JP2003125787A - Method for differentiation culture of cartilage - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for easily and quickly obtaining cartilage cells used in cartilage transplantation which is carried out as a surgical technique for restoring the cartilage in the case of arthropathy or the like. SOLUTION: This cartilage differentiation method comprises culturing marrow-derived cells or articular cartilage-derived cells differentiable into the cartilage in a serum-free medium containing TGFβ, BMP or the like under low-oxygen conditions. This method is applicable to screening cartilage differentiation-related genes, arthropathy-related genes and cartilage differentiation promoters or inhibitors.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a cartilage differentiation culture method, a method for producing cartilage cells or cartilage in each differentiation process, and the like.

[0002]

2. Description of the Related Art Cartilage tissue has a poor regenerative power, and once damaged, it tends to progress to joint diseases such as osteoarthritis. When these joint diseases progress further, it is often the case that the joint must be replaced with an artificial joint.
y of life). An autologous cartilage transplantation treatment is being performed to compensate for such a cartilage damage site. This is because a part of cartilage tissue is cut out from a normal cartilage site, cells are taken out from the tissue using an appropriate digestion method, the cells are cultured using some kind of culture instrument, the cells are increased in a considerable amount, and then the original patient's It is to restore the cartilage damage site and to heal it. However, chondrocytes are generally known to undergo so-called dedifferentiation and lose the properties peculiar to cartilage when subjected to plate culture which is usually performed with adherent cells. Therefore, attempts have been made to maintain the cartilage morphology by using a special culture substrate or culture method, but with these culture methods, it is rather unnecessary or adversely affected when returning to the joint part of the patient again. In order to obtain it, improvement in consideration of cost and the like has been strongly desired. In addition, the method of isolating chondrocytes from joint tissue is complicated and burdens the patient, and therefore modification has been desired. Recently, it has been known that mesenchymal stem cells derived from bone marrow can be differentiated into adipocytes, chondrocytes in addition to osteocytes, and mesenchymal stem cells isolated from adult bone marrow extract are undifferentiated. It has been reported that it is possible to differentiate into chondrocytes by proliferating while maintaining the state and then culturing under certain specific conditions. For example, Pittenger et al. (Science. 1999 284: 143-14
7) has found a method of causing cartilage differentiation by precipitating cells in a serum-free medium containing TGF-β and the like by centrifugal force to form a cell mass, and mesenchyme sold by BIOWHITTAKER A similar method for cartilage differentiation is also described in the attached document of stem cells. If it is possible to easily and quickly differentiate into chondrocytes using such cells, it is expected to be applicable to autologous transplantation aiming at healing of joint diseases as described above. However, these methods
It is characterized by cartilage differentiation by high-density culture of cells in a centrifuge tube using a serum-free medium containing TGFβ3, but it has a long period of time until a cartilage marker is expressed, and cells exist in a mass. Therefore, the degree of cartilage differentiation between the inside and the surface is completely different, and it is difficult to disperse the cell clusters.Therefore, it is not easy to move to the next operation for autologous transplantation and other experiments. there were. Lennon
Et al. (J. Cell. Physiol. 2001 187: 345-355) are cultured in a hypoxic environment using rat-derived mesenchymal stem cells,
We reported an increase in the accumulation of markers of bone formation but did not find cartilage differentiation. Moreover, Lennon et al. Used a serum-containing medium, and have not yet identified the molecule having a (bone) differentiation promoting action contained therein. J. Cell Scie
nce (2000) 113, 1161-1166, cloned using human bone marrow stromal fibroblasts as a starting material and reported that some of the clones differentiate into cartilage, bone and adipocytes. There is. However, the progenitor cells of the bone marrow are determined in the direction of differentiation at an early stage, and some cells can differentiate into cartilage, while some cells cannot.

[0003]

DISCLOSURE OF THE INVENTION The present invention provides a cartilage differentiation method with dramatically improved cartilage efficiency, a cartilage differentiation method using the method, a method for searching for a gene or protein associated with a joint disease, a cartilage differentiation medicine for a joint disease. The problem is to provide a search method of.

[0004]

[Means for Solving the Problems] As a result of intensive studies to solve the above problems, the present inventors have added BMP to a serum-free medium containing TGFβ and the like, and further cultured under hypoxic conditions. As a result, it was found that the cartilage efficiency is dramatically improved even when the plate culture method is used. Furthermore, it was also found that, using this culture method, even normal chondrocytes that have been dedifferentiated by plating can start to express cartilage marker specific to chondrocytes again, that is, they can be redifferentiated. By using the culture method described in the present invention, as described above, in the case of autologous transplantation of joint-derived chondrocytes or bone marrow-derived mesenchymal stem cells, the drawbacks thus far are compensated for, and the treatment of the field of joint diseases is performed. In particular, we think that it can bring epoch-making progress to regenerative medicine,
The present invention has been completed.

That is, the present invention provides a method for conveniently and rapidly obtaining chondrocytes used for cartilage transplantation performed as a surgical technique for repairing cartilage in joint diseases and the like. More specifically, the present invention provides (1)
A method for cartilage differentiation, which comprises culturing cells capable of differentiating into cartilage under hypoxic conditions, (2) Bone marrow-derived cells, articular cartilage-derived cells, skin-derived cells, embryo The method according to (1) above, which is a cell or a fetal-derived cell, (3) the method according to (1) above, wherein the cartilage-differentiating cell is a human mesenchymal stem cell or a dedifferentiated chondrocyte, (4) low Oxygen conditions are oxygen partial pressure of about 0.1% to about 10%
(5) TGFβ or BM, which is
The method according to (1) above, which is cultured in the presence of P, (6) The method according to (5) above, which is further cultured in the presence of ascorbic acid, proline, dexamethasone, insulin, transferrin or selenite, (7) The method according to (1) above, which comprises culturing in a serum-free medium, (8)
The method according to (7) above, wherein the serum-free medium contains TGFβ or BMP, (9) the above-mentioned (8) wherein the serum-free medium further contains ascorbic acid, proline, dexamethasone, insulin, transferrin or selenite.
The method described in (1) above, characterized in that (10) a plate culture is performed, and (11) the culture is performed in a serum-free medium containing TGFβ and BMP4 at a hypoxic condition of an oxygen partial pressure of about 1 to 5%. The method according to (1) above, (12) a method for producing chondrocytes or cartilage, which comprises culturing cells capable of differentiating into cartilage under hypoxic conditions, (13) chondrocytes are cartilage precursor cells, and proliferate The method according to (12) above, which is a chondrocyte, a mature chondrocyte or a hypertrophied chondrocyte, (14) above (1).
2) Chondrocytes or cartilage produced by the production method described above,
(15) Chondrocytes or cartilage according to (14) above, which can be cultured on a plate, (16) Chondrocytes or cartilage containing the cartilage produced by the production method according to (12) above, (17)
To transplant an effective amount of the chondrocytes or cartilage produced by the production method according to (12) above into the medicament according to (16) above, which is a preventive / therapeutic agent for joint diseases, and (18) mammals. Preventive and therapeutic methods for characteristic joint diseases, (1
9) The above (1) for producing a prophylactic / therapeutic agent for joint diseases
2) Use of chondrocytes or cartilage produced by the production method described in (2) above, (20) The method described in (1) above or (12) above.
A method for screening a cartilage differentiation-related gene or a joint disease-related gene, which comprises using the manufacturing method described above,
(21) A method of detecting a gene whose expression is increased or decreased in chondrocytes or cartilage in each differentiation process obtained by using the method described in (1) above or the production method described in (12) above. (22) A cartilage differentiation-related gene or a joint disease-related gene obtained by the screening method according to (20) or (21) above, (23) A cartilage differentiation-related according to (22) above A cartilage differentiation-related protein encoded by a gene or a joint disease-related gene, a joint disease-related protein or a salt thereof, (24) The method according to (1) above or (1) above.
2) A method for screening a substance that promotes or inhibits cartilage differentiation, which comprises using the production method described in (2)
5) A method for screening a preventive / therapeutic agent for joint diseases, which comprises using the method described in (1) above or the production method described in (12) above, (26) cartilage differentiation-related in the case of adding a test compound (24) or (2) using the expression level of a gene or a joint disease-related gene as an index
5) The screening method described in (27) above (24)
(28) a substance obtained by the screening method described above which promotes or inhibits cartilage differentiation, (28) a medicament comprising the substance obtained by the screening method described in (24) above which promotes or inhibits cartilage differentiation, (29) a joint disease The drug according to (28) above, which is a preventive / therapeutic agent for (30) above.
5) Prophylactic / therapeutic agent for joint disease obtained by the screening method described in (31), (31) Pharmaceutical comprising the prophylactic / therapeutic agent for joint disease obtained by the screening method described in (25) above, (32) Joint disease To the medicament according to (31) above, which is a prophylactic / therapeutic agent for (3) above, or (33) a mammal, is to administer an effective amount of the substance according to (27) above or the prophylactic / therapeutic agent according to (30) above. A method for preventing and / or treating a joint disease characterized by the following: (34) Use of the substance according to (27) or the prophylactic / therapeutic agent according to (30) for producing a prophylactic / therapeutic agent for a joint disease. .

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The cells used in the method of the present invention may be either purified cells or cells in a mixed state as long as they can differentiate into chondrocytes.
Human or warm-blooded animal cells (eg, guinea pig, rat, mouse, chicken, rabbit, pig, sheep, cow, monkey, etc.) are used. More specifically, human or warm-blooded animal bone marrow-derived cells, articular cartilage-derived cells, skin-derived cells, embryonic cells,
Fetal-derived cells and the like are used, and they can be further separated and classified by the expression of surface antigen (eg, TGFβ receptor) and used. More specifically, as cells that can be differentiated into chondrocytes, human mesenchymal stem cells, dedifferentiated chondrocytes (eg, dedifferentiated human normal chondrocytes) and the like are used. The culture is Transforming Growth Factor β (TGF
β) or (and) Bone Morphogenetic Protein (B
MP) is preferable, and the culture may be performed in the presence of additives such as ascorbic acid, proline, dexamethasone, insulin, transferrin and selenious acid. The medium composition used for culture is a medium for animal cell culture, which is commercially available as a basic medium, and more preferably Dulb.
ecco's Modified Eagle's Medium (DMEM) can be used, and it can also be used by appropriately improving it. Further, a serum medium containing 10% fetal bovine serum or the like can be used, but a basal medium is preferable. It is preferable to add TGFβ or / and BMP to the basal medium, and further additives such as ascorbic acid, proline, dexamethasone, insulin, transferrin and selenious acid may be added. Examples of ascorbic acid include ascorbic acid 2-phosphoric acid. As TGFβ, T
Any one may be used as long as it belongs to the TGFβ family such as GFβ1, TGFβ2 and TGFβ3, and a low molecular compound having the same action as TGFβ can be used for substitution. As BMP, BMP2, BMP4 and the like are used, and among them, BMP4 is the most preferable, and a protein that exhibits the same action as BMP (for example, CDMP) or a low molecular weight compound can be used instead.
Ascorbic acid to be added (preferably ascorbic acid 2-
The amount of phosphoric acid is about 5 to 500 μg / ml, preferably about 10
˜100 μg / ml, more preferably about 50 μg / ml. The amount of proline added is about 5 to 500 μg / ml, preferably about 10 to 10 μg / ml, more preferably about 40 μg.
g / ml. The amount of dexamethasone added is about 1 to
It is 1000 nM, preferably about 10 to 500 nM, more preferably about 100 nM. The insulin, transferrin, and selenous acid to be added are added to a commercially available ITS solution at a concentration of about 0.1 to about 10 times, preferably about 1 time. T to add
The amount of GFβ is about 0.1-100 ng / ml, preferably about 1-50.
ng / ml, more preferably about 10 ng / ml.
The amount of BMP added is about 20 to 2000 ng / ml, preferably about 100 to 500 ng / ml, more preferably about 200 ng / ml.
ml. When a recombinant protein is used as an additive, the above-mentioned addition amount may be changed depending on the degree of purification. For example, if the quality of the recombinant protein is not good and the actual concentration is low, the amount added may be increased.

The above-mentioned cells are treated with a general medium (for example, 10
% Fetal bovine serum-containing DMEM, etc., culture conditions (eg 37 ℃,
After culturing and proliferating with 5% CO ₂ ) or after separating the above cells directly, they are transplanted to the above serum-free medium to start the plate culture. The amount of cells can be from about 10% to 100% confluent, and the culture can be performed in a high-density, multi-layered state exceeding 100% confluent. Immediately after transplantation, or after leaving it for a while, transition to hypoxia. For hypoxic culture, for example, a commercially available nitrogen gas or the like may be mixed to use a low oxygen incubator of a type that lowers the oxygen partial pressure, or nitrogen gas may be blown into an appropriate space to lower the oxygen partial pressure. It is also possible to culture. Oxygen partial pressure is about 0.1%
To about 10%, more preferably about 1% to about 5%, particularly preferably about 3%. The culture temperature is about 25 ° C to about 40 ° C, preferably about 32 ° C to about 38 ° C. p
In order to maintain H, the final concentration should be around 5% CO
You may blow in ₂ gas. Cultivation time is usually about 1 day to about 2 weeks, more preferably about 2 days to about 10 days. Usually, the medium is exchanged for about 1 day to about 1 week, more preferably about 2 days to about 4 days. Culture conditions include TGFβ (eg, TGFβ3, TGFβ2, TGFβ1), BMP (especially BM) in the medium.
P4) is added, and oxygen partial pressure is about 1-5% (especially about 3%)
Is most preferable. In this case, the medium is
A serum-free medium is preferred. More specifically, when human mesenchymal stem cells are used as cells that can differentiate into cartilage,
It is most preferable that TGFβ3 and BMP4 are added and the oxygen partial pressure is 3%. When dedifferentiated chondrocytes (eg, dedifferentiated human normal chondrocytes) are used as cells capable of differentiating into cartilage, TGFβ3 and BMP4 are added to the medium, and the oxygen partial pressure is 3%.
Is most preferable.

When cultured under such conditions, expression of type 2 collagen mRNA, which is a cartilage marker, is usually observed at least 3 days after shifting to hypoxia, and the expression level reaches a peak in about 10 days. Such cartilage markers are for example
It can be confirmed by the expression of mRNA using the RT-PCR method, or can be performed by measuring the amount of protein using an appropriate antibody.

[0009] Chondrocytes are classified into cartilage progenitor cells, proliferating chondrocytes, mature chondrocytes, hypertrophic chondrocytes, etc. according to the degree of differentiation. However, the present invention allows these differentiation processes to be performed more rapidly than conventional methods. Can be reproduced. Further, according to the present invention, since the differentiation process uniformly takes place, the cultured chondrocytes at the required differentiation stage can be used for recovery, treatment, etc. using each differentiation marker as an index. Examples of proliferating chondrocyte markers include aggrecan gene, Type II
Examples include collagen gene. Examples of mature chondrocyte markers include aggrecan gene, Type II collagen gene, and PTHrP receptor gene. Examples of markers of hypertrophic chondrocytes include Indian hedgehog gene. Furthermore, since the chondrocytes or cartilage obtained using the method of the present invention can be cultured on a plate, the cells can be recovered by a simple method such as ordinary trypsin treatment or using a scraper. .

The chondrocytes or cartilage produced by the method of the present invention can be used, for example, in joint diseases (eg, fractures in the orthopedic region, re-fractures, bone deformities / degenerative spondylosis, osteosarcoma, myeloma,
Non-metabolic bone diseases such as osteogenesis imperfections and scoliosis; metabolism of bone defects, osteoporosis, osteomalacia, rickets, fibrous osteomatitis, renal osteodystrophy, bone Pecet's disease, ankylosing myelitis, etc. Bone disease; cartilage disease such as osteoarthritis and rheumatoid arthritis)
Etc., can be used for cartilage transplantation performed as a surgical technique for repairing cartilage. The cartilage transplantation method can be performed using a conventional method. Therefore, chondrocytes or cartilage produced by the method of the present invention,
It can be used as a safe and low-toxicity prophylactic / therapeutic agent for the above-mentioned joint diseases. It is also possible to search for genes related to cartilage differentiation and search for pharmaceuticals using the main culture system. For example, when a recombinant protein such as TGFb or BMP is used as a drug, it has problems in price, stability, administration method, etc., but it is also possible to search for a compound that substitutes the function of these proteins.

Further, the present invention provides (1) a cartilage differentiation-related gene (eg, a cartilage differentiation promoting gene, a cartilage differentiation suppressing gene) characterized by using the cartilage differentiation method or the chondrocyte or cartilage production method of the present invention Or a method A for screening a gene associated with a joint disease (eg, a gene for inducing a joint disease, a gene for suppressing a joint disease), more specifically, (2) a method obtained by using the cartilage differentiation method or the chondrocyte or cartilage production method of the present invention. Provided is a screening method A for a joint disease-related gene, which comprises detecting a gene whose expression is increased or decreased in a chondrocyte or cartilage undergoing differentiation. Examples of the chondrocytes in each differentiation process include the above-mentioned chondrocyte precursor cells, proliferating chondrocytes, mature chondrocytes, and enlarged chondrocytes. A gene whose expression is increased or decreased can be detected using the expression amount of mRNA and the production amount of gene product as indicators. The expression level of mRNA can be determined by a method known per se, such as Northern blotting or Reverse transcription-polymerase chain reaction.
(RT-PCR), TaqMan polymerase chain reaction or the like or a method similar thereto can be used for the measurement. The production amount of the gene product can be measured by a method known per se, for example, a method using an antibody or a method using chromatography. The cartilage differentiation-related gene or joint disease-related gene obtained by this screening method A, or the cartilage differentiation-related protein encoded by it, the joint disease-related protein or a salt thereof is, for example, itself, a joint disease (eg, in the orthopedic area). Non-metabolizing bone diseases such as bone fracture, re-fracture, bone deformity and spondylosis, osteosarcoma, myeloma, osteogenesis imbalance, scoliosis; bone defect, osteoporosis, osteomalacia, rickets, fibrous osteomyelitis, kidney Osteodystrophy,
It can be used for the prevention and treatment of metabolic bone diseases such as bone Pecchet's disease and ankylosing myelitis; cartilage diseases such as osteoarthritis and rheumatoid arthritis), and also promotes or inhibits cartilage differentiation described below. It can also be used in screening method B for preventive / therapeutic agents for substances and joint diseases.

The present invention further provides (1) a method B for screening a substance for promoting or inhibiting cartilage differentiation, which comprises using the method for cartilage differentiation or the method for producing cartilage cells or cartilage of the present invention, and (2) the present invention. A method C for screening a prophylactic / therapeutic agent for joint diseases, which comprises using the method for cartilage differentiation or the method for producing chondrocytes or cartilage of the present invention;
More specifically, (3) the screening method according to (1) or (2) above, wherein the expression level of a cartilage differentiation-related gene or a joint disease-related gene when a test compound is added is used as an index. Specifically, this screening method B or C is a method in which a test compound is added to the chondrocytes or cartilage of each differentiation process obtained using the cartilage differentiation method or the chondrocyte or cartilage production method of the present invention. Promote cartilage differentiation by controlling the expression of cartilage differentiation-related genes or joint disease-related genes by measuring the expression level of cartilage differentiation-related genes, joint disease-related genes, etc., and the cartilage differentiation state without addition Alternatively, it is a method of selecting a substance that inhibits or a substance that prevents / treats a joint disease.

Examples of test compounds include non-peptidic compounds of biological origin (sugars, lipids, etc.), synthetic compounds (including peptides), microbial cultures, cell extracts, plant extracts, animal tissue extracts, genes (CDNA, genomic DN
A) and the like are used, and these compounds may be novel compounds or known compounds. The cartilage differentiation-related gene (eg, cartilage differentiation promoting gene, cartilage differentiation suppressing gene) or joint disease-related gene (eg, joint disease inducing gene, joint disease suppressing gene) may be any new or known gene, For example, Connective Tissue Research (Connect Tissue Res Volume 41,
175-184, 2000), the genes obtained by the screening method A of the present invention described above, and the like. The gene whose expression is increased or decreased is mRN.
The expression level of A and the production level of the gene product can be detected as an index. The expression level of mRNA can be determined by a method known per se,
For example, Northern blotting and Reverse transcript
ion-polymerase chain reaction (RT-PCR) and TaqMan poly
It can be measured according to a method such as merase chain reaction or a method similar thereto. The production amount of the gene product can be measured by a method known per se, for example, a method using an antibody or a method using chromatography. For example, as compared with the case where the test compound is not administered, the mR of the cartilage differentiation suppressor gene when the test compound is administered
When the amount of NA or the amount of gene product is increased by about 20% or more, preferably about 30% or more, more preferably about 50% or more, the test compound can be selected as a substance that suppresses cartilage differentiation, while the test MRN of cartilage differentiation promoting gene when the compound is administered as compared to when the compound is not administered
A amount, gene product amount is about 20% or more, preferably about 30
% Or more, more preferably about 50% or more, the test compound can be selected as a substance that promotes cartilage differentiation. The cartilage differentiation state can be observed under a microscope or the expression of the cartilage differentiation marker gene described above. Quantity (mRN
The amount of A expression and the amount of gene product produced) can be used as indicators.

The substance which promotes or inhibits cartilage differentiation or the prophylactic / therapeutic drug for joint disease, which is obtained by the screening method B or C of the present invention, can regulate joint cartilage differentiation and the like, whereby joint diseases (eg, in the orthopedic area) Non-metabolizing bone diseases such as bone fracture, re-fracture, bone deformity and spondylosis, osteosarcoma, myeloma, osteogenesis imbalance, scoliosis; bone defect, osteoporosis, osteomalacia, rickets, fibrous osteomyelitis, kidney It is possible to prevent and treat metabolic bone diseases such as osteodystrophy, bone Pecet's disease, ankylosing myelitis; cartilage diseases such as osteoarthritis and rheumatoid arthritis. Furthermore, the substance that promotes or inhibits cartilage differentiation or the prophylactic / therapeutic drug for joint diseases obtained by the screening method B or C of the present invention is a bone tissue repair agent after surgery for multiple myeloma, lung cancer, breast cancer and the like. Also, in the field of dentistry, it can be used for treatment of periodontal diseases, repair of periodontal tissue defects in periodontal diseases, stabilization of artificial roots, ridge formation and repair of cleft palate.

When the substance obtained by the screening method B or C of the present invention is used as the above-mentioned preventive / therapeutic agent, it can be formulated by a conventional method.
For example, the substance is a sugar-coated tablet if necessary,
Orally used as capsules, elixirs, microcapsules, etc., or parenterally in the form of injectable solutions such as sterile solutions with water or other pharmaceutically acceptable liquids or suspensions it can. For example, a known physiologically acceptable carrier, flavoring agent, excipient,
It can be prepared by mixing with a vehicle, preservative, stabilizer, binder and the like in a unit dose form generally required for the practice of the preparation. The amount of active ingredient in these preparations is such that a suitable dose within the indicated range can be obtained. Examples of additives that can be mixed with tablets, capsules and the like include gelatin,
Binders such as corn starch, tragacanth, acacia, excipients such as crystalline cellulose, swelling agents such as corn starch, gelatin, alginic acid, lubricants such as magnesium stearate, sucrose, lactose or saccharin. Such sweetening agents, peppermint, red oil or flavoring agents such as cherry are used. When the unit dosage form is a capsule, a liquid carrier such as fat may be included in addition to materials of the above type. Sterile compositions for injection can be formulated according to conventional pharmaceutical practices such as dissolving or suspending the active substance in a vehicle such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As the aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used, and a suitable solubilizing agent is used. , For example, alcohol (eg ethanol),
You may use together with polyalcohol (eg, propylene glycol, polyethylene glycol), a nonionic surfactant (eg, polysorbate 80 ^™ , HCO-50) and the like. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.

Further, the above-mentioned pharmaceutical composition includes, for example, a buffer (eg, phosphate buffer, sodium acetate buffer), a soothing agent (eg, benzalkonium chloride, procaine hydrochloride), a stabilizer ( For example, human serum albumin, polyethylene glycol, etc.), preservatives (eg,
(Benzyl alcohol, phenol, etc.), antioxidant, etc. The prepared injection solution is usually filled in a suitable ampoule. Since the pharmaceutical composition thus obtained is safe and has low toxicity, for example, mammals (for example, humans, rats, mice, rabbits, sheep, pigs,
(Cattle, cats, dogs, monkeys, etc.). The dose of the substance varies depending on the administration subject, target organ, symptom, administration method, etc. However, in the case of oral administration, it is generally used, for example, in patients with rheumatoid arthritis (60 kg).
As), about 0.1 mg to 100 per day
mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc., but usually in the form of injection, for example, rheumatoid arthritis patient (as 60 kg)
In the above, it is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day by intravenous injection. In the case of other animals, the dose converted per 60 kg can be administered.

In the present specification and drawings, when abbreviations are used for bases, amino acids, etc., IUPAC-IUB is used.
It is based on the abbreviations by the Commission on Biochemical Nomenclature or the abbreviations commonly used in this field, and examples are given below. When amino acids may have optical isomers, the L form is shown unless otherwise specified. DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine M: A or CR: G or A W: A or T S: G or CY: T or C K: G Or TV: A or G or CH: A or C or TD: A or G or TB: G or C or TN: A or C or G or T The following sequences are shown. [SEQ ID NO: 1] This shows the DNA sequence of primer 1. [SEQ ID NO: 2] This shows the DNA sequence of primer 2. [SEQ ID NO: 3] This shows the DNA sequence of primer 3. [SEQ ID NO: 4] This shows the DNA sequence of primer 4. [SEQ ID NO: 5] This shows the DNA sequence of primer 5. [SEQ ID NO: 6] This shows the DNA sequence of primer 6. [SEQ ID NO: 7] This shows the DNA sequence of primer 7. [SEQ ID NO: 8] This shows the DNA sequence of primer 8.

[0018]

The present invention will be described in more detail with reference to the following examples, which are not intended to limit the scope of the present invention.

Example 1 Cartilage Differentiation of Human Mesenchymal Stem Cells Human mesenchymal stem cells (BIOWHITTAKER) were used to examine the conditions for cartilage differentiation. Subculture is based on the manual attached at the time of cell purchase, and is based on the medium for human mesenchymal stem cells (BIOWHITTA
KER). The subcultured cells were dispersed by trypsinization, and ascorbic acid was added to DMEM (GIBCO).
Diphosphoric acid (Wako Pure Chemical Industries) 50 μg / ml, L-proline (Wako Pure Chemical Industries) 40 μg / ml, dexamethasone (Wako Pure Chemical Industries) 0.1 μM, insulin, transferrin, selenite etc. ITS + premix liquid (manufactured by BECTON DICKINSON)
Suspend in 100% diluted medium and add 10
The cell solution was prepared so as to have 10,000 cells / ml. 24 this liquid
Dispense 1 ml each into a well plate (Falcon), then add TGFβ
3 (Genzyme) at 10 ng / ml or / and hrBMP4 (R & D
systems) was added to 200 ng / ml to make a group, and the culture was started. Cultivation was carried out by injecting nitrogen gas into a normal atmosphere to adjust the oxygen partial pressure to 3%, or an incubator using a normal atmosphere (both Na
pco) was used, and carbon dioxide gas was blown into each of them at 5% to carry out at 37 ° C. A 24-well plate was collected after 3 days of culture, and RNA was extracted immediately after removing the medium. RNA
Was extracted using RNeasy (QIAGEN) and its protocol was followed. As a primer, a primer 1 having a base sequence represented by SEQ ID NO: 1 and a primer 2 having a base sequence represented by SEQ ID NO: 2 were used. The obtained total RNA was reverse-transcribed using RNA PCR kit (Takara), and then type II collagen which is a cartilage differentiation marker.
Gene expression was confirmed by PCR. As a result,
As shown in, human mesenchymal stem cells were added with TGFβ3 and BMP4,
And when the three conditions of oxygen partial pressure of 3% are complete, most Type II
A band of collagen gene was confirmed, and it was found that cartilage differentiation occurs.

Example 2 Chondrogenic differentiation of dedifferentiated human normal chondrocytes (1) Human normal chondrocytes (Cell Applications, Inc.)
Were cultured according to the manual attached at the time of purchase. As the medium, a dedicated medium (Cell Applications, Inc.) was also used, but with repeated passages, the expression of the type II collagen gene, which is a cartilage differentiation marker, was not observed at all, and as reported in the literature, etc. Were found to be dedifferentiated by plating. Therefore,
The dedifferentiated cells were used to observe the expression of Type II collagen gene on day 3 of culture using the same medium and culture conditions as in Example 1. As a result, even when using dedifferentiated chondrocytes, T
It was found that Type II collagen gene was most expressed and redifferentiated into chondrocytes when the three conditions of addition of GFβ3 and BMP4 and oxygen partial pressure of 3% were complete. (2) In order to examine the factors essential as the redifferentiation conditions for the further dedifferentiated chondrocytes, the conditions for lacking various factors were set from the medium culture conditions described in Example 1, and on the 3rd day of culture. The expression of the Type II Collagen gene was observed. As a result, as shown in FIG. 2, chondrocytes can be efficiently redifferentiated by adding ascorbic acid or dexamethasone in addition to the three conditions of adding TGFβ3 and BMP4 and oxygen partial pressure of 3%. Do you get it.

Example 3 Chondrogenic Differentiation of Human Mesenchymal Stem Cells Using human mesenchymal stem cells (BIOWHITTAKER), 50 μg / ml of ascorbic acid diphosphate and 40 μg of L-proline (Wako Pure Chemical Industries) were added to DMEM. / ml, dexamethasone 100 nM, TGFβ3 10 ng / ml, BMP4 200 ng / ml, insulin, transferrin, selenite etc. ITS + pre
Mix medium (BECTON DICKINSON) was added at a 100-fold dilution to obtain 100,000 cells / ml, and add 1 to 24-well plate.
The solution was dispensed by ml and the culture was started under the condition of oxygen partial pressure of 3%. 3
Start the culture by changing the medium with the same composition every day.
Total RNA was extracted on days 3, 3, 6, 13 and 20. Example
Reverse transcription is performed in the same manner as in 1, and then agglutin (Ag
grecan; SEQ ID NOs: 3, 4), type II collagen (Ty
peII collagen; SEQ ID NO: 1, 2), Indian hedgehog (SEQ ID NO: 5, 6), P
THrP Receptor (Sequence No. 7,8)
PCR using the primers designed based on the sequence of
Agarose gel electrophoresis was performed. Perform ethidium bromide staining to detect the intensity of the detected band as Intrelligent
Comparison was performed using Quantifier software (BIOIMAGE). As a control, a value corrected with the β-actin gene expression level detected in the same manner, and the point with the highest expression level during the course of culturing being 1 is shown in Table 1.

[Table 1] It is known that chondrocytes express a unique gene along with differentiation, and this result is consistent with such findings, and it is possible to faithfully perform cartilage differentiation even in plate culture using the main culture conditions. It was confirmed that it was possible.

Further, using the total RNA extracted above as a material, oligonucleotide microarray (Human Genome U95
A; Affymetrix) was used for gene expression analysis.
The experimental method is described in the Affymetrix experimental guidebook (Expression
analysis technical manual). Sox9, a transcription factor essential for cartilage differentiation (Deng et al., Nat. Genet. 1999
, 22: 85-89) showed a pattern similar to the expression pattern of Aggrecan over time (Table 2).

[Table 2] The value with 1 being the highest expression level during the course of culture is shown. Sox9, which is thought to be involved in cartilage differentiation, also showed an expression pattern very similar to that of Aggrecan. This further confirmed that the main culture system is suitable, and was confirmed to be a very useful system when searching for genes involved in cartilage differentiation and joint diseases by using a microarray or GeneChip. .

Example 4 Using human mesenchymal stem cells (BIOWHITTAKER), 50 μg / ml of ascorbic acid diphosphate and 40 μl of L-proline were added to DMEM.
μg / ml, dexamethasone 100 nM and BMP4 200 ng /
ITS + premix solution (manufactured by BECTON DICKINSON) containing ml, insulin, transferrin, selenite, etc. was added to a 100-fold dilution concentration to obtain 100,000 cells / ml.
Dispense 1 ml each into a well plate, and use TGFβ1, TGFβ2 or TGF
β3 (manufactured by Genzyme) was added to each to 10 ng / ml, and the culture was started under the conditions of oxygen partial pressure of 3% and 37 ° C. Total RNA was extracted 3 days after the start of culture, reverse transcription was performed in the same manner as in Example 1, and PCR was performed using primers designed based on the sequence of Type II collagen (SEQ ID NO: 1, 2). Then, agarose gel electrophoresis was performed. Ethidium bromide staining was performed to compare the expression levels of Type II collagen to see the effect of adding TGFβ (Fig. 3). As a result, T
It was found that GFβ showed a similar effect with any of TGFβ1, TGFβ2 and TGFβ3.

[0024]

According to the method of the present invention, (1) the differentiation process can be reproduced more quickly than the conventional method. Since the differentiation process occurs uniformly, the cultured chondrocytes at the required differentiation stage can be collected and used for treatment by using each differentiation marker as an index. (2) Genes involved in cartilage differentiation and joint diseases Or you can search for proteins, (3)
Compounds used for cartilage differentiation and joint diseases can be searched.

[0025]

[Sequence list]

[Sequence Listing] <110> Takeda Chemical Industries, Ltd. <120> Culture methods of chondrogenic differentiat
ion <130> B02191 <150> JP 2001-193503 <151> 2001-06-26 <160> 8 <210> 1 <211> 24 <212> DNA <213> Artificial sequence <220><223> Primer <400 > 1 ctccccggca ctcctggcac tgat 24 <210> 2 <211> 25 <212> DNA <213> Artificial sequence <220><223> Primer <400> 2 ccttgggcac ctcgggctcc tttag 25 <210> 3 <211> 21 <212> DNA <213> Artificial sequence <220><223> Primer <400> 3 gttcgtggag ggtgttactg a 21 <210> 4 <211> 19 <212> DNA <213> Artificial sequence <220><223> Primer <400> 4 caagcctggt gcctctgtc 19 <210> 5 <211> 24 <212> DNA <213> Artificial sequence <220><223> Primer <400> 5 gaggccggct ttgactgggt gtat 24 <210> 6 <211> 20 <212> DNA <213> Artificial sequence <220><223> Primer <400> 6 ctttgtgagc ggggcgtagg 20 <210> 7 <211> 21 <212> DNA <213> Artificial sequence <220><223> Primer <400> 7 cgcgccgtga gcatcttcgt c 21 <210> 8 <211> 17 <212> DNA <213> Artificial sequence <220><223> Primer <400> 8 ccggccggcg ttggtct 17

[Brief description of drawings]

[Fig. 1] Ty when human mesenchymal stem cells are differentiated under each condition
The result of having confirmed the expression level of peII collagen gene by PCR is shown. Lane 1 is differentiation medium (DM), lane 2 is TGF
β3-non-added DM, lane 3 is ascorbic acid-free D
M shows the results of culturing in lane 4, DM without dexamethasone, lane 5 in DM without BMP-4, and lane 6 in DM. Lane 7 is a molecular weight marker (Takara
(Made by the company). Lanes 1 to 5 show the results of culturing at an oxygen partial pressure of 3%, and lane 6 shows the results at a normal oxygen partial pressure.

[Fig. 2] Results of PCR confirmation of the expression level of Type II collagen gene when human dedifferentiated chondrocytes were redifferentiated under each condition.
(Upper), and βActin gene expression (lower) subjected to PCR as a control are shown. Lanes 1 to 6 have normal oxygen concentration and lanes 7 to 12 have 3% culture. Lanes 1 and 7 are media containing all the additives described in Example 1, lanes 2 and 8 are media containing the additives described in Example 1 except TGFβ3, and lanes 3 and 9 are Examples. A medium containing ascorbic acid from the additive described in 1.
Lanes 4 and 10 contain a medium containing the additive described in Example 1 without insulin, transferrin and selenite, and Lanes 5 and 11 contain the additive described in Example 1 without dexamethasone. The medium, lanes 6 and 12, show the results of culturing in the medium containing the additive described in Example 1 except that BMP4 was removed. Lane M shows a molecular weight marker (Takara).

[Fig. 3] Ty when human mesenchymal stem cells are differentiated under each condition
The result of having confirmed the expression level of the peII collagen gene by PCR, and the βActin gene expression which was subjected to PCR as a control are shown. Lane 1 is TGFβ-free medium, lane 2 is TGF
Lane 1 shows the results of culture with β1-added medium, lane 3 with TGFβ2-added medium, and lane 4 shows the results of culture with TGFβ3-added medium. lane
0 indicates a molecular weight marker (Takara).

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Claims (34)

[Claims] 1. A method for cartilage differentiation, which comprises culturing cells capable of differentiating into cartilage under hypoxic conditions. 2. The cells capable of differentiating into cartilage are derived from bone marrow,
The method according to claim 1, which is a cell derived from articular cartilage, a cell derived from skin, an embryonic cell, or a cell derived from a fetus. 3. The method according to claim 1, wherein the cells capable of differentiating into cartilage are human mesenchymal stem cells or dedifferentiated chondrocytes. 4. A hypoxic condition is an oxygen partial pressure of about 0.1% to about 10.
%. The method of claim 1 which is%. 5. The method according to claim 1, which comprises culturing in the presence of TGFβ or BMP. 6. The method according to claim 5, further comprising culturing in the presence of ascorbic acid, proline, dexamethasone, insulin, transferrin or selenite. 7. The method according to claim 1, which comprises culturing in a serum-free medium. 8. The method according to claim 7, wherein the serum-free medium contains TGFβ or BMP. 9. The method according to claim 8, wherein the serum-free medium further contains ascorbic acid, proline, dexamethasone, insulin, transferrin or selenious acid. 10. The method according to claim 1, wherein plating is performed. 11. A hypoxic condition is an oxygen partial pressure of about 1 to 5%, and TG
The method according to claim 1, wherein the culture is performed in a serum-free medium containing Fβ and BMP4. 12. A method for producing cartilage cells or cartilage, which comprises culturing cells capable of differentiating into cartilage under hypoxic conditions. 13. The chondrocyte is a chondrocyte precursor cell, a proliferating chondrocyte,
The method according to claim 12, wherein the chondrocytes are mature chondrocytes or hypertrophied chondrocytes. 14. A chondrocyte or cartilage produced by the production method according to claim 12. 15. The chondrocyte or cartilage according to claim 14, which can be cultured on a plate. 16. A medicament containing chondrocytes or cartilage produced by the production method according to claim 12. 17. A preventive / therapeutic agent for joint diseases.
6. The medicine according to 6. 18. A method for preventing or treating a joint disease, which comprises transplanting an effective amount of chondrocytes or cartilage produced by the production method according to claim 12 into a mammal. 19. Use of chondrocytes or cartilage produced by the production method according to claim 12 for producing a prophylactic / therapeutic agent for joint diseases. 20. A method for screening a cartilage differentiation-related gene or a joint disease-related gene, which comprises using the method according to claim 1 or the production method according to claim 12. 21. A gene whose expression level is increased or decreased is detected in chondrocytes or cartilage in each differentiation process obtained by using the method according to claim 1 or the production method according to claim 12. The screening method according to claim 20. 22. A cartilage differentiation-related gene or a joint disease-related gene obtained by the screening method according to claim 20 or 21. 23. A cartilage differentiation-related protein encoded by the cartilage differentiation-related gene or joint disease-related gene according to claim 22, a joint disease-related protein or a salt thereof. 24. A method for screening a substance that promotes or inhibits cartilage differentiation, which comprises using the method according to claim 1 or the production method according to claim 12. 25. Prevention of joint diseases, characterized by using the method according to claim 1 or the production method according to claim 12.
Therapeutic drug screening method. 26. The screening method according to claim 24 or 25, wherein the expression level of a cartilage differentiation-related gene or a joint disease-related gene when a test compound is added is used as an index. 27. A substance which promotes or inhibits cartilage differentiation, which is obtained by the screening method according to claim 24. 28. A medicine comprising a substance which promotes or inhibits cartilage differentiation, which is obtained by the screening method according to claim 24. 29. A preventive / therapeutic agent for joint diseases.
8. The medicine according to 8. 30. A preventive / therapeutic agent for joint diseases, which is obtained by the screening method according to claim 25. 31. A medicine comprising a prophylactic / therapeutic agent for a joint disease, which is obtained by the screening method according to claim 25. 32. A preventive / therapeutic agent for joint diseases.
The medicine according to 1. 33. A method for preventing / treating a joint disease, which comprises administering an effective amount of the substance according to claim 27 or the prophylactic / therapeutic drug according to claim 30 to a mammal. 34. The substance according to claim 27 or the prophylaxis according to claim 30 for producing a prophylactic / therapeutic agent for a joint disease.
Use of therapeutic drugs.