JP2006089395A - Prophylactic and/or curative agent for disease caused by production increase of genetic product of collagen gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a prophylactic and/or curative agent for diseases caused by the increase in collagen production, particularly to find new applications of DMP1(Dentinmatrix protein 1). <P>SOLUTION: In order to solve the above problem, a means for specifically expressing DMP1 in bone tissue has been established, and based on examining the function of DMP1, it has been found that the production of DMP1 inhibits the expression of I-type collagen gene. Thus, as the new applications of DMP1, an inhibitor for producing the genetic product of the collagen gene and/or a method for inhibiting producing the genetic product of the collagen gene, the prophylactic and/or curative agent for diseases caused by the increase in collagen production, and method(s) for preventing and/or treating such diseases, are provided, respectively. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a preventive and / or therapeutic agent for diseases caused by increased production of collagen gene products.

  Collagen is essential for the proper formation of connective tissue. Therefore, excessive or underproduction of collagen, or abnormal production of collagen (including misprocessed collagen) causes many connective tissue diseases and disorders. Currently, as many as 20 types of collagen have been identified, and these collagens, including type I, II, and type III fibrillar collagens, are synthesized as procollagen precursors containing amino and carboxy terminal peptide extensions. . These peptide extensions, termed “proregions”, are referred to as N- and C-propeptides, respectively. Typically, upon secretion of procollagen triple helix precursor molecules from cells, the pro region is cleaved to produce mature triple helix collagen molecules. When cleaved, “mature” collagen molecules have the ability to associate into, for example, highly structured collagen fibers (Non-Patent Documents 1-5).

  There are a number of serious diseases associated with inadequate or uncontrolled production of collagen. These diseases include, for example, endocardial sclerosis, idiopathic interstitial fibrosis, interstitial pulmonary fibrosis, perimuscular fibrosis of arteries, simmers fibrosis, pericentral fibrosis (Pericentral fibrosis), hepatitis, dermal fibroma, biliary cirrhosis, alcoholic cirrhosis, acute pulmonary fibrosis, idiopathic pulmonary fibrosis, acute respiratory distress syndrome, renal fibrosis / glomerulonephritis, renal fibrosis / diabetic kidney , Scleroderma / systemic, scleroderma / local, keloid, hyperplastic scar, severe joint adhesion / arthritis, myelofibrosis, corneal scar formation, cystic fibrosis, muscular dystrophy (Duchene dystrophy), cardiac fibrosis, Pathological fibrosis such as myofibrosis / retinal detachment, esophageal stenosis, Peyronie's disease or scar formation is included. In addition, fibrotic diseases may be induced or developed by surgery, such as scar repair / plastic surgery, glaucoma, cataract fibrosis, corneal scar formation, joint adhesion, graft-versus-host disease. , Tendon surgery, nerve entrapment, dupuytren contracture, OB / GYN adhesion / fibrosis, pelvic adhesion, peridural fibrosis, restenosis. One effective method for treating these diseases is to suppress pathological overproduction of collagen. Thus, identification and isolation of molecules that control, inhibit and / or regulate collagen production is important from a medical point of view.

  Bone, dentin, and cementum are calcified with calcium phosphate deposited in the extracellular matrix. The extracellular matrix is mainly collagen, but also includes a non-collagenous matrix, and most of the non-collagenous matrix is an acidic phosphorylated protein. It has been believed that the collagen fibers function as a scaffold on which hydroxyapatite crystals are deposited, and that acidic phosphorylated proteins are involved in calcification of the extracellular matrix. The acidic phosphorylated protein identified by recombinant DNA technology was first named AG1 (Non-patent document 6) and later changed to DMP1 (Dentinmatrix protein 1) (Non-patent document 7). The cDNA clone of DMP1 has been identified from a rat tooth cDNA library and has been considered to be specific for tooth dentin (Non-patent Document 8), but its expression was later expressed in other calcified tissues. It was shown to exist.

Furthermore, the present inventor has found that in bone tissues of birds and mammals, the DMP1 gene is not expressed in osteoblasts that secrete bone matrix and is not expressed in calcified bone matrix regardless of the animal species. It was specifically expressed in existing bone cells, and the protein was found to be distributed only in the bone matrix surrounding the bone cells (Non-patent Document 4). The characteristic aspect of DMP1 is that it has a very high acidic amino acid content and is highly phosphorylated in the tissue, so it is highly negatively charged in the tissue. Furthermore, the present inventor found a relationship between the negative ion state at the bone formation site and the bone mineralization rate, and a patent application for a bone mineralization promoting factor has been submitted as a novel use of DMP1 ( Patent Document 1).
Japanese Patent Application 2003-059130 Annu. Rev. Biochem. 1978; 47: 129-162 The Proteins. Academic Press, New York: 412-632 Extracellular Matrix Biochemistry. ElsevierScience Publishing Co., New York 1984: 83-118 N. Engl.J. Med. 1984; 311: 376-383 Structure and Functionof Collagen Types.Academic Press Inc., Orlando, Florida 1987: 1-42 J. Biol Chem1993; 268: 12624-12630 J. Histochem Cytochem1994; 42: 1527-1531 J. Histochem Cytochem1994; 42: 1527-1531 J. Bone Miner Res 2001; 16: 2017-2026

  An object of the present invention is to provide a preventive and / or therapeutic agent for a disease caused by increased production of collagen, and particularly to find a new use of DMP1.

  In order to solve the above problems, the present inventor has established means for specifically expressing DMP1 in bone tissue, and from the examination of its function, the production of DMP1 suppresses the expression of the collagen gene, and consequently the production of collagen. As a result, the present invention was completed.

The present invention comprises the following.
1. A gene product production inhibitor of a collagen gene comprising an effective amount of DMP1.
2. A collagen gene gene product production inhibitor comprising a DMP1 gene carrying vector.
3. 3. The inhibitor according to 1 or 2 above, wherein the collagen is type I collagen.
4). A method for inhibiting production of a gene product of a collagen gene, comprising an effective amount of DMP1.
5. 5. The method according to item 4 above, wherein the presence of DMP1 is due to filling.
6). 6. The method according to 4 or 5 above, wherein a DMP1 gene carrying vector is used.
7). 7. The method according to any one of items 4 to 6, wherein the collagen is type I collagen.
8). A method for preventing and / or treating a disease caused by increased production of a gene product of a collagen gene, comprising using the inhibitor according to any one of the preceding items 1 to 3.
9. 9. The method according to item 8 above, wherein the disease caused by increased production of the gene product of the collagen gene is fibrosis.
10. A pharmaceutical composition comprising an effective amount of the inhibitor according to any one of 1 to 3 above.
11. A preventive and / or therapeutic agent for a disease caused by increased production of a gene product of a collagen gene, which comprises using the inhibitor according to any one of items 1 to 3.
12 An agent for preventing and / or treating a disease caused by an increase in production of a gene product of a collagen gene comprising an effective amount of the inhibitor according to any one of 1 to 3 above.
13. 13. The prophylactic and / or therapeutic agent according to the above item 11 or 12, wherein the disease caused by increased production of the gene product of the collagen gene is fibrosis.

  In the present invention, it was found that the expression of the collagen gene is suppressed by specifically expressing DMP1 in bone tissue. Therefore, it is possible to inhibit collagen production by DMP1, and thus it is possible to prevent and / or treat diseases caused by increased production of collagen, more specifically fibrosis. Thus, the present invention is very useful for the prevention and / or treatment of diseases caused by increased production of collagen.

  Technical and scientific terms used herein have meanings commonly understood by one of ordinary skill in the art unless otherwise defined. Hereinafter, embodiments of the present invention will be described in more detail. The following detailed description is exemplary and illustrative only and is not intended to limit the invention in any way.

  DMP1 is an acidic protein that is an extracellular matrix protein having 400 to 550 amino acids. The amino acid sequence and gene sequence are disclosed by Toyozawa et al. (JMol Evol 1999; 48: 160-166, Gene 1999; 234: 307-314, JMol Evol 2000; 50: 31-38, etc.). The DMP1 gene is a known gene, and the sequence of DMP1 has been reported from 10 kinds of animals including humans. From amino acid sequence comparisons, it is known that all species of DMP1 begin with a hydrophobic leader sequence containing 16-21 amino acids. The cell-adhesive peptide RGD motif is not specific to the DMP1 molecule, but has been confirmed to be present in many other acidic phosphorylated proteins such as osteopontin, bone sialoprotein, and dentin sialophosphoprotein. The Arg-Gly-Asp sequence is strictly conserved with the mammalian DMP1 sequence and may have its important biological function. In addition, DMP1 contains a very large amount of amino acids that show acidity, and since many motif sequences including Ser are phosphorylated, it is judged to be highly negatively charged in tissues. This suggests that DMP1 has a strong binding ability with calcium ions and is necessary for calcification of the extracellular matrix.

  One feature of the present invention is that it has been found that expression of a collagen gene is suppressed by specifically expressing DMP1 in bone tissue. The inventor selectively expressed the DMP1 gene in bone tissue and accumulated the protein in the bone matrix. From FIG. 1 showing the results of the Examples, in transgenic mice with increased production of DMP1, the thickness of the skull decreased, the amount of cancellous bone in the long canal bone decreased, and the thickness of the cortical bone at the metaphysis. Is decreasing. Furthermore, from FIG. 4, the expression level of the type I collagen gene is reduced in the transgenic mouse. Since type I collagen is a major protein that constitutes these connective tissues, DMP1 regulates transcription of type I collagen genes and inhibits production of type I collagen gene products, thereby forming these connective tissues. It is thought that it is inhibited.

  One embodiment of the present invention achieved by the above findings is a collagen gene product production inhibitor and inhibition method characterized by containing an effective amount of DMP1. A preferred example of collagen is type I collagen. It has already been confirmed that DMP1 can be produced by genetic engineering, and it is possible to use recombinant DMP1 that has been mass-produced in a system using a known general-purpose E. coli host promoter system. In particular, post-translational modifications such as sugar chain structure are not necessary, and DMP1 produced by Escherichia coli is sufficient. In the present invention, DMP1 is a substitution, deletion, addition, or insertion of one to several amino acids that maintain the gene product production inhibitory function of the collagen gene of DMP1, in particular, a conserved sequence confirmed in mammals. Synthetic polypeptides consisting of several tens of amino acids that partially mimic the sequence variation and the amino acid sequence of DMP1 are also targeted. Means for introducing mutations such as substitution, deletion, addition or insertion are known per se, and for example, Ulmer's technique (Ulmer, K. M. Science 1983, 219: 666-671) can be used. DMP1 may be a mixture with a biodegradable synthetic resin or the like.

  For example, in order to fill DMP1 into a target site, it is preferably prepared in a paste form by general-purpose means by blending with a known composition. For example, a blended composition with a polylactic acid derivative or the like can be used. The vector carrying the DMP1 gene can exert the gene product production inhibitory function of the collagen gene of the present invention by means of specific gene introduction into the target tissue. The object of the present invention can also be achieved by means for carrying a gene in a self-propagating vector. That is, a vector carrying the DMP1 gene can also be included in the embodiment of the present invention.

  Desirably, the gene introduction means is based on an integration method, for example, a liposome method, a calcium phosphate method, an electroporation method, or the like. However, a transient expression method such as a liposome method, a calcium phosphate method, an electroporation method, a viral vector method, an atelocollagen method, or the like may be used. The vector to be used is not particularly limited, and applies to all recombinant vectors capable of introducing an inserted DNA fragment by a usual recombination experiment, such as a known plasmid, phage, cosmid, BAC, YAC, recombinant virus, transposon, etc. be able to. The vector can be constructed together with a promoter and an enhancer which are naturally suitable for a combination known per se. For example, a commercially available protein expression vector in which a promoter suitable for a normal host is inserted can be used. Specific examples include ZAPExpress (manufactured by Stratagene), pSVK3 (manufactured by Amersham Pharmacia Biotech), pEGFP-C1 (manufactured by Clontech), and atelocollagen. In the examples of the present invention, the mouse pro-α1 (I) collagen promoter was used in the examples of the present invention, but other promoters such as osteocalcin promoter may be used. Since this promoter is a promoter of a protein that is specifically secreted by osteogenic cells, it provides a powerful means for specifically expressing DMP1 in bone tissue.

  The DMP1 polynucleotide is inserted into the vector by ligating the polynucleotide or a DNA fragment containing the polynucleotide so as to be placed under the control of the promoter downstream of the promoter in the vector. In addition, a structure in which a Kozak sequence (Kozak, M., Gene, 234, 187, (1999)) is inserted between the promoter and DMP1, or a DNA encoding a tag polypeptide is inserted downstream of DMP1. A vector having the same is also preferably used. The polypeptide that serves as a tag is not particularly limited, and examples thereof include a FLAG tag (BioTechniques, 7, 580, (1989)). A homologous recombination technique (AAVertes et al., Biosci. Biotechnol. Biochem., 57, 2036 (1993)) that directly inserts a promoter-linked DMP1 polynucleotide into the target cell chromosome, or a transposon or insertion sequence (AA Vertes et al., Molecular Microbiol., 11,739 (1994)) and the like. DMP1 thus prepared can be directly loaded into the target site where inhibition of production of the gene product of the collagen gene is desired. Moreover, the vector carrying the DMP1 gene can be administered by direct injection, gene gun, injection or the like.

  Another embodiment of the present invention relates to a prophylactic and / or therapeutic agent, a prophylactic method and / or a therapeutic method for a disease caused by increased production of a gene product of a collagen gene. The preventive and / or therapeutic agent for the disease contains the inhibitor. The prevention method and / or treatment method of the disease can be achieved by using the inhibition method.

  Specific examples of diseases caused by increased production of collagen gene products include fibrosis, and more specifically, but not limited to, endocardial sclerosis, idiopathic interstitial fibrosis, stroma. Pulmonary fibrosis, perimuscular fibrosis of arteries, perimuscular fibrosis, pericentral fibrosis, hepatitis, dermal fibroma, biliary cirrhosis, alcoholic cirrhosis, acute lung fibrosis Disease, idiopathic pulmonary fibrosis, acute respiratory distress syndrome, renal fibrosis / glomerulonephritis, renal fibrosis / diabetic nephropathy, scleroderma / systemic, scleroderma / local, keloid, hyperplastic scar, severe Pathologic fibrosis or scarring such as joint adhesion / arthritis, myelofibrosis, corneal scar formation, cystic fibrosis, muscular dystrophy (Duchenu dystrophy), cardiac fibrosis, myofibrosis / retinal detachment, esophageal stenosis, Peyronie's disease Formation is included. In addition, fibrotic diseases may be induced or developed by surgery, such as scar repair / plastic surgery, glaucoma, cataract fibrosis, corneal scar formation, joint adhesion, graft-versus-host disease. , Tendon surgery, nerve entrapment, Dupuytren contracture, OB / GYN adhesion / fibrosis, pelvic adhesion, peridural fibrosis, restenosis.

  The collagen gene product production inhibitor can be prepared as a pharmaceutical composition by selecting in consideration of the balance between biological usefulness and toxicity. The pharmaceutical composition according to the present invention is usually preferably produced using one or more pharmaceutical carriers. Examples of pharmaceutical carriers include diluents and excipients such as fillers, extenders, binders, moisturizers, disintegrants, lubricants and the like that are usually used according to the form of use of the formulation. These are appropriately selected and used depending on the administration form of the resulting preparation. For example, water, pharmaceutically acceptable organic solvent, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerin , Propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose and the like. These may be used alone or in combination of two or more according to the dosage form according to the present invention. If necessary, various ingredients that can be used in normal protein preparations, for example, stabilizers, bactericides, buffers, isotonic agents, chelating agents, pH adjusters, surfactants, etc. are used as appropriate. You can also.

  The dose range of the pharmaceutical composition is not particularly limited, and the effectiveness of the contained components, the dosage form, the administration route, the type of disease, the nature of the subject (weight, age, medical condition and use of other medicines, etc.), It is appropriately selected according to the judgment of the doctor in charge. In general, an appropriate dose is, for example, in the range of about 0.01 μg to 100 mg, preferably about 0.1 μg to 1 mg, per kg of the subject's body weight. However, dosages can be varied using general routine experimentation for optimization well known in the art. The above dose can be divided into 1 to several times a day, and may be administered intermittently at a rate of once every several days or weeks.

  When administering a pharmaceutical composition according to the present invention, the pharmaceutical composition may be used alone or in combination with other compounds or medicaments required for treatment. As the administration route, either systemic administration or local administration can be selected. In this case, an appropriate administration route is selected according to the disease, symptoms and the like. For example, the parenteral route includes normal intravenous administration and intraarterial administration, as well as subcutaneous, intradermal and intramuscular administration. Or oral administration is also possible. Furthermore, transmucosal administration or transdermal administration is also possible. As the dosage form, various forms can be selected according to the therapeutic purpose, and representative examples include solid dosage forms such as tablets, pills, powders, powders, fine granules, granules, capsules and the like. , Aqueous preparations, ethanol solution preparations, suspensions, fat emulsions, liposome preparations, inclusions such as cyclodextrins, liquid dosage forms such as syrups and elixirs. Depending on the route of administration, these can be further administered orally, parenterally (instillation, injection), nasal, inhalation, vaginal, suppository, sublingual, eye drops, ear drops, ointments, creams And can be prepared, molded and prepared according to ordinary methods.

  Powders, pills, capsules and tablets are excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, polyvinyl alcohol, hydroxypropyl It can be produced using a binder such as cellulose and gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin. For the production of tablets and capsules, solid pharmaceutical carriers are used. The suspension can be produced using water, sugars such as sucrose, sorbitol, and fructose, glycols such as PEG, and oils. Injectable solutions can be prepared using a carrier consisting of a salt solution, a glucose solution, or a mixture of saline and glucose solution.

  Liposomeization involves, for example, adding a solution obtained by dissolving the substance in a solvent (such as ethanol) to a solution obtained by dissolving phospholipid in an organic solvent (such as chloroform), and then distilling off the solvent. Additionally, after shaking, sonication and centrifugation, the supernatant can be filtered and collected. For fat emulsification, for example, the substance, oil components (vegetable oil such as soybean oil, sesame oil, olive oil, MCT, etc.), emulsifier (phospholipid, etc.), etc. are mixed and heated to form a solution, and then the required amount of water is added. It can be carried out by emulsification and homogenization using an emulsifier (homogenizer such as a high-pressure jet type or ultrasonic type). It can also be lyophilized. When emulsifying a fat, an emulsification aid may be added. Examples of the emulsification aid include glycerin and saccharides (for example, glucose, sorbitol, fructose, etc.). For cyclodextrin inclusion, for example, a solution in which cyclodextrin is heated and dissolved in water or the like is added to a solution in which the substance is dissolved in a solvent (such as ethanol), and then cooled and the deposited precipitate is filtered and sterilized and dried. It can be done by doing. At this time, the cyclodextrin to be used may be appropriately selected from cyclodextrins (α, β, or γ type) having different pore diameters according to the size of the substance.

  EXAMPLES Hereinafter, although an Example demonstrates this invention, an Example shows an example of this invention and does not limit the technical scope.

Example 1
Evaluation of DMP1 transgenic mouse skeleton by microfocus X-ray photography

  From the start codon to the stop codon of the translational region of the rat DMP1 gene, pNASSβ (CLONTECHLab.) Is selectively expressed in bone tissue with the pro-α1 (I) collagen promoter (Reference: J CellBiol.1995; 129: 1421-1432). , Inc.) using a restriction enzyme to construct a construct for producing a transgenic mouse. Using this construct, a gene was introduced into a C57B / L fertilized egg to produce a DMP1 transgenic mouse. The expression level of DMP1 in bone tissue was examined by Northern blotting, the expression site was confirmed again by in situ hybridization, and DMP1 that was forcibly expressed about 5 times the normal value was used as a DMP1 transgenic mouse. Compared to mice. After killing the DMP1 transgenic mouse with an anesthetic, the soft tissue was removed, stored in 70% alcohol, and a microfocus X-ray photograph was taken.

  As a result, as shown in FIG. 1A, it was found that the 3 month old DMP1 transgenic mouse (Tg) did not show a large abnormality but was slightly smaller than the wild type. As shown in FIG. 1B, the skull of the 2 month old DMP1 transgenic mouse (Tg) was slightly stronger in X-ray permeability than the normal mouse (WT) (upper part of FIG. 1B). As a result of strong enlargement of the bone fragment punched out from the center of the parietal bone, it was found that the parietal bone of the DMP1 transgenic mouse (Tg) was stronger in X-ray permeability than the normal mouse (WT) (bottom of FIG. 1B) ).

  Further, as shown in FIGS. 1C to 1E, the femurs of DMP1 transgenic mice (Tg) at 1 month, 3 months and 6 months were generally stronger in X-ray permeability than normal mice (WT). As shown in FIGS. 1D and E, the femurs of 3 and 6 month old DMP1 transgenic mice (Tg) showed a decrease in cancellous bone at the distal end of the distal diaphysis compared to normal mice (WT). Further, as shown in FIG. 1E, the femur of the 6 month old DMP1 transgenic mouse (Tg) was shorter than that of the normal mouse (WT). From these results, it was found that bone formation was inhibited in the DMP1 transgenic mice as compared with normal mice, and in particular, formation of cancellous bones in the femur was inhibited.

Example 2
Evaluation of the distal metaphysis of the femur by pQCT

DMP1 transgenic mice prepared by the same method as in Example 1 were sacrificed with anesthetics, soft tissues were removed, stored in 70% alcohol, and cancellous bone area (pQCT) at the metaphyseal part (perpendicular quantitative computed tomography (pQCT)). mm ² ), cortical bone thickness (mm) and bone density (mg / cm ³ ) were measured. The horizontal axis was sliced in 0.5 mm increments from the distal side, and each value was measured for slices with slice numbers 1-15.

  As a result of analysis of the cancellous bone area at the metaphysis at 3 months and 6 months after birth, as shown in FIGS. 2B and C, the cancellous bone area is normal in the vicinity of the epiphyseal cartilage (slice number: 2-4). DMP1 transgenic mice showed a significant increase compared to, but canopy bone area was significantly reduced in DMP1 transgenic mice in the majority of the metaphysis (slice numbers: 8-15). As a result of analyzing the thickness of the cortical bone at the metaphysis at 3 months and 6 months after birth, as shown in FIGS. 2D and E, the thickness of the cortical bone is near the epiphyseal cartilage (slice number: 1-4). Was significantly decreased in DMP1 transgenic mice compared to normal mice. Furthermore, as a result of analyzing the bone mineral density of the cortical bone at the metaphysis, as shown in FIGS. 2F and G, the bone density of the cortical bone at the end of the femoral metaphysis at the age of 3 months is DMP1 transgenic compared to that of the normal mouse. Although the number of mice increased significantly, it was found that the change was smaller at 6 months of age. From these results, it was found that the DMP1 transgenic mice had a reduced cancellous bone area and cortical bone thickness compared to normal mice.

Example 3
Histopathological evaluation of the distal metaphysis of the distal femur

  DMP1 transgenic mice prepared by the same method as in Example 1 were anesthetized with anesthetics, fixed with perfusion with 4% paraformaldehyde for 15 minutes, decalcified with EDTA, and embedded in paraffin. The sample was sliced into 5 μm, stained with hematoxylin and eosin, and the distal end of the femur distal side was observed with an optical microscope.

  As a result, as shown in FIGS. 3A and 3E, a remarkable change between normal mice (FIG. 3A) and DMP1 transgenic mice (FIG. 3E) was observed at the distal metaphysis of the femur at 1 month after birth. There wasn't. However, in the femoral distal metaphysis at 3 months after birth, as shown in FIGS. 3B, C, F and G, from the side and front images, the DMP1 transgenic mouse (FIG. 3B and C) compared to the normal mouse (FIG. 3B and C). A clear decrease in cancellous bone at the metaphysis of FIGS. 3F and G) was observed (see strong inset in FIGS. 3C and G). In addition, at the distal end of the femoral distal side of the femur at 6 months of age, as shown in FIGS. Although it decreased, it was not a significant decrease compared to 3 months after birth. From these results, it was found that the cancellous bone decreased especially in the distal extremity of the distal femur of DMP1 transgenic mice at 3 months of age compared to normal mice.

Example 4
Evaluation of bone matrix gene expression by Northern blot and in situ hybridization of femur

Experiment 4.1 Northern blot of each bone matrix gene in the femur Total RNA was extracted from the femur of a transgenic mouse (1 month old) prepared by the same method as in Example 1, using the guanidine / cesium chloride extraction method. After electrophoresis on a denaturing gel (1% agarose gel, 1 × MOPS, 20% formaldehyde), the gel was immobilized on Hybond-N ⁺ membrane (Amersham) with UV cross-linking (150 J). Was then ³² P labeled by the multi-labeling kit (Amersham Corp.), I-type collagen gene, osteopontin gene, osteocalcin gene, alkaline phosphatase gene, and the film at the probe glyceraldehyde phosphate dehydrogenase (GAPDH) gene 42 After hybridizing overnight at 0 ° C., it was washed and exposed to an X-ray film.

  As a result, as shown in FIG. 4, the gene expression level of the type I collagen gene in the DMP1 transgenic mouse (Tg) was significantly decreased as compared with the normal mouse (WT). There was no significant difference in gene expression of other bone matrix proteins.

Experiment 4.2 In situ hybridization of each bone matrix gene in the femur Transfused with 4% paraformaldehyde for 15 minutes after anesthesia of a transgenic mouse (1 month old) prepared in the same manner as in Example 1 In the RNase free state, it was fixed, decalcified with EDTA, and embedded in paraffin. After slicing the sample to 5 μm, using the DIG labeling kit (RocheDiagnostics), hybridization was carried out overnight at 55 ° C. with an RNA probe of each bone matrix protein gene labeled with DIG, washed the next day, and NBT / BCIP solution ( RocheDiagnostics).

  Similar to the results of the Northern blot in Experiment 4.1, in the in situ hybridization, as shown in FIG. 5, the expression level of the type I collagen gene in the DMP1 transgenic mouse (Tg) is higher than that in the normal mouse (WT). Although there was a decrease, no change in expression distribution was observed. In addition, no changes were observed in the expression level and expression distribution of osteopontin and osteocalcin genes.

  From these results, it was found that the gene expression level of the type I collagen gene was decreased in the DMP1 transgenic mice. From this, by suppressing the production of the gene product of type I collagen gene by DMP1, strong skull X-ray permeability in the DMP1 transgenic mouse observed in the above analysis, bone area reduction in cancellous bone, It was speculated that phenomena such as a decrease in cortical bone thickness occurred. In addition, no decrease in the expression of bone matrix protein genes (osteopontin gene, osteocalcin) gene other than the type I collagen gene was observed. From this, it is considered that the number of osteoblasts in the DMP1 transgenic mouse is not decreased as compared with the normal mouse. Therefore, the possibility that the above phenomenon occurred due to a decrease in osteogenic action due to a decrease in osteoblasts in DMP1 transgenic mice was denied.

Example 5
Evaluation of bone resorption level by osteoclasts

Experiment 5.1 Measurement of Deoxypyridinoline Levels Transgenic mice (2 months and 3 months old) prepared in the same manner as in Example 1 and normal mice were collected for 3 days to measure deoxypyridinoline levels (( (Consigned to Mitsubishi BLC).

  As a result, as shown in FIG. 6, it was found that bone resorption was significantly reduced in both 2 months and 3 months.

Experiment 5.2 Measurement of the number of osteoclasts The femurs of transgenic mice (2 months and 3 months old) and normal mice prepared by the same method as in Example 1 were embedded in non-decalcified resin and the sample was 15 μm. After slicing, the cells were stained with toluidine blue, and the number of osteoclasts was measured.

  As a result, as shown in FIG. 7, there was no significant difference in the number of osteoclasts in the cancellous bone at the metaphysis of the distal femur between normal and DMP1 transgenic mice at 2 and 3 months of age. I couldn't. From this result, in osteoclastic cells increased in DMP1 transgenic mice, the increased osteoclast action resulted in strong skull X-ray permeability, decreased bone area in cancellous bone, decreased cortical bone thickness, etc. The possibility was denied.

  As described above, by the collagen gene gene product production inhibitor and / or inhibition method provided by DMP1 provided in the present invention, diseases caused by increased production of collagen gene product, specifically, prevention of fibrosis and / Or treatment becomes possible. Thus, the present invention is very useful for the prevention and / or treatment of diseases caused by increased production of collagen gene products.

The result of analyzing the skeleton by microfocus X-ray photograph is shown. (Example 1) WT represents a normal mouse and Tg represents a DMP1 transgenic mouse. A shows the results for the whole body skeleton, B for the skull, C for the 1 month old femur, D for the 3 month old femur, and E for the 6 month old femur. The result of having performed pQCT in the femur distal side metaphyseal part is shown. (Example 2) ● indicates the result in a DMP1 transgenic mouse, ○ indicates the result in a normal mouse, and the test of the significance of the data was performed by ANOVA. When the risk rate p <0.005 with the control group, it is indicated by ★, and when the risk rate p <0.001, it is indicated by ★★. The bar indicates standard error. A shows the femoral site corresponding to the scan number of pQCT, B, D, and F show the results of the femur at 3 months of age, and C, E, and G show the results of the femur at 6 months of age. B and C are cancellous bone areas at the metaphysis, D and E are cortical bone thicknesses at the metaphysis, and F and G are bone densities of cortical bone at the metaphysis. The result of having performed hematoxylin and eosin dyeing | staining using the tissue slice of the femur distal side metaphysis is shown. (Example 3) A to D show results in normal mice, and E to H show results in DMP1 transgenic mice. A, B, D, E, F, G, and H are side views, C and G are front views, and insets of C and G are enlarged views of the metaphysis. A and E show results at 1 month of age, B, C, F and G show results at 3 months of age, and D and H show results at 6 months of age. The result of having analyzed each bone matrix gene expression in a femur by a Northern blot is shown. (Example 4) Type I is a result of analysis of expression of type I collagen gene, OPN is an osteopontin gene, OC is an osteocalcin gene, ALP is an alkaline phosphatase gene, and GAPDH is a result of analyzing expression of a glyceraldehyde phosphate dehydrogenase gene. The result of having analyzed each bone matrix gene expression in a femur by in situ hybridization is shown. (Example 4) a and d are the type I collagen gene, b and e are the osteopontin gene, and c and f are the results of analyzing the expression of the osteocalcin gene. The result of having analyzed the bone resorption level by an osteoclast is shown. (Example 5) White bars show results in normal mice, and black bars show results in DMP1 transgenic mice. Data significance was tested with ANOVA. When the risk rate p <0.001 with the control group, it is indicated by ★★. The bar indicates standard error. The result of having measured the number of osteoclasts is shown. (Example 5) White bars show results in normal mice, and black bars show results in DMP1 transgenic mice. Data significance was tested by ANOVA, but no significant difference was found between the control group and each group. The bar indicates standard error.

Claims (13)

A gene product production inhibitor of a collagen gene comprising an effective amount of DMP1. A collagen gene gene product production inhibitor comprising a DMP1 gene carrying vector. The inhibitor according to claim 1 or 2, wherein the collagen is type I collagen. A method for inhibiting production of a gene product of a collagen gene, comprising an effective amount of DMP1. The method of claim 4, wherein the presence of DMP1 is due to filling. 6. The method according to claim 4 or 5, wherein a DMP1 gene carrying vector is used. The method according to any one of claims 4 to 6, wherein the collagen is type I collagen. A method for preventing and / or treating a disease caused by increased production of a gene product of a collagen gene, comprising using the inhibitor according to claim 1. The method according to claim 8, wherein the disease caused by increased production of a gene product of a collagen gene is fibrosis. A pharmaceutical composition comprising an effective amount of the inhibitor according to claim 1. A preventive and / or therapeutic agent for a disease caused by increased production of a gene product of a collagen gene, wherein the inhibitor according to claim 1 is used. A preventive and / or therapeutic agent for a disease caused by increased production of a gene product of a collagen gene comprising an effective amount of the inhibitor according to claim 1. The prophylactic and / or therapeutic agent according to claim 11 or 12, wherein the disease caused by increased production of the gene product of the collagen gene is fibrosis.

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