JP3737532B2 - Cartilage disorder treatment - Google Patents

Description

[0001]
[Industrial application fields]
The present invention relates to a drug useful for the treatment and prevention of cartilage disease, and more particularly to a cartilage disorder therapeutic agent, a chondrocyte proliferation promoter and a proteoglycan production promoter containing HGF (Hepatocyte Growth Factor) as an active ingredient.
[0002]
[Prior art]
Cartilage is a connective tissue composed of chondrocytes and the surrounding matrix, and is present in joints, spinal discs, costal cartilage, pinna, external auditory canal, pubic bone, pharyngeal palate and the like. Cartilage is composed of chondrocytes and cartilage matrix produced by chondrocytes. The cartilage matrix is mainly composed of fiber components such as collagen fibers, proteoglycan and water. Cartilage is composed of hyaline cartilage (肋Cartilage, throat cartilage, articular cartilage, etc.), elastic cartilage (auricular cartilage etc.) and fibrocartilage (intervertebral disc cartilage, pubic cartilage, articular cartilage etc.). In the above cartilage matrix, collagen fibers are involved in rigidity and strength against cartilage tension and shear force, proteoglycan is involved in strength against compressive force, and water is considered to be a characteristic of viscoelastic body as a living tissue. For example, in the case of articular cartilage, 78.6% of the cartilage mass is water, collagen is 20%, and proteoglycan is 7%.
Examples of the action of cartilage include reduction of friction at the epiphysis (interchondral cartilage), retention of elasticity (auricular cartilage, etc.), and motor function (costal cartilage, pubic cartilage, etc.).
[0003]
[Problems to be solved by the invention]
As described above, cartilage has an important effect on maintaining the function of the living body, and various diseases caused by cartilage disorders have been conventionally known, such as cartilage dysplasia, osteoarthritis, deformity, etc. Examples include intervertebral disc disease and fracture repair / healing failure. In particular, the number of patients with joint disorders has increased remarkably due to the arrival of an aging society, the increase of trauma due to sports, the appearance of occupational diseases such as key puncher disease, and the advancement of medical care in this area is desired.
Various treatments have been tried to treat cartilage disorders, but they are not intended to directly eliminate the cause, but for example, by administering an anti-inflammatory agent or the like, It was only symptomatic such as a method of suppressing disorders such as pain based on disease; a method of injecting hyaluronic acid preparations into joints to lubricate joint movement.
Thus, no radical cure for joint disorders has been found, and in particular, osteoarthritis has a large number of patients, and an effective treatment method is eagerly desired.
[0004]
As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that HGF has an action of promoting the proliferation of chondrocytes and promoting the production of proteoglycans and treating various diseases caused by cartilage disorders. Thus, the present invention was completed. The above HGF is a protein found as a factor that proliferates hepatocytes in vitro (Biochem Biophys Res Commun,122, 1450, 1984, Proc. Natl. Acad. Sci. USA,83, 6489, 1986, FFBS Letter,twenty two, 311, 1987, Nature,342, 440, 1989, Proc. Natl. Acad. Sci. USA,87, 3200, 1990). HGF, which was discovered as a factor that specifically proliferates liver parenchymal cells, exhibits various activities such as tissue injury healing in vivo according to recent research results by many researchers including the present inventors. As a result, it is expected to be applied not only as a research target but also to therapeutic drugs for humans and animals.
It has been elucidated that HGF is mainly produced by mesenchymal cells, and it has been clarified that a so-called paracrine mechanism in which HGF is supplied from neighboring cells as needed is established. However, when the liver or kidney is injured, the production of HGF is also increased in non-injured organs, such as the lung, so it is considered that HGF is also supplied by the so-called endocrine mechanism.
Regarding such HGF receptors, recent studies have established that the c-Met proto-oncogene encodes the HGF receptor (Bottaro et al., Science251, 802-804, 1991; Naldini et al., Oncogene6, 501-504, 1991).
As described above, a lot of knowledge about HGF has been obtained. However, the chondrocyte proliferation promoting action and proteoglycan production promoting action of HGF are novel findings that have not been known so far, and the present invention made based on such findings. Is to provide a drug useful for the treatment of various diseases caused by cartilage disorders.
[0005]
[Means for Solving the Problems]
The present invention made to solve the above problems
(1) A therapeutic agent for cartilage disorder characterized by containing HGF as an active ingredient;
(2) A chondrocyte proliferation promoter comprising HGF as an active ingredient;
(3) A proteoglycan production promoter characterized by containing HGF as an active ingredient.
[0006]
As the HGF used in the present invention, those prepared by various methods can be used as long as they are purified to the extent that they can be used as pharmaceuticals.
Various methods are known as methods for preparing HGF. For example, extracted and purified from liver, spleen, lung, bone marrow, brain, kidney, placenta and other organs such as rats, cows, horses and sheep, blood cells such as platelets and leukocytes, plasma and serum (FEBS Letters,224, 312, 1987, Proc. Natl. Acad. Sci. USA,86, 5844, 1989 etc.).
Further, HGF can also be obtained by culturing primary cultured cells or established cells that produce HGF, and separating and purifying them from the culture (culture supernatant, cultured cells, etc.). Alternatively, the gene encoding HGF may be incorporated into an appropriate vector by genetic engineering techniques, transformed into a suitable host, and the desired recombinant HGF may be obtained from the culture supernatant of the transformant. (E.g. Nature,342, 440, 1989, JP-A-5-111383, Biochem. Biophys. Res. Commun.,163, 967, 1989, etc.). The host cell is not particularly limited, and various host cells conventionally used in genetic engineering techniques such as Escherichia coli, Bacillus subtilis, yeast, filamentous fungi, plant or animal cells can be used.
[0007]
More specifically, as a method for extracting and purifying HGF from a living tissue, for example, carbon tetrachloride is intraperitoneally administered to a rat, and the liver of a rat in a hepatitis state is extracted and pulverized, and S-sepharose, heparin The protein can be purified by usual protein purification methods such as gel column chromatography such as Sepharose and HPLC.
In addition, using gene recombination methods, an animal cell such as a Chinese hamster ovary (CHO) cell, mouse C127 cell, an expression vector in which a gene encoding the amino acid sequence of human HGF is incorporated into a vector such as bovine papillomavirus DNA, Monkey COS cells and the like can be transformed and obtained from the culture supernatant.
[0008]
As long as the HGF thus obtained has substantially the same effect as HGF, a part of the amino acid sequence is deleted or substituted with another amino acid, a part of the other amino acid sequence is inserted, or NGF One or more amino acids may be bonded to the terminal and / or the C terminal, or the sugar chain may be similarly deleted or substituted.
[0009]
The therapeutic agent and the promoter of the present invention contain the above-mentioned HGF as an active ingredient, and HGF has an action of promoting the growth of chondrocytes and promoting the production of proteoglycans as shown in Test Examples described later. Further, HGF has the advantage that it does not show any effect on unaffected cartilage tissue, and acts only on the damaged cartilage tissue, so that it is less likely to cause side effects. Therefore, the therapeutic agent and the promoter of the present invention are effective for treatment / prevention of various diseases caused by cartilage disorders, and for example, the following diseases are included.
[0010]
Osteoarthritis
Cartilage dysplasia
Fracture healing and repair
Repair of articular cartilage and disc due to trauma
Acute suppurative arthritis
Tuberculosis arthritis
Syphilitic arthritis
Rheumatoid arthritis
Rheumatic fever, systemic lupus erythematosus
Degenerative spondylosis
Intervertebral hernia
Bone graft repair
[0011]
The therapeutic agent and promoter of the present invention are applied to the treatment and prevention of various diseases caused by cartilage disorders in mammals (eg, cows, horses, pigs, sheep, dogs, cats, etc.) in addition to humans.
[0012]
The therapeutic agent and accelerator of the present invention can take various preparation forms (for example, liquids, solids, capsules, etc.), but in general, only the active ingredient HGF or a conventional carrier and injection, inhalation Suppository, suppository or oral preparation. The injection can be prepared by a conventional method, for example, HGF is dissolved in an appropriate solvent (for example, sterilized water, buffer solution, physiological saline, etc.), then filtered through a filter or the like, and sterilized. It can then be prepared by filling a sterile container. The HGF content in the injection is usually adjusted to about 0.0002 to 0.2 (W / V%), preferably about 0.001 to 0.1 (W / V%). Oral medicines are formulated into dosage forms such as tablets, granules, fine granules, powders, soft or hard capsules, solutions, emulsions, suspensions, syrups, etc. It can be prepared according to the conventional method of chemical conversion. Suppositories can also be prepared by a conventional method using a conventional base (for example, cacao butter, lauric butter, glycerogelatin, macrogol, witepsol). Inhalants can also be prepared according to conventional means on pharmaceutical preparations.
The HGF content in the preparation can be appropriately adjusted according to the dosage form, applicable disease and the like.
[0013]
In formulating, a stabilizer is preferably added, and examples of the stabilizer include albumin, globulin, gelatin, glycine, mannitol, glucose, dextran, sorbitol, ethylene glycol and the like. Furthermore, the preparation of the present invention may contain additives necessary for formulation, such as excipients, solubilizers, antioxidants, soothing agents, tonicity agents and the like. In the case of a liquid preparation, it is desirable to store it after removing moisture by freeze storage or freeze drying. The freeze-dried preparation is used by re-dissolving and adding distilled water for injection at the time of use.
[0014]
The therapeutic agent and promoter of the present invention can be administered by an appropriate administration route according to the formulation form. For example, it can be administered in the form of an injection into vein, artery, subcutaneous, intramuscular or the like. The dosage is appropriately adjusted according to the patient's symptoms, age, body weight, etc., but is usually 0.05 mg to 500 mg, preferably 1 mg to 100 mg as HGF, which is administered once to several times a day. Is appropriate.
[0015]
【The invention's effect】
In the present invention, HGF, which is an active ingredient, has an action of promoting chondrocyte proliferation and promoting the production of preteoglycan. Therefore, the therapeutic agent and the promoter of the present invention are useful for the treatment / prevention of various diseases caused by the above-mentioned cartilage disorder. Furthermore, since HGF acts only on damaged cartilage tissue, it has an effect that a drug with few side effects can be obtained.
[0016]
【Example】
EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example and a manufacture example, this invention is not limited to these examples. The materials and methods used in the following experiments are as follows.
[0017]
Materials and methods
▲ 1 ▼ in situ Hybridization
Rat HGF-cDNA (RBC1 clone) (Proc. Natl. Acad. Sci. USA,87, 3200, 1990) was subcloned into pGEM7 vector [a-³⁵Antisense and sense RNA probes labeled with S] UTP (400 Ci / mmol, Amersham) were prepared. The labeled transcript was alkaline hydrolyzed to 50-150 bases as a riboprobe.
In situ hybridization is described in the literature (Biochem. Biophys. Res. Commun.,173, 42, 1990). The sample was fixed with 4% paraformaldehyde-phosphate physiological saline solution, dehydrated with ethanol, washed with toluene, and embedded in paraffin. A 5 μm section was cut out and mounted on a slide glass coated with poly-L-lysine. The sections were deparaffinized with glycine and acetic anhydride and hybridized with the probe at 50 ° C. for 16 hours. Thereafter, the sections were washed with 0.1 × SSC solution at 50 ° C. for 1 hour, treated with RNAase A (20 μg / ml) at 37 ° C. for 30 minutes, and then washed twice with 2 × SSC solution at 37 ° C. for 10 minutes. did. The sections were immersed in emulsion (1: 1 Kodak NBT-2 dilution) and exposed for 2 weeks. The sections were developed and fixed on Kodak D-19 and stained with hematoxylin and eosin.
[0018]
(2) Cells and cell culture
Chondrocytes are described in the literature (J. Cell. Physiol.,133, 491, 1987) from New Zealand White rabbits from 23-day-old fetuses and 4-week-old newborns. Articular cartilage was isolated from femoral articular cartilage of the knee, and costal cartilage was isolated from hyaline cartilage of the ribs (Dev. Biol.,136, 500, 1989). Synovial fibroblasts were isolated from synovial tissue of the knee joint. The minced synovial tissue fragments were cultured in DMEM containing 10% FBS for 10 days, and cells grown by trypsin treatment were collected. Literature (Exp. Cell Res.,157, 483, 1985), embryonic mesenchymal cells were isolated from the leg muscle tissue of 20-day-old rat fetuses. Leg bud mesenchymal cells were isolated from 10.5 day old rat fetuses. The leg buds were cut out under a surgical microscope, treated with 0.25% trypsin in 30 minutes, pipetted, and isolated cells were obtained with nylon gauze. All cells other than leg bud cells were treated with DMEM (hereinafter referred to as medium A) containing 10% FBS and 60 μg / ml kanamycin at 37 ° C., 5% CO 2.₂/ 95% maintained under air.
[0019]
(3) Measurement of DNA synthesis
The rate of DNA synthesis is reduced to 10% TCA insoluble cell pellet [^ThreeH] -thymidine ([6-^ThreeH] -thymidine, Amersham, 20 Ci / mmol) (J. Clin. Invest.,85, 626, 1990). Cells are 1.5 x 10 per 6 mm well in a 96 well plate.^FourThe seeds were seeded at a density of 1, and cultured until confluent. To stop growth, cells were preincubated with 0.1 ml of DMEM containing 0.3% FBS. Various concentrations of HGF were added to the medium. Incubation continued for 24 hours. 1 μCi / ml [^Three-H] thymidine was added 3 hours before the incubation was stopped. After labeling, the cells were washed three times with ice-cold PBS, twice with 5% TCA containing 3 mM thymidine, and once with ethanol: dimethyl ether (3: 1). The residue in the well was solubilized with 100 μl of 0.1N NaOH, transferred to a liquid scintillation vial, neutralized with 1N HCl, and then the radioactivity was measured with a scintillation counter (Rack-beta, Pharmacia).
[0020]
(4) Measurement of proteoglycan synthesis
Chondrocytes are 1.5 x 10 per 6mm well^FourThe seeds were seeded at a density of 1, and maintained in 0.1 ml of medium A. When cells reached confluence, they were preincubated with 0.1 ml DMEM containing 0.3% FBS for 24 hours. Thereafter, the cells were incubated for 24 hours with 0.1 ml of DMEM containing 0.3% FBS and HGF. 1 μCi / ml [³⁵S] -sulfate groups were added 20 hours before the end of incubation. Proteoglycan synthesis can be performed on the precipitate with cetylpyridinium chloride after protease digestion [³⁵S] -sulfate group uptake was measured (Exp. Cell Res.,130, 73, 1980).
[0021]
(5) Total RNA preparation and reverse transcription PCR
Total RNA from cartilage can be obtained from literature (Anal. Biochem.,203, 352, 1992). Freshly isolated tissue fragments (0.1 g wet weight) were quickly homogenized with 2 ml of 4M GITC solution of 4M guanidine thiocyanate, 0.1M Tris hydrochloric acid (pH 7.5), 1% 2-mercaptoethanol. The homogenate was mixed with 100 μl of 10% SDS and centrifuged for 5 minutes in a microcentrifuge. 2 ml of the supernatant was overlaid with the same volume of 1.6 g cesium trifluoroacetate and 1 mM EDTA (pH 8.0) in a Beckman polyallomer centrifuge tube (13 × 51 mm). The sample was centrifuged at 35,000 rpm (147,000 × g) at 18 ° C. for 20 hours. After removing the supernatant by aspiration, the precipitate was dissolved in 200 μl of 4M GITC solution, extracted with phenol: chloroform: isoamyl alcohol (25: 24: 1), mixed with 20 μl of 3M sodium acetate (pH 4.8), and doubled. Precipitated with a volume (440 μl) of ethanol. The sediment was dissolved in DEPC-treated water.
First, first strand cDNA was synthesized from 0.5 μg total RNA using SuperScript reverse enzyme (Gibco-BRL) and a downstream antisense primer. Subsequent PCR growth was performed. Growth was performed under conditions of 35 cycles (in the case of chondrocytes) or 40 cycles (in the case of cartilage tissue) at 94 ° C. for 30 seconds, 58 ° C. for 1 minute, and 72 ° C. for 1.5 minutes. Primer base sequences for PCR growth are rat and mouse c-Met (Oncogene,2, 593, 1988), a 725 bp fragment was prepared with 5'-CAGT (A / G) ATGATCTCAATGGGCAT-3 'and 5'-AATGCCCTCTTCCTATACTACT-3'.
[0022]
Example 1
HGF mRNA expression in the developing limb
Expression of HGF mRNA in the limb buds of nascent mice was examined by in situ hybridization. The results are shown in FIG. 1, FIG. 2 and FIG.
FIG. 1 shows the expression of HGF mRNA in the limb buds of early-early mice, and is a photomicrograph of a longitudinal section of the hind limb. It was taken after the graphic and staining. In the figure, A to D are sections of a 10.5 day old fetus, and E to H are sections of an 11 day old fetus.
FIG. 2 shows HGF mRNA expression in the limb buds of finger-forming mice, and is a photomicrograph of a longitudinal section of the hind limb. The bright field (left side) and the corresponding dark field (right side) are in situ hybridization and autoradio. It was taken after the graphic and staining. In the same figure, A and B are 12.5 day old fetuses, C to F are 13 day old fetuses, and G to J are 14 day old fetus sections. Fe is the femur, Fi is the rib, Ta is the tarsal bone, and I to V are finger numbers.
FIG. 3 is a photomicrograph showing the expression of HGF mRNA in the limb bud and thorax of the nascent mouse. The bright field (left side) and the corresponding dark field (right side) were taken after in situ hybridization, autoradiography and staining. Is. In the figure, A and B are cross-sectional sections of the hind limbs of a 16-day-old fetus; C and D are longitudinal sections of the thorax of a 13-day-old fetus, and E and F are 14-day-old fetuses. Ta represents tarsal bone, Ti represents tibia, and Rib represents prechondral accumulation of radial cartilage.
[0023]
As shown in FIG. 1, diffuse expression of HGF mRNA was detected around the bottom region of the limb on day 11. At this stage, no cartilage accumulation occurred in the limbs. As cartilage accumulation proceeds, the expression site of HGF mRNA has become more limited. HGF mRNA expression was observed in the wrist / heel and elbow / knee joint regions when the basal limbs, joined legs, and self-legs were formed on day 12.5 (see FIGS. 2A and B. For convenience, knees). And 踝). In the late phase (13-14 days), HGF mRNA was expressed in adjacent and limited mesenchymal cells of the cartilage accumulation in the wrist / heel and elbow / knee joint regions (see FIGS. 2C-J). On day 16, HGF mRNA was localized to limited mesenchymal cells adjacent to tarsal cartilage (see FIGS. 3A and B). The level of HGF mRNA expression in the extremities decreased with differentiation. No HGF mRNA was detected on the growth plates of the limbs throughout the study.
[0024]
Example 2
HGF mRNA expression in the developing thorax
Expression of HGF mRNA in the thorax of nascent mice was examined by in situ hybridization. The results are shown in FIGS.
As shown in FIGS. 3C-F, HGF mRNA was expressed in the surrounding intercostal mesenchymal tissue at the tip of the elongated prochondral cluster. No hybridization signal was detected in prechondral accumulation.
[0025]
Example 3
Scatter activity test of HGF on chondrocytes
Chondrocytes from knee and cartilage cartilage, synovial cells from knee joint, and fibroblasts grown from limb muscle tissue to determine which cells are the target cells of HGF around the differentiated cartilage tissue And the effect of exogenously added HGF on these cells was examined.
That is, 3 × 10 rabbit articular chondrocytes in a 16 mm well^ThreeSeeded at cell density and maintained in medium A for 2 days. Thereafter, treatment with HGF was performed for 2 days. At the end of the incubation, a phase contrast photomicrograph was taken. The result is shown in FIG.
As shown in FIG. 4, polygonal chondrocytes proliferated into islands when HGF was not treated (control) (FIG. 4A). On the other hand, in the culture treated with HGF (3 ng / ml), chondrocytes did not form islands in a single cell state (FIG. 4B). Thus, HGF was found to stimulate chondrocyte migration. HGF was not dispersed in fibroblasts and synoviocytes.
[0026]
Example 4
Effect of HGF on chondrocyte proliferation
The effect of HGF on chondrocyte proliferation was examined.
That is, articular chondrocytes collected from 4-week-old rabbits were cultured. The confluent cells were serum-removed for 24 hours and then treated with various concentrations of HGF, according to the methods described in the Materials and Methods section [^ThreeH] -thymidine incorporation was measured. A similar test was performed on rabbit synovial fibroblasts. The results are shown in FIGS. 5A (articular chondrocytes) and B (synovial fibroblasts). In addition, a result shows the average value +/- standard deviation of 3 times of a test (it is the same also in FIG. 5C, FIG. 6 and FIG. 7A and B).
As shown in FIG. 5A, HGF is directed to rabbit articular chondrocytes [^ThreeIt was shown that the uptake of H] -thymidine was increased in a dose-dependent manner and promoted DNA synthesis, that is, the effect of promoting proliferation on articular chondrocytes. A 3 fold increase in DNA synthesis relative to the control was observed at 1 ng / ml HGF. On the other hand, as shown in FIG. 5B, synovial fibroblasts did not respond to HGF.
[0027]
In another experiment, the effect of HGF on the number of articular cartilage cells was examined.
That is, 1 x 10 rabbit articular chondrocytes in a 16 mm well^FourCells were seeded and maintained in DMEM medium containing 10% FBS. Subsequently, 10 ng / ml HGF was added and incubated for 48 hours. After the incubation was completed, the number of cells was measured. The result is shown in FIG. 5C.
As shown in FIG. 5C, 10 ng / ml HGF increased the cell number by approximately 1.8 times compared to the control.
[0028]
Example 5
Effect of HGF on proteoglycan production
As described above, HGF promoted chondrocyte proliferation. Next, the effect of articular chondrocytes on proteoglycan production was examined by the method described in the above section of materials and methods. Proteoglycan synthesis can be achieved by converting the protein into a macromolecule (glycosaminoglycan) that precipitates with cetylpyridinium chloride after protease digestion.³⁵S] -Sulfate group uptake measurement (Exp. Cell Res.,130, 73, 1980). Note that the following factors were also tested instead of HGF.
Insulin-like growth factor (IGF) -I: concentration 100 ng / ml
IGF-II: concentration 100 ng / ml
Parathyroid hormone (PTH): concentration 10^-7M
TGF-β: concentration 3 ng / ml
The result is shown in FIG. As shown in FIG. 6, HGF is dose-dependent [³⁵Increased uptake of S] -sulfate groups. Maximum increase was obtained with 1 ng / ml HGF. This effect is exhibited by TGF-β (J. Cell Physiol.,138, 329, 1989) and PTH (J. Clin. Invest.,85, 626, 1990), but similar to IGF-I and II (Exp. Cell Res.,130, 73, 1980).
[0029]
Example 6
Effect of HGF on DNA synthesis and proteoglycan production in the presence of anti-HGF antibody
As described above, HGF is generally considered to act on target cells by the paracrine mechanism, and the results of the in situ hybridization described above seem to support this idea. Therefore, in order to confirm this point, it was examined whether an HGF polyclonal antibody changes the function of chondrocytes.
That is, confluent rabbit articular chondrocytes were treated or not treated with 25 μg / ml anti-HGF polyclonal antibody (affinity-purified IgG fraction) in the presence or absence of 3 ng / ml HGF. Then, according to the method shown in the above-mentioned materials and methods,^ThreeH] -thymidine or [³⁵Labeled with S] -sulfate groups and measured for DNA synthesis or proteoglycan production. The results are shown in FIGS. 7A (DNA synthesis) and B (proteoglycan production). In the figure, Ab represents an anti-HGF polyclonal antibody.
As shown in FIG. 7, the addition of anti-HGF polyclonal antibody alone did not change DNA synthesis or proteoglycan production in articular chondrocytes. However, the anti-HGF polyclonal antibody completely inhibited the effect of exogenously added HGF. This indicates that chondrocytes do not produce enough HGF to regulate the function of the cartilage itself.
[0030]
Example 7
Expression of HGF receptor mRNA in chondrocytes
The expression of HGF receptor (c-Met) in chondrocytes in vivo and in vitro was examined by reverse transcription PCR. Joint tissue and costal cartilage sections were excised from 4 week old rats under a surgical microscope and total RNA was extracted as described in Methods and Materials section. The extracted RNA (0.5 μg) was reverse transcribed, amplified using c-Met primers, and then analyzed by 1.5% agarose gel electrophoresis. The result is shown in FIG.
As shown in FIG. 8, a small amount of c-Met expression was detected after 40 times amplification of articular cartilage tissue and costal cartilage tissue, and cultured chondrocytes showed significant c-Met expression after 35 times amplification. Admitted.
[0031]
Production Example 1
Production example of HGF preparation
(1) HGF 20μg
Human serum albumin 100mg
The above substance was dissolved in 0.01 M PBS having a pH of 7.0, and the total volume was adjusted to 20 ml. After sterilization, 2 ml each was dispensed into a vial, and freeze-dried and sealed.
(2) HGF 40μg
Tween 80 1mg
Human serum albumin 100mg
The above substance was dissolved in physiological saline for injection, and the total volume was adjusted to 20 ml. After sterilization, 2 ml was dispensed into vials, and freeze-dried and sealed.
[Brief description of the drawings]
FIG. 1 is a photomicrograph (form of organism) showing the expression of HGF mRNA in the limb bud of an early-onset mouse (left side is bright field, right side is corresponding dark field). In the figure, A to D are 10.5 day-old fetuses, and E to H are longitudinal section slices of 11-day-old fetuses.
FIG. 2 is a photomicrograph (form of organism) showing the expression of HGF mRNA in the limb buds of finger-forming mice (left side is bright field, right side is corresponding dark field). In the same figure, A and B are 12.5 day old fetuses, C to F are 13 day old fetuses, and G to J are 14 day old fetus sections. Fe is the femur, Fi is the rib, Ta is the tarsal bone, and I to V are finger numbers.
FIG. 3 is a photomicrograph (form of organism) showing the expression of HGF mRNA in the limb bud and thorax of a developing mouse (the left side is bright field, the right side is the corresponding dark field). In the figure, A and B are cross-sectional sections of the hind limbs of a 16-day-old fetus; C and D are 13-day-old fetuses, and E and F are longitudinal sections of the thorax of a 14-day-old fetus. Ta represents tarsal bone, Ti represents tibia, and Rib represents prechondral accumulation of radial cartilage.
FIG. 4 is a photomicrograph (organism morphology) showing the scatter activity of HGF on chondrocytes. In the same figure, A shows control (HGF non-treatment) and B shows HGF treatment.
FIG. 5 shows the effect of HGF on chondrocyte proliferation. In the figure, A shows the effect on articular chondrocyte DNA synthesis, B shows the effect on synovial cell DNA synthesis, and C shows the effect on articular chondrocyte proliferation (cell number).
FIG. 6 shows the effect of HGF on proteoglycan production.
FIG. 7 shows the effect of HGF on DNA synthesis (FIG. 7A) and proteoglycan production (FIG. 7B) in the presence of anti-HGF antibody.
FIG. 8 is an electrophoresis photograph showing the expression of HGF receptor mRNA in chondrocytes.

Claims (3)

A therapeutic agent for cartilage disorders, comprising HGF as an active ingredient. A chondrocyte growth promoter comprising HGF as an active ingredient. A proteoglycan production promoter comprising HGF as an active ingredient.

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