the labeling agent.

 For the insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing proteins or enzymes may be used. As the carrier, for example, insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, and glass are used.

 In the sandwich method, the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and the labeled monoclonal antibody of the present invention is further reacted (secondary reaction). By measuring the activity of the protein, the amount of the receptor protein of the present invention in the test solution can be determined. The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at staggered times. The labeling agent and the method of insolubilization can be in accordance with those described above.

 In the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. May be used.

 In the method for measuring the gene product by the sandwich method of the present invention, the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is preferably an antibody having a different site to which the gene product binds. That is, the antibody used in the primary reaction and the secondary reaction is preferably, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the receptor protein, the antibody used in the primary reaction is preferably An antibody that recognizes other than the C-terminal, for example, the N-terminal, is used.

The monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, or a nephrometry. In the competition method, the antigen in the test solution and the labeled antigen were allowed to react competitively with the antibody. The unreacted labeled antigen is separated from (F) and the labeled antigen (B) bound to the antibody (BZF separation), and the amount of B or F label is measured, and the amount of antigen in the test wave is determined. Quantify. In this reaction method, a soluble antibody is used as the antibody, BZF separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the above antibody, or a solid phase antibody is used as the first antibody. An immobilization method using a soluble antibody as the first antibody and using an immobilized antibody as the second antibody is used.

 In the immunometric method, an antigen in a test wave and a solid-phased antigen are subjected to a competitive reaction with a fixed amount of a labeled antibody, and then the solid phase and the liquid phase are separated. The antigen is allowed to react with an excess amount of the labeled antibody, and then the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in either phase is measured to determine the amount of antigen in the test wave.

 In nephelometry, the amount of insoluble sediment generated as a result of an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test wave is small and only a small amount of sediment is obtained, laser-nephrometry utilizing laser scattering is preferably used.

In applying each of these immunological assay methods to the assay method of the present invention, no special conditions, operations, and the like need to be set. A measurement system for the gene product may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For details of these general technical means, it is possible to refer to reviews and compendiums [for example, Hiroshi Irie “Radio Noatssey” (Kodansha, published in 1974), Irie III ” "Continuing Radio Imnoatssey" (Kodansha, issued in 1979), "Enzyme Immunoassay" edited by Eiji Ishikawa et al. (Medical Publishing, published in 1978), "Enzyme Immunoassay" edited by Eiji Ishikawa et al. (Ed.) (Medical Shoin, published in 1982), Eiji Ishikawa et al., “Enzyme Immunoassay” (3rd edition) (Medical Publishing, published in 1987), “Methods in ENZYMOLOGY” Vol. 70 (Immunochemical Techniques (Part A)), ibid Vo 73 (Immunochemical Techniques (Part B)), ibid Vol. 74 (Immunochemical Techniques (Part 0), ibid Vol. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), ibid.Vol. 92 (Immunochemical Techniques (Part E: Monocl onal Ant ibodies and General Immunoassay Methods)), 121 (Immunochemi ca technicques (Part I: Hybrid technology and Monoclonal Ant ibod ies)) (see Academic Press, etc.)).

 As described above, the gene product can be quantified with high sensitivity by using the antibody of the present invention.

 Furthermore, by quantifying the gene product in vivo using the antibody of the present invention, it is possible to diagnose various diseases associated with the gene product. In other words, since the antibody of the present invention can detect a gene product whose function has been elucidated, it is useful as a diagnostic agent for a disease involving the gene product. For example, when the gene product is involved in joint function, the antibody of the present invention is useful as a diagnostic agent for joint function, more specifically as a diagnostic agent for bone or joint disease. Examples of bone or joint diseases include non-metabolic bone diseases such as bone fractures, refractures, bone deformities and osteoporosis, osteosarcoma, myeloma, osteogenesis imperfections, and scoliosis in the field of orthopedic surgery; bone defects, osteoporosis Metabolic bone diseases such as osteomalacia, rickets, fibroostitis, renal osteodystrophy, bone Petiet's disease, ankylosing myelitis; cartilage diseases such as osteoarthritis and rheumatoid arthritis Joint disease.

 Further, the antibody of the present invention can be used for specifically detecting the gene product present in a subject such as a body fluid or a tissue. It is also used for preparing an antibody column used for purifying the gene product, detecting the gene product present in each fraction during purification, and analyzing the behavior of the gene product in test cells. Can be

 Furthermore, the antibody of the present invention has low toxicity and can regulate the function of the gene product in vivo. Therefore, for example, a pharmaceutical composition such as a mammalian joint function regulator (preferably a therapeutic agent for joint disease) It is useful as an agent for preventing and treating bone or joint diseases. Specific examples of bone or joint diseases include non-metabolic bone diseases such as bone fractures, refractures, bone deformities and osteoporosis, osteosarcoma, myeloma, osteogenesis imperfecta, and scoliosis in the field of orthopedic surgery; Metabolic bone diseases such as osteoporosis, osteomalacia, rickets, fibrous osteomyelitis, renal osteodystrophy, bone Behcet's disease, ankylosing myelitis; typical cartilage diseases such as osteoarthritis and chronic rheumatoid arthritis Joint disease.

Furthermore, the antibody of the present invention can be used for skeletons after surgical operations such as multiple myeloma, lung cancer, and breast cancer. As a tissue restorative, and in the dental field, it can be used for treatment of periodontal disease, repair of periodontal tissue defects in periodontal disease, stabilization of artificial dental roots, repair of ridges and cleft palate.

 When the antibody of the present invention is used as the above-mentioned pharmaceutical composition, it can be prepared by a conventional method. For example, it can be produced and used in the same manner as a pharmaceutical composition containing a compound having a joint function promoting action or a joint function reducing action described above.

 (Antisense nucleic acid)

 The present invention provides an antisense nucleic acid for a gene whose function has been elucidated using the function elucidation method of the present invention.

 DNA encoding a gene product that has been cloned or determined from an antisense polynucleotide (nucleic acid) capable of inhibiting the replication or expression of a gene whose function has been elucidated (hereinafter abbreviated as the gene). Can be designed and synthesized based on the nucleotide sequence information of Such a polynucleotide (nucleic acid) can hybridize to the RNA of the gene, inhibit the synthesis or function of the RNA, or interact with the RNA related to the gene product. It can regulate and control gene expression.

 Polynucleotides complementary to the selected sequence of the gene product-related RNA and capable of specifically hybridizing with the gene product-related RNA regulate the expression of the gene in vivo and in vitro · It is useful for controlling, and is also useful for treating or diagnosing diseases.

 The term "corresponding" means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. The “correspondence” between a nucleotide, a nucleotide sequence or a nucleic acid and a peptide (protein) means that the amino acid of the peptide (protein) specified by the sequence derived from the nucleotide (nucleic acid) sequence or its complement is usually used. pointing.

5 'end hairpin loop of the gene, 5' end 6—base pair 'repeat, 5' end untranslated region, polypeptide translation initiation codon, protein coding region, ORF translation initiation codon, 3 'end untranslated region, 3 'End palindrome region, and 3' end to aviation The loop may be selected as a preferred target region, but any region within the gene may be selected as a target.

The relationship between the target nucleic acid and the polynucleotide complementary to at least a part of the target region can be said to be that the relationship between the target nucleic acid and the polynucleotide that can hybridize with the target is “antisense”. Antisense polynucleotides may be 2-deoxy-D-ribose-containing polydeoxynucleotides, D-report-containing polydeoxynucleotides, N-glycosides of purine or pyrimidine bases, and others. Or other polymers having a non-nucleotide backbone (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special bonds, provided that the polymer is Pairing of bases as found in DNA and RNA (contains nucleotides having a configuration permitting base attachment)). They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and even DNA: RNA hybrids, and can further comprise unmodified polynucleotides (or unmodified oligonucleotides). Nucleotides), as well as those with known modifications, e.g., those with labels, capped, methylated, or one or more naturally occurring nucleotides with analogs, as known in the art Modified, intramolecularly nucleotide-modified, such as those having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, olebamate, etc.), a charged bond or a sulfur-containing bond (eg, Those having phosphorothioate, phosphorodithioate, etc., such as proteins (nucleases, nucleases' inhibitors, toxins) Antibodies, signal peptides, poly-L-lysine, etc.) or sugars (for example, monosaccharides), etc., which have side-chain groups, or interacting compounds (for example, acridine, psoralen, etc.), chelates Those containing compounds (eg, metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, those with modified bonds (eg, alpha-anomer type nucleic acids) Etc.). Here, the “nucleoside”, “nucleotide” and “nucleic acid” may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. These modifications are It may contain methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced with halogens, aliphatic groups, etc., or functionalities such as ethers, amines, etc. It may be converted to a group.

 The antisense polynucleotide (nucleic acid) of the present invention is an RNA, a DNA or a modified nucleic acid (RNA, DNA). Specific examples of the modified nucleic acid include, but are not limited to, sulfur derivatives of nucleic acids, thiophosphate derivatives, and those resistant to degradation of polynucleoside amides and oligonucleoside amides. The antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, to make the antisense nucleic acid more stable in the cell, to increase the cell permeability of the antisense nucleic acid, to have a greater affinity for the target sense strand, and to be more toxic if it is toxic. Make sense nucleic acid less toxic.

 Thus, many modifications are known in the art, for example, J. Kawakami et al., Pharm Tech Japan, Vol. 8, pp. 247, 1992; Vol. 8, pp. 395, 1992; ST, Crooke et. al. ed., Ant isense Research and Applicat ions, CRC Press, 1993.

The antisense nucleic acid of the present invention may contain a modified or modified sugar, base, or bond, provided in a special form such as ribosome, microsphere, or applied by gene therapy. Or can be given in additional form. Such additional forms include polycations, such as polylysine, which act to neutralize the charge on the phosphate backbone, and lipids, which enhance interaction with cell membranes or increase nucleic acid uptake. For example, crude water-based substances such as phospholipids and cholesterol are exemplified. Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate, cholic acid, etc.). Such a substance can be attached to the 3 'end or 5' end of a nucleic acid, and can be attached via a base, a sugar, or an intramolecular nucleoside bond. Other groups include 3 'or 5' ends of nucleic acids. Specific capping groups for preventing degradation by nucleases such as exonuclease and RNase. Such capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.

 Since the antisense nucleic acid of the present invention has low toxicity and can regulate the function of the gene or the gene product in a living body, it can be used, for example, in regulating a joint function of a mammal (preferably a drug for treating a joint disease). It is useful as a pharmaceutical composition, as an agent for preventing or treating bone or joint diseases. Specific examples of bone or joint diseases include non-metabolic bone diseases such as fractures, refractures, bone deformities and osteomyelitis, osteosarcoma, myeloma, osteogenesis incomplete, scoliosis in the orthopedic field; Metabolic bone diseases such as osteoporosis, osteomalacia, rickets, fibrous ostitis, renal osteodystrophy, bone Behcet's disease, stiff myelitis; and cartilage diseases such as osteoarthritis and rheumatoid arthritis Representative joint diseases are mentioned. Furthermore, the antisense nucleic acid of the present invention can be used as a bone tissue repair agent after surgery for multiple myeloma, lung cancer, breast cancer, etc., and in the dental field, treatment of periodontal disease, periodontal tissue in periodontal disease It can be used to repair defects, stabilize artificial dental roots, repair ridge formation and cleft palate.

When the antisense nucleic acid of the present invention is used as the above pharmaceutical composition, the antisense nucleic acid is used alone or in an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, or the like. After insertion, it can be performed according to conventional means. The antisense nucleic acid can be administered as it is or together with an auxiliary for promoting uptake, using a gene gun or a catheter such as a hydrogel catheter. For example, the antisense nucleic acid can be sterilized orally as a sugar-coated tablet, capsule, elixir, or micron-capsule, etc., as necessary, or with water or other pharmaceutically acceptable liquids. It can be used parenterally in the form of injections, such as aqueous solutions or suspensions. For example, the antisense nucleic acid can be combined with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like in a unit dosage form generally required for the practice of the formulation. Can be manufactured by mixing You. The amount of the active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.

 Examples of additives that can be mixed with tablets, capsules, etc. include binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid, etc. Swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry. When the unit dosage form is a capsule, the above type of material can further contain a liquid carrier such as an oil or fat. Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. it can. Examples of the aqueous liquid for injection include physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like. Agents, for example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene daricol), non-ionic surfactant (eg, Polysorbate 80 ™, HCO-50) May be. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. In addition, the above-mentioned pharmaceutical composition includes, for example, a buffer (eg, phosphate buffer, sodium acetate buffer), a soothing agent (eg, benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (eg, human serum It may be combined with preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc. The prepared injection solution is usually filled in a suitable ampoule.

 Since the pharmaceutical composition thus obtained is safe and has low toxicity, it can be used, for example, in mammals (for example, humans, rats, mice, rabbits, sheep, bush, horses, cats, dogs, monkeys, etc.). Can be administered.

The dose of the antisense nucleic acid varies depending on the administration subject, target organ, symptoms, administration method, and the like. However, in the case of oral administration, for example, in an osteoarthritis patient (as 60 kg), About 0.1 mg to 100 mg per day, preferably about It is 1.0-5 O mg, more preferably about 1.0-2 O mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptoms, administration method and the like. 60 kg) per day, about 0.01 to 3 Omg per day, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 1 Omg by intravenous injection. It is convenient to administer. For other animals, the equivalent dose per 60 kg can be administered.

 Furthermore, the antisense nucleic acid can also be used as a diagnostic oligonucleotide probe for examining the presence of DNA developed in tissues or cells using the method of the present invention and the state of expression thereof.

 Furthermore, since the antisense nucleic acid of the present invention can detect a gene whose function has been elucidated, it is useful as a diagnostic agent for a disease involving the gene. For example, when the gene is involved in joint function, the antisense nucleic acid of the present invention is useful as a diagnostic agent for joint function and as a diagnostic agent for bone or joint disease. Specific examples of bone or joint diseases include non-metabolic bone diseases such as fractures, refractures, bone deformities and osteoporosis, osteosarcoma, myeloma, osteogenesis imperfections, and scoliosis in the field of orthopedic surgery; bone defects, osteoporosis Metabolic bone diseases such as osteomalacia, rickets, fibrous osteomyelitis, renal osteodystrophy, bone Behcet's disease, ankylosing myelitis; represented by cartilage diseases such as osteoarthritis and rheumatoid arthritis Joint disease.

 (Genetic diagnostic agent)

 Genes whose functions have been elucidated using the method for elucidating gene functions of the present invention (DNA or mRNA) can be used, for example, as probes to obtain human or warm-blooded animals (for example, rats, mice, guinea pigs, (Eg heron, bird, sheep, widow, bush, horse, dog, dog, cat, monkey, chimpanzee, etc.) in the gene (abnormality), such as damage, mutation or reduced expression of the gene, It can be used as a gene diagnostic agent because it can detect an increase in the gene or overexpression of the gene.

The above-described genetic diagnosis can be performed by, for example, the known Northern hybridization or PCR-SSCP method. For example, when overexpression is detected by Northern hybridization or when the mutation of the gene is detected by PCR-SSCP method, for example, it is possible that the disease is a disease such as joint disease accompanied by decreased joint function. It can be diagnosed as high. [Screening method for Promoter activity modulator]

 The present invention

 (1) A reporter gene assay using a promoter that controls the expression of a gene whose function or efficacy has been elucidated using the function elucidation method or efficacy evaluation method of the present invention, in which a test compound is administered. Provided is a method for screening a compound that regulates the promoter activity, which comprises measuring the promoter activity when a test compound is not administered.

 More specifically, a primary cell or H9c2 cell line that constitutes a joint, or a primary cell or H9c2 cell line that constitutes a joint into which a gene produced using the meniscal ligament resection model animal of the present invention has been introduced, is used as a host cell. Perform the reporter gene assay. The repo overnight gene accession can be performed by a method known per se, for example, The Journal of Biological Chemistry (JB), Vol. 272, pp. 22800-22808, 1997, Development, Vol. 124, 793- 804, 1997) can be used.

 Test compounds include, for example, peptides, proteins, non-peptidic compounds derived from living organisms (such as carbohydrates and lipids), synthetic compounds, microorganism cultures, cell extracts, plant extracts, and animal tissue extracts. However, these compounds may be novel compounds or known compounds.

 Culture of the cells constituting the joint can be performed in the same manner as described above.

 Promoter activity can be measured by examining the expression level of a reporter gene. For example, the amount of reporter gene expression is measured according to a method such as Northern blotting or Reverse transcrip tion-polymerase chain reaction (RT-PCR)-^ TaqMan polymerase chain reaction or a method analogous thereto. be able to.

In this method, the expression level of the reporter gene when the test compound is administered is about 20% higher than that when the test compound is not administered, preferably 30% or more, More preferably, a compound that enhances about 50% or more can be selected as a compound that enhances the activity of a gene whose function has been elucidated or a gene product thereof.

 On the other hand, the expression level of the reporter gene when the test compound is administered is about 20% or more, preferably 30% or more, and more preferably about 50% or more as compared with the case where the test compound is not administered. The compound that inhibits can be selected as a compound that inhibits the activity of the gene whose function has been elucidated or the gene product thereof.

 Therefore, a compound that enhances the activity of a gene whose function has been elucidated or its gene product is useful as a drug such as a function enhancer for the gene or its gene product.

 On the other hand, a compound that inhibits the activity of a gene whose function has been elucidated or its gene product is useful as a drug such as a function inhibitor of the gene or its gene product. These medicaments can be produced and used in the same manner as the above-mentioned pharmaceutical composition. Example

 Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto. Example 1 Preparation for surgery

Male SD rats (9 weeks old; body weight 320-370 g) were anesthetized with sodium pentobarbital (50 mg / kg, ip). Either the right or left hind limb knee hair was shaved with a clipper and laid on the operating table in a supine position. The surgical site was disinfected with an 80% ethanol solution. The disinfected hind limb knee skin was incised in the median direction, and the connective tissue immediately below was dissected, exposing the knee joint. To avoid cutting the tendon located on the side of the body from around the patella, make a midline incision separately from the left and right sides of the tendon and muscle located above it, and leave the patella in the outward direction with the remaining tendon and muscle attached. Moved. After moving the patella, bend the knee joint and incise the boundary between the femur and tibia from the frontal direction.Follow this incision and place it at the boundary between the femur and tibia, taking care not to damage the joint The tendons and muscles were cut inward in a circle. Example 2 medial collateral ligament, anterior cruciate ligament and posterior cruciate ligament amputation

 The medial collateral ligament is hit during the medial incision. The medial collateral ligament was cut, taking care not to damage the joint surface. To open the joint cavity, the femur and tibia were cut down to the rear of the knee. Next, ophthalmic scissors were inserted from the front of the knee joint, and the anterior cruciate ligament and the posterior cruciate ligament connecting the femur and tibia while expanding the joint cavity were confirmed and cut in order. At this time, care should be taken not to damage the blood vessels in the branches of the femoral artery and vein existing around the joint. Example 3 Resection of the medial meniscus

 In Example 2, three knee ligaments, the medial collateral ligament, the anterior cruciate ligament, and the posterior cruciate ligament, are cut, whereby the knee joint can be greatly opened laterally and posteriorly. The knee joint was greatly opened posteriorly and posteriorly, and it was confirmed that the meniscus was connected to the tibia by connective tissue. The medial side of the meniscus was excised with ophthalmic scissors. The meniscus is formed symmetrically from the center of the joint, but this resection is limited to the meniscus inside the center. Thus, the outer meniscus will remain on the proximal tibia articular surface, connected to the connective tissue. Example 4 Knee joint closure and suture

 After resection of the meniscus, close the joints so that the femur and tibia are in the same positional relationship as before the operation, and take care not to impair the mobility of the joints. It was sutured in order. Example 5 Evaluation of efficacy

Using the meniscal ligament resection model rat produced in Example 4, a known over-the-counter drug, ARU (ARTZ; a joint function improving drug containing sodium hyaluronate component; Seikagaku Corporation), and Ro-3-2-3 5 5 5 (a compound reported to be effective in the treatment of osteoarthritis; Arthritis Rheum, 1998 Sep; 41 1639-44) was administered to the knee joint, and the drug was treated for osteoarthritis. The effect was confirmed, and it was confirmed that the animal model created by the production method of the present invention can be used for evaluating drug efficacy. The meniscus of the right limb knee of a 9-week-old male SD rat purchased from Nippon Chills River was resected. The drug was administered to the joint space of the right limb from which the meniscus was removed. Aru used a dissolved formulation of 10 mg Zm1. Ro-32-355-55 was dissolved in DMSO and ethanol, and the final drug concentration was set to 20 nmo1. The solution was used in a state of being sufficiently mixed with the waltz (10 mg Zm1). The administration method was 1001 twice a week for 2 weeks, 2 times a week for each joint. The administration drug was prepared at the time of use.

 After completion of the administration for 2 weeks, the rats were sacrificed by exsanguination under ether anesthesia, and the knee joints were collected. The knee joint was fixed with 4% paraformaldehyde at 4 ° C. After washing and washing the knee joint tissue, the knee joint was demineralized with decalcifying solution B (Wako Pure Chemical Industries) for 2 weeks. Next, paraffin embedding was performed in accordance with the usual method (Histology Research Method, Yutaka Sano, Nanzando, 1985). Micro made paraffin block! And sliced to a thickness of 5 m. After slicing, the sections were stretched in a warm bath, mounted on a slide glass, and dried at 37 with a dryer. After deparaffinization, the prepared sections were stained with hematoxylin-eosin and safranin O according to standard methods, and mounted (Histology Research, Yutaka Sano, Nanzando, 1985). The joint site to which the drug was administered was observed histologically in bright field and compared with the control group.

 As a result of microscopic observation, joint destruction was not suppressed in the group administered with Waltz, but the joint cavity was maintained. In addition, in the group that was administered in combination with Waltz and Ro-32-355, articular cartilage destruction was suppressed throughout the entire articular cartilage layer, and the joint cavity was maintained. From the above, the production method of the present invention efficiently produces a joint disease model animal such as osteoarthritis, and the meniscal ligament resection model non-human mammal obtained by the production method of the present invention has a function. It has been shown that it can be used for methods for elucidating the functions of unknown genes, screening methods for drug candidate compounds, and methods for evaluating drug efficacy. Industrial applicability

 ADVANTAGE OF THE INVENTION The manufacturing method of the meniscal ligament resection model non-human mammal of the present invention can efficiently produce a joint disease model animal such as osteoarthritis. The meniscal ligament resection model non-human mammal obtained by the production method of the present invention can be used for a method for elucidating the function of a gene whose function is not known, a method for screening a drug candidate compound, a method for evaluating the efficacy, and the like.

Claims

The scope of the claims 1. It is characterized in that joint destruction is substantially induced by cutting the medial collateral ligament, anterior cruciate ligament and posterior cruciate ligament of a non-human mammal, and then resecting the medial meniscus of the same limb. Meniscal ligament resection model non-human mammal, its joints, its joint tissue or a method of producing cells constituting the joints.  2. The method according to claim 1, wherein the non-human mammal is a rat.  3. A meniscal ligament resection model non-human mammal containing the gene, wherein the gene is administered to the non-human mammal obtained by the production method according to claim 1, a joint thereof, a joint tissue thereof, or a joint tissue thereof. A method for producing cells composing a joint.  4. A non-human mammal having the ability to produce the gene product, characterized in that the gene is administered to the non-human mammal obtained by the production method according to claim 1, its joint, its joint tissue or its joint. A method for producing the constituent cells.  5. The method according to any one of claims 1 to 4, wherein the non-human mammal is an animal from which the drug effect or gene function of a drug, gene or gene product can be estimated.  6. The method according to claim 3, wherein the gene is (1) a sense DNA, (2) an antisense DNA, (3) a viral vector or (2) a plasmid vector.  7. The method according to claim 3 or 4, wherein the gene is a joint disease-specific variable gene. 8. A method for clarifying the function of a gene or a gene product thereof, which comprises administering a gene whose function is unknown to a non-human mammal obtained by the production method according to claim 1. 9. The method according to claim 8, wherein changes in the non-human mammal, its joint, joint tissue, or cells constituting the joint are searched for when the gene whose function is unknown is administered and when it is not administered. .  10. The method according to claim 8, wherein the function of the non-human mammal, the joint thereof, the joint tissue or the cell constituting the joint is searched for when the gene whose function is unknown is administered and when the gene is not administered. . 11. A kit for elucidating the function of a gene or a gene product thereof, comprising a non-human mammal obtained by the production method according to claim 1, a joint thereof, a joint tissue thereof, or a cell constituting the joint. 12. A function of the gene or the gene product thereof, including a non-human mammal containing the gene obtained by the production method according to claim 3, a joint thereof, a joint tissue thereof, or a cell constituting the joint. Elucidation kit.  13. A non-human mammal capable of producing a gene product obtained by the production method according to claim 4, a joint thereof, a joint tissue thereof, or a cell constituting the joint, wherein the gene or A kit for elucidating the function of the gene product.  14. A medicament comprising a gene whose function has been elucidated using the method according to any one of claims 8 to 10 or the kit according to any one of claims 11 to 13.  15. A medicament comprising a gene product of a gene whose function has been elucidated using the method according to any one of claims 8 to 10 or the kit according to any one of claims 11 to 13.  16. The medicament according to claim 14 or 15, which is a joint disease regulator.  1 7 .Fractures, refractures, bone deformities and osteoporosis, osteosarcoma, myeloma, osteogenesis imperfecta, scoliosis, bone loss, osteoporosis, osteomalacia, rickets, fibrous osteomyelitis, renal osteoarthritis 17. The medicament according to claim 16, which is an agent for preventing or treating nutrition, osteoporosis, osteomyelitis, osteoarthritis or rheumatoid arthritis.  18. A non-human mammal, a joint thereof, a joint tissue thereof, or a cell constituting the joint, which comprises the joint disease-specific variable gene obtained by the production method according to claim 3, wherein the disease is regulated. Drug screening method.  19. A non-human mammal capable of producing a joint disease-specific variable gene product obtained by the production method according to claim 4, a joint thereof, a joint tissue thereof, or a cell constituting the joint. Screening method for a joint disease regulator. 20. The amount of mRNA corresponding to the joint disease-specific gene in the case of adding or not adding a test compound to the non-human mammal, the joint, the joint tissue, or the cells constituting the joint is determined. 10. The screening method according to claim 18, wherein the measurement is performed. 21. Measure the production amount of the joint disease-specific gene gene product with and without the test compound added to the non-human mammal, its joint, its joint tissue, or cells constituting the joint. The screening method according to claim 18 or 19. Law.  2 2. The non-human mammal, its joint, its joint tissue, or its joint, with or without the addition of a test compound to the non-human mammal, its joint, its joint tissue, or cells constituting the joint. 10. The screening method according to claim 18 or 19, wherein a function of a cell constituting the cell is searched.  23. A joint disease comprising a non-human mammal, a joint thereof, a joint tissue thereof, or a cell constituting the joint, comprising a joint disease-specific variable gene obtained by the production method according to claim 3. Kit for screening for modulators.  2 4. A non-human mammal capable of producing a joint-related disease gene product obtained by the production method according to claim 4, a joint thereof, a joint tissue thereof, or a cell constituting the joint. A screening kit for a joint disease regulator.  25. An agent for regulating a joint disease obtained by using the method according to any one of claims 18 to 22 or the screening kit according to claim 23 or 24.  26. It comprises a joint disease regulator obtained by using the method according to any one of claims 18 to 22 or the screening kit according to claim 23 or 24. 2 7 .Fractures, refractures, bone deformities and osteomyelitis, osteosarcoma, myeloma, osteogenesis imperfecta, scoliosis, bone loss, osteoporosis, osteomalacia, rickets, fibrous osteomyelitis, renal osteoarthritis 27. The medicament according to claim 26, which is a prophylactic and / or therapeutic agent for nutrition, osteoporosis, stiff myelitis, osteoarthritis or rheumatoid arthritis.  28. An effective amount of a joint disease-modulating agent obtained by using the method according to any one of claims 18 to 22 or the screening kit according to claim 23 or 24 to a mammal. A method for preventing and treating joint diseases, characterized in that:  29. A method for evaluating the efficacy of a compound, comprising administering a test compound to a non-human mammal, a joint thereof, a joint tissue thereof, or a cell constituting the joint obtained by the production method according to claim 1. 30. The method according to claim 29, wherein changes in the non-human mammal, its joints, joint tissues, or cells constituting the joints are searched for with and without the administration of the test compound. 31. The method according to claim 29, wherein a function of a non-human mammal, a joint thereof, a joint tissue or a cell constituting the joint is searched for in a case where the test compound is administered and a case where the test compound is not administered.  32. The method of claims 29-31, wherein the test compound is a gene product.  33. A medicament comprising a compound whose efficacy has been evaluated using the method according to any one of claims 29 to 31.  34. The medicament according to claim 33, which is a joint disease regulator.  3 5 .Fractures, refractures, bone deformities and osteoporosis, osteosarcoma, myeloma, osteogenesis imperfecta, scoliosis, bone loss, osteoporosis, osteomalacia, rickets, fibrous osteomyelitis, renal osteoarthritis 35. The pharmaceutical according to claim 34, which is a prophylactic and / or therapeutic agent for nutrition, osteoporosis, stiff myelitis, osteoarthritis or rheumatoid arthritis.  36. An antibody against a gene product, a partial peptide or a salt thereof of which gene or function has been elucidated by the method of claim 8 or 29.  37. An antisense nucleic acid against a gene whose function has been elucidated by the method according to claim 8. 38. A medicament comprising the antibody according to claim 36.  39. A medicament comprising the antisense nucleic acid according to claim 37.  40. The medicament according to claim 38 or 39, which is a joint disease regulator.  4 1 .Fractures, refractures, bone deformities and osteoporosis, osteosarcoma, myeloma, osteogenesis imperfecta, scoliosis, bone loss, osteoporosis, osteomalacia, rickets, fibrous osteomyelitis, renal osteoarthritis 41. The medicament according to claim 40, which is a therapeutic agent for * nutrition, prevention of osteoporosis, osteoarthritis, osteoarthritis, or rheumatoid arthritis.  42. A diagnostic agent for joint disease comprising the antibody according to claim 36. 43. A diagnostic agent for joint disease comprising the antisense nucleic acid according to claim 37. 4 4.Fracture, refracture, bone deformity, osteomyelitis, osteosarcoma, myeloma, osteogenesis imperfecta, scoliosis, bone loss, osteoporosis, osteomalacia, rickets, fibrous osteomyelitis, renal osteoarthritis 43. The diagnostic agent according to claim 42 or 43, which is a diagnostic agent for nutrition, osteoarthritis, osteoarthritis, osteoarthritis, or rheumatoid arthritis.