JP2017112836A - Preparation methods of osteoporosis model animals - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide preparation methods of osteoporosis model animals in which osteoblastic activity is inhibited, and to provide methods of evaluating the therapeutic effect for osteoporosis of a test substance.SOLUTION: A preparation method of nonhuman osteoporosis model animals comprises inhibiting the expression or function of synoviolin proteins in nonhuman animals using an osteoblastic activity-inhibiting composition containing a carrier and, as an active ingredient, a synoviolin inhibitor that inhibits the expression or function of synoviolin proteins in nonhuman animals. The therapeutic effect for osteoporosis of a test substance is evaluated using the nonhuman osteoporosis model animals.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method for producing an osteoporosis model animal. More specifically, the present invention relates to a method for producing an osteoporosis model animal that inhibits the activity of osteoblasts by inhibiting the activity of Synoviolin.

  In developing osteoporosis therapeutics, it is important to create osteoporosis model animals (especially non-human mammals).

  Further, for example, Japanese Patent No. 4998880 discloses an osteoporosis model animal from which an ovary has been removed. In the osteoporosis model animal obtained by removing this ovary, the activity of osteoclasts increases, and as a result, the bone becomes weak and the bone density decreases.

  Japanese Patent Application Laid-Open No. 2010-51238 discloses a method for producing an osteoporosis model animal by administering a feed containing homocystin. The osteoporosis model animal created by the method disclosed in this publication increases the fracture frequency without decreasing the bone density.

Patent No. 4998880 JP 2010-51238 A

  An osteoporosis model animal obtained by activating osteoclasts often has a disorder other than bone, and as a result, may not be suitable as an osteoporosis model.

  Then, an object of this invention is to provide the preparation method of the osteoporosis model animal which inhibited the activity of the osteoblast.

  The present invention is based on the findings of the examples that the activity of osteoblasts can be inhibited by inhibiting the activity of Synoviolin, and as a result, an osteoporosis model animal in which biological functions are maintained can be created. That is, in the examples of the present specification, a non-human synoviolin knockout animal was prepared and its bone density and osteoblasts were detected. As a result, osteoblasts were significantly reduced, and a model animal for osteoporosis could be obtained. . The present invention is based on the embodiments and related technologies.

The first aspect of the present invention relates to a method for producing a non-human osteoporosis model animal. This method comprises the step of inhibiting the expression or function of Synoviolin protein in a non-human mammal.
An example of the step of inhibiting the expression or function of synoviolin protein in a non-human mammal is a step including a step of knocking out or knocking down a synoviolin gene. An example of a non-human osteoporosis model animal is a non-human osteoporosis model animal in which the activity of osteoblasts is inhibited.

The second aspect of the present invention relates to a method for evaluating the therapeutic effect of a test substance on osteoporosis.
In this method, first, a non-human osteoporosis model animal is prepared according to the method described above.
Next, the test substance is administered to a non-human osteoporosis model animal.
Then, the non-human osteoporosis model animal to which the test substance is administered is analyzed. By doing in this way, it can be evaluated whether a test substance has the therapeutic effect of osteoporosis (or whether there is a serious side effect).

The third aspect of the present invention relates to a composition for inhibiting osteoblast activity. An osteoblast activity-inhibiting composition is a composition used to inhibit osteoblast activity. In addition to the carrier, this composition contains a synoviolin inhibitor that is an active ingredient for inhibiting the activity of osteoblasts.
Examples of synoviolin inhibitors are
Synoviolin siRNA, Synoviolin decoy nucleic acid or Synoviolin antisense nucleic acid;
One base sequence selected from SEQ ID NOs: 2 to 6, a base sequence complementary to these base sequences, or a base in which 1 or 2 bases are substituted, inserted, deleted or added from any of these base sequences Synoviolin siRNA having the sequence; and a base sequence represented by SEQ ID NO: 7 or a synoviolin decoy having a base sequence in which one or two bases are substituted, inserted, deleted or added from the base sequence represented by SEQ ID NO: 7 It contains any one or more of nucleic acids.

  The RNA having the nucleotide sequences shown in SEQ ID NOs: 2 to 4 is Synoviolin siRNA, as described in Izumi T, et al. , Arthritis Rheum. 2009; 60 (1): 63-72, Yamazaki S, et al. , EMBO J. et al. 2007; 26 (1): 113-22. Are known as disclosed using experiments. It is known that RNA having the nucleotide sequences shown in SEQ ID NOs: 5 and 6 is Synoviolin siRNA, as disclosed in WO 2005/074988 using experiments.

SEQ ID NO: 5'-GCUGUGACAGAUGCCAUCA-3 '
SEQ ID NO: 5'-GGUGUUCUUUGGGCAACUG-3 '
SEQ ID NO: 5'-GGUUCUGCUGUACAUGGCC-3 '
SEQ ID NO: 5: 5′-CGUUCCUGUGUACCGCCGUCA-3 ′
Sequence number 6: 5'-GUUUTGGGUGACUGGUGUA-3 '

  “RNA having a base sequence in which one or two bases are substituted, inserted, deleted or added from any of these base sequences” means one base sequence selected from SEQ ID NOs: 2 to 6 and these bases RNA having a base sequence in which one or two bases are substituted, inserted, deleted or added from any base sequence of the base sequence complementary to the sequence. Any one kind of substitution, insertion, deletion or addition may occur, or two or more may occur.

In one embodiment of the third aspect of the present invention, the active ingredient is substituted, inserted, deleted or added by one or two bases from the nucleotide sequence represented by SEQ ID NO: 7 or the nucleotide sequence represented by SEQ ID NO: 7. Synoviolin decoy nucleic acid having a base sequence. The fact that the nucleic acid having the base sequence represented by SEQ ID NO: 7 is a synoviolin decoy nucleic acid is described in, for example, Tsuchimochi K, et al. Mol Cell Biol. 2005; 25 (16): 7344-56. Are known as shown in the examples.
SEQ ID NO: 7: 5′-AUGGUGACGUGUGCUAGA-3 ′

  The fourth aspect of the present invention relates to a composition for producing an osteoporosis model animal, comprising the osteoblast activity inhibiting composition of the third aspect.

  Although this osteoporosis model animal may have increased osteoclast activity as a result of inhibiting osteoblast activity, its ecology is maintained compared to conventional osteoporosis model animals. Appropriate.

FIG. 1 is a graph showing the measurement results of total bone density, cancellous bone density, cortical bone density, and cortical bone thickness by the pQCT method using femurs extracted from osteoblast-specific conditional KO mice. Fig. 2 shows cortical epicardial circumference, cortical intima circumference, X-axis bone strength index (strength in the three-point bending direction) by pQCT method using femurs extracted from osteoblast-specific conditional KO mice It is a graph which shows the measurement result of a polar coordinate bone strength parameter | index (strength in the direction of torsion bending). FIG. 3 shows the ratio of bone mass per tissue mass (BD / TV), trabecular width (Tb.Th), by two-dimensional micro-CT method using femurs extracted from osteoblast-specific conditional KO mice. It is a graph which shows the measurement result of the number of trabeculae (Tb.N) and the trabecular gap (Tb.Sp). Fig. 4 shows the bone marrow cavity in a range that can be seen in all directions without being blocked by the trabecular bone from the point in the bone marrow cavity by the two-dimensional micro CT method using the extracted femur of an osteoblast-specific conditional KO mouse. Marlou Star volume (V * m.space), which is the average of the volume, Traverse star volume (V * tr), which is the average of the volume ranging from the point in the trabecular bone to the end of the bone mass in all directions The number of Nd per tissue mass and the total length of the skeletal line per tissue mass when the connection point of the beam is defined as a nodule (Nd) and the free end not coupled with other bone mass is defined as the end It is a graph which shows the measurement result of (length). FIG. 5 is an image obtained by photographing a femur extracted from an osteoblast-specific conditional KO mouse by a two-dimensional micro CT method. FIG. 6 is a graph showing the measurement results of articular cartilage width using a knee joint-preserved slice of the femur and tibia of an osteoblast-specific conditional KO mouse. FIG. 7 is a graph showing the measurement results of subchondral bone width using a knee joint-preserved slice of the femur and tibia of an osteoblast-specific conditional KO mouse. FIG. 8 is a graph showing the measurement results of joint gaps using knee joint-preserved sliced femur and tibia of osteoblast-specific conditional KO mice. FIG. 9 is an image obtained by observing, with a microscope, a knee joint-preserved slice of the femur / tibia of an osteoblast-specific conditional KO mouse. FIG. 10 is a graph showing growth rate measurement, growth cartilage width, and number of hypertrophic chondrocytes using an osteoblast-specific conditional KO mouse excised femur / tibia knee joint-preserved slice. . FIG. 11 is an image obtained by observing, with a microscope, a knee joint-preserved slice of the femur / tibia of an osteoblast-specific conditional KO mouse. FIG. 12 is an image obtained by observing a region in the vicinity of the growth plate cartilage of the knee joint-preserved slice of the femur and tibia of an osteoblast-specific conditional KO mouse. FIG. 13 is a graph showing the results of measurement of the number of osteoclast cells using a knee joint-preserved slice of femur and tibia of osteoblast-specific conditional KO mice. FIG. 14 is a graph showing the measurement results of total columnar, bone marrow mass and cortical bone width using an excised femoral shaft polished specimen of an osteoblast specific conditional KO mouse. FIG. 15 shows the resorption surface (ES), osteoid surface (OS) and osteoid surface (OS) on the cortical bone opening surface and intimal surface, using a polished femoral shaft polished specimen of an osteoblast-specific conditional KO mouse. It is a graph which shows the measurement result of the ratio of a static surface (QS), and the roughening surface per cortical bone surface. FIG. 16 is a graph showing the measurement results of bone mineralization rate, bone formation rate, and calcification surface per bone surface using an excised femoral shaft polished specimen of an osteoblast-specific conditional KO mouse. It is. FIG. 17 is an image obtained by observing a polished femoral shaft polished specimen of an isolated femur of an osteoblast-specific conditional KO mouse with a microscope. FIG. 18 shows the resorption surface (ES), osteoid surface (OS), and resting surface (secondary cancellous bone surface) using a knee joint-preserved slice of the femur isolated from an osteoblast-specific conditional KO mouse. It is a graph which shows the measurement result of the ratio of QS), the number of osteoclasts, the osteoid width, and the number of osteoblasts. FIG. 19 is a graph showing the results of measurement of bone mineralization rate, bone formation rate, and calcified surface per bone surface using a knee joint-preserved slice of the femur of an osteoblast-specific conditional KO mouse. It is. FIG. 20 is a graph showing the measurement results of the bone mass and the bone mass width on the surface of the secondary cancellous bone using the knee joint-preserved sliced femur of an osteoblast-specific conditional KO mouse. FIG. 21 is an image obtained by microscopically observing the surface of the secondary cancellous bone using a knee joint-preserved slice of a femur extracted from an osteoblast-specific conditional KO mouse. FIG. 22 is a graph showing the measurement results of total bone density, cancellous bone density, cortical bone density, and cortical bone thickness by the pQCT method using the femur extracted from a systemic conditional KO mouse. FIG. 23 shows the cortical epicardial circumference, cortical intima circumference, X-axis bone strength index (strength in the three-point bending direction) and polar coordinate bone by pQCT method using the extracted femur of systemic conditional KO mice. It is a graph which shows the measurement result of an intensity | strength parameter | index (strength in the direction of torsion bending). FIG. 24 shows the ratio of bone mass per tissue mass (BD / TV), trabecular width (Tb.Th), and number of trabeculae by two-dimensional micro CT method using the femurs of systemic conditional KO mice. It is a graph which shows the measurement result of (Tb.N) and trabecular gap (Tb.Sp). FIG. 25 shows the average volume of the bone marrow cavity in a range that can be seen without being blocked by the trabecular bone from all points in the bone marrow cavity by the two-dimensional micro CT method using the extracted femur of a systemic synoviolin knockout mouse. A certain Marurou Star volume (V * m.space), a trabecular star volume (V * tr) that is the average of the volume in the range from a point in the trabecular bone to the end of the bone mass in all directions, a trabecular joint point Is defined as a nodule (Nd), and the free end that is not connected to other bone mass is defined as the end, and the Nd number per tissue mass and the total length of the skeletal line per tissue mass (Total strut length) It is a graph which shows a result. FIG. 26 is an image obtained by photographing the femur extracted from the systemic conditional KO mouse by the two-dimensional micro CT method. FIG. 27 is a graph showing the measurement result of articular cartilage width using a knee joint-preserving sliced femur and tibia of systemic conditional KO mice. FIG. 28 is a graph showing the measurement results of growth rate measurement, growth cartilage width, and number of hypertrophic chondrocytes using a knee joint-preserved slice of the femur and tibia of systemic conditional KO mice. FIG. 29 is an image obtained by observing a knee joint-preserved slice of the femur and tibia of a systemic conditional KO mouse with a microscope. FIG. 30 is a graph showing the results of measurement of the number of osteoclast cells using a knee joint-preserving sliced femur and tibia of systemic conditional KO mice. FIG. 31 is a graph showing the measurement results of total columnar, bone marrow mass, and cortical bone width using a polished femoral shaft polished specimen of the femur of a generalized conditional KO mouse. FIG. 32 shows the resorption surface (ES), osteoid surface (OS), and resting surface (on the cortical bone opening surface and intimal surface) using the excised femoral shaft polished specimen of the systemic conditional KO mouse. It is a graph which shows the measurement result of the ratio and the roughening surface per cortical bone surface. FIG. 33 is a graph showing measurement results of bone mineralization rate, bone formation rate, and calcification surface per bone surface, using a polished femoral shaft polished specimen of the femur of systemic conditional KO mice. FIG. 34 is an image obtained by observing, with a microscope, a polished specimen of the femoral shaft of a removed femur of a systemic conditional KO mouse. FIG. 35 shows the resorption surface (ES), osteoid surface (OS), and resting surface (QS) on the surface of the secondary cancellous bone using the knee joint-preserved slice of the femur of a generalized conditional KO mouse. It is a graph which shows the measurement result of a ratio and the number of osteoclasts, osteoid width, and the number of osteoblasts. FIG. 36 is a graph showing measurement results of bone mineralization rate, bone formation rate, and calcification surface per bone surface, using a knee joint-preserving sliced femur of a systemic conditional KO mouse. FIG. 37 is a graph showing the measurement results of the bone mass and the bone mass width on the surface of the secondary cancellous bone using the knee joint-preserved sliced femur of the systemic conditional KO mouse. FIG. 38 is an image obtained by microscopically observing the surface of the secondary cancellous bone using a knee joint-preserved slice of the femur of a systemic conditional KO mouse. FIG. 39 is a design diagram of a gene targeting vector used for the preparation of a systemic conditional knockout (KO) mouse (synoviolin knockout mouse (syvn1 cKO mouse)).

  Hereinafter, embodiments for carrying out the present invention will be described with reference to the drawings. The present invention is not limited to the embodiments described below, but includes those appropriately modified by those skilled in the art from the following embodiments.

  The first aspect of the present invention relates to a method for producing a non-human osteoporosis model animal. The non-human osteoporosis model animal is a non-human mammal that is a model for osteoporosis. By using a non-human osteoporosis model animal, it is possible to develop an osteoporosis therapeutic agent and a therapeutic method.

  This method for producing a model animal includes a step of inhibiting the expression or function of Synoviolin protein in a non-human mammal. As will be described later, Synoviolin, Synoviolin protein and Synoviolin gene are known. A method for inhibiting the expression and function of a gene is also known. In the present invention, a known method may be appropriately used to inhibit the expression or function of synoviolin protein in a non-human mammal. An example of the step of inhibiting the expression or function of synoviolin protein in a non-human mammal is a step including a step of knocking out or knocking down a synoviolin gene. An example of a non-human osteoporosis model animal is a non-human osteoporosis model animal in which the activity of osteoblasts is inhibited.

The second aspect of the present invention relates to a method for evaluating the therapeutic effect of a test substance on osteoporosis.
In this method, first, a non-human osteoporosis model animal is prepared according to the method described above.
Next, the test substance is administered to a non-human osteoporosis model animal. The test substance may be administered orally together with a known carrier, or may be administered as an injection around a bone tissue such as a joint or around the bone tissue. Then, the non-human osteoporosis model animal to which the test substance is administered is analyzed. Methods for analyzing non-human osteoporosis model animals are known. Examples of methods for analyzing non-human osteoporosis model animals include methods of measuring bone density, methods of measuring bone strength, methods of measuring components such as salt contained in bone, and measuring the amount of collagen and the cross-linked state of collagen. A method for measuring cartilage density, and a method for image analysis of the state of bone and cartilage. By doing in this way, it can be evaluated whether a test substance has the therapeutic effect of osteoporosis (or whether there is a serious side effect).

The third aspect of the present invention relates to a composition for inhibiting osteoblast activity. An osteoblast activity-inhibiting composition is a composition used to inhibit osteoblast activity. In addition to the carrier, this composition contains a synoviolin inhibitor that is an active ingredient for inhibiting the activity of osteoblasts. The carrier is a pharmaceutically acceptable carrier.
Examples of synoviolin inhibitors are
Synoviolin siRNA, Synoviolin decoy nucleic acid or Synoviolin antisense nucleic acid;
One base sequence selected from SEQ ID NOs: 2 to 6, a base sequence complementary to these base sequences, or a base in which 1 or 2 bases are substituted, inserted, deleted or added from any of these base sequences Synoviolin siRNA having the sequence; and a base sequence represented by SEQ ID NO: 7 or a synoviolin decoy having a base sequence in which one or two bases are substituted, inserted, deleted or added from the base sequence represented by SEQ ID NO: 7 It contains any one or more of nucleic acids.

  The fourth aspect of the present invention relates to a composition for producing an osteoporosis model animal, comprising the osteoblast activity inhibiting composition of the third aspect. A non-human osteoporosis model animal can be prepared by administering the above-described osteoblast activity inhibiting composition to a target non-human mammal.

  The accession number in the public gene database Genbank of human Synoviolin gene is AB024690 (SEQ ID NO: 1).

  SEQ ID NO: 1 shows the base sequence of the gene encoding synoviolin in humans. Even proteins other than those encoded by the nucleotide sequence have high homology (usually 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more). And the protein which has the function which Synoviolin protein has is contained in Synoviolin of this invention.

  “Synoviolin gene” in the present invention includes, for example, an endogenous gene (such as a homolog of a human synoviolin gene) in another organism corresponding to the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1.

  In addition, the endogenous DNA of other organisms corresponding to the DNA consisting of the base sequence described in SEQ ID NO: 1 generally has high homology with the DNA described in SEQ ID NO: 1. High homology means 50% or more, preferably 70% or more, more preferably 80% or more, more preferably 90% or more (for example, 95% or more, further 96%, 97%, 98% or 99% or more). ) Homology. This homology is determined by the mBLAST algorithm (Altschul et al. (1990) Proc. Natl. Acadsci. USA 87: 2264-8; Karlin and Altschul (1993) Proc. Natl. Acadsci. USA 90: 5873-7). be able to. In addition, when isolated from the living body, the DNA is considered to hybridize with the DNA described in SEQ ID NO: 1 under stringent conditions. Here, as “stringent conditions”, for example, “2 × SSC, 0.1% SDS, 50 ° C.”, “2 × SSC, 0.1% SDS, 42 ° C.”, “1 × SSC, 0.1 % SDS, 37 ° C. ”and more stringent conditions are“ 2 × SSC, 0.1% SDS, 65 ° C. ”,“ 0.5 × SSC, 0.1% SDS, 42 ° C. ”and“ 0. 2 × SSC, 0.1% SDS, 65 ° C. ”. Those skilled in the art can appropriately obtain an endogenous gene corresponding to a synoviolin gene in other organisms based on the base sequence of the synoviolin gene. In this specification, a protein (gene) corresponding to a synoviolin protein (gene) in a non-human organism or a protein (gene) functionally equivalent to the above-mentioned synoviolin is simply referred to as “synoviolin protein (gene)”. May be written.

  “Synoviolin” of the present invention can be prepared not only as a natural protein but also as a recombinant protein using a gene recombination technique. The natural protein can be prepared by, for example, a method using affinity chromatography using an antibody against synoviolin protein against an extract of a cell (tissue) considered to express synoviolin protein. On the other hand, the recombinant protein can be prepared by culturing cells transformed with DNA encoding synoviolin protein. The “Synoviolin protein” of the present invention is suitably used, for example, in the screening method described below.

  In the present invention, “expression” includes “transcription” from a gene or “translation” into a polypeptide and “degradation inhibition” of a protein. “Expression of Synoviolin protein” means that transcription and translation of a gene encoding Synoviolin protein occurs, or Synoviolin protein is generated by transcription and translation thereof.

  Those skilled in the art can appropriately evaluate (measure) the various functions described above using general techniques. Specifically, the method described in the examples described later, or the method can be modified as appropriate.

  Thus, “inhibiting the expression and / or function of a synoviolin protein” refers to reducing or eliminating the amount, function or activity of the wild-type synoviolin gene or protein as compared to the amount, function or activity of the wild-type synoviolin gene or protein. The “inhibition” includes both inhibition of both function and expression, and inhibition of either one.

  Specifically, ubiquitination is performed by repeating a cascade reaction in which enzymes such as ubiquitin activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3) cooperate to form a ubiquitin molecule on a substrate protein. A process of forming a polyubiquitin chain by binding in a branch. This polyubiquitin chain is formed via the ε-amino group of the 48th lysine residue in the ubiquitin molecule, and becomes a degradation signal to the 26S proteasome, leading to degradation of the target protein.

  As a method for confirming the influence of the test substance on the expression of the Synoviolin gene or the influence of the Synoviolin protein activity, for example, a method disclosed in International Publication WO 2006-137514 may be used as appropriate.

  Effect of test substance on expression of Synoviolin gene

  A test compound is brought into contact with cells expressing the synoviolin gene. Examples of “cells” used in this case are cells derived from humans, mice, or rats. It is also possible to use microbial cells such as Escherichia coli and yeast transformed to express Synoviolin. As an example of “a cell expressing a synoviolin gene”, a cell expressing an endogenous synoviolin gene or a cell into which an exogenous synoviolin gene is introduced and expressing the gene can be used. A cell in which an exogenous synoviolin gene is expressed can be usually prepared by introducing an expression vector into which a synoviolin gene has been inserted into a host cell. The expression vector can be prepared by general genetic engineering techniques.

  The test compound used in this method is not particularly limited. For example, natural compounds, organic compounds, inorganic compounds, proteins, peptides and other single compounds, compound libraries, gene library expression products, cell extraction Product, cell culture supernatant, fermented microorganism product, marine organism extract, plant extract and the like.

  The “contact” of the test compound to the cell expressing the synoviolin gene is usually performed by adding the test compound to the culture medium of the cell expressing the synoviolin gene, but is not limited to this method. When the test compound is a protein or the like, the “contact” can be performed by introducing a DNA vector expressing the protein into the cell.

  Next, in this method, the expression level of the synoviolin gene is measured. Here, “gene expression” includes both transcription and translation. The gene expression level can be measured by methods known to those skilled in the art.

  For example, mRNA is extracted from cells expressing the synoviolin gene according to a standard method, and the transcription amount of the gene is measured by performing Northern hybridization method, RT-PCR method, DNA array method, etc. using this mRNA as a template. be able to. In addition, the amount of translation of the gene can also be measured by recovering the protein fraction from cells expressing the Synoviolin gene and detecting the Synoviolin protein expression by electrophoresis such as SDS-PAGE. Furthermore, it is also possible to measure the translation amount of a gene by detecting the expression of the protein by carrying out Western blotting using an antibody against Synoviolin protein. The antibody used for the detection of synoviolin protein is not particularly limited as long as it is a detectable antibody. For example, both a monoclonal antibody and a polyclonal antibody can be used.

  In this method, a compound that reduces the expression level is then selected as compared with the case where the test compound is not contacted (control). The compound to be reduced becomes an agent for a method for producing an osteoporosis model animal in which osteoblast activity is inhibited.

  Further, a compound that reduces (suppresses) the activity of the protein is selected as compared with the case where the test compound is not contacted (control). A compound that lowers (suppresses) becomes a drug for cancer treatment. An example of the effect of synoviolin protein on the activity is the effect of synoviolin protein on self-ubiquitination.

Effect of Synoviolin Protein on Self-Ubiquitination For example, JP 2008-74753 (Patent No. 5008932) discloses plumbagin (2-methyl-5-hydroxy-1,4-naphthoquinone) and quercetin (2- (3 , 4-dihydroxyphenyl) -3,5,7-trihydroxy-4H-1-benzopyrano-4-one) is disclosed to inhibit the self-ubiquitination of synoviolin protein. Synoviolin self-ubiquitination means ubiquitination of a protein caused by interaction between synoviolins, as disclosed in Japanese Patent Application Laid-Open No. 2008-74753. Protein ubiquitination occurs by synoviolin binding to the protein.

  The effect of synoviolin protein on self-ubiquitination may be confirmed using the method disclosed in Japanese Patent Application Laid-Open No. 2008-74753 (Patent No. 5008932). For example, a test substance is added to an in vitro self-ubiquitination reaction solution of MBP-Syvn1 ΔTM-His and reacted at 37 ° C. for 30 minutes. After the reaction, ubiquitinated protein is detected by Western blotting using an anti-HA antibody. MBP-Syvn1 ΔTM-His means synoviolin deficient in the transmembrane region in which maltose binding protein (MBP) is fused on the N-terminal side and His tag is fused on the C-terminal side.

  In an embodiment of the present invention, the Synoviolin inhibitor is that of Synoviolin siRNA. A nucleic acid having an inhibitory action due to the RNAi effect is generally also called siRNA or shRNA. RNAi is a short double-stranded RNA (hereinafter abbreviated as “dsRNA”) consisting of a sense RNA consisting of a sequence homologous to the mRNA of a target gene and an antisense RNA consisting of a complementary sequence to the cell. This is a phenomenon that induces destruction by specifically and selectively binding to a target gene mRNA, and efficiently inhibiting (suppressing) the expression of the target gene by cleaving the target gene. For example, when dsRNA is introduced into a cell, the expression of a gene having a sequence homologous to that RNA is suppressed (knocked down). As described above, RNAi can suppress the expression of a target gene, so that it can be applied as a simple gene knockout method instead of the conventional complicated and low-efficiency gene disruption method by homologous recombination, or a method applicable to gene therapy. It is attracting attention as. The RNA used for RNAi is not necessarily completely identical to the Synoviolin gene or a partial region of the gene, but preferably has perfect homology.

siRNA can be designed as follows.
(A) There is no particular limitation as long as it is a gene encoding Synoviolin, and any region can be used as a target candidate. For example, in the case of a human, an arbitrary region of GenBank accession number AB024690 (SEQ ID NO: 1) can be a candidate.
(B) A sequence beginning with AA is selected from the selected region, and the length of the sequence is 19 to 25 bases, preferably 19 to 21 bases. The GC content of the sequence may be selected, for example, from 40 to 60%.
(A) a step of bringing a test compound into contact with a cell expressing the synoviolin gene (b) a step of measuring the expression level of the synoviolin gene in the cell (c) as compared with the case where the test compound is measured in the absence of the test compound And selecting a compound that reduces the expression level

  Examples of Synoviolin siRNA include one nucleotide sequence selected from SEQ ID NOs: 2 to 6, nucleotide sequences complementary to these nucleotide sequences, or substitution or insertion of one or two bases from any of these nucleotide sequences , RNA having a deleted or added base sequence. The RNA having the nucleotide sequences shown in SEQ ID NOs: 2 to 4 is a Synoviolin siRNA. Izumi T, et al, Arthritis Rheum. 2009; 60 (1): 63-72., EMBO, Yamazaki S, et al. . , EMBO J. et al. 2007; 26 (1): 113-22. As known using experiments. It is known that RNA having the base sequences shown in SEQ ID NOs: 5 and 6 is Synoviolin siRNA, as disclosed in WO 2005/074988 using experiments.

SEQ ID NO: 5'-GCUGUGACAGAUGCCAUCA-3 '
SEQ ID NO: 5'-GGUGUUCUUUGGGCAACUG-3 '
SEQ ID NO: 5'-GGUUCUGCUGUACAUGGCC-3 '
SEQ ID NO: 5: 5′-CGUUCCUGUGUACCGCCGUCA-3 ′
Sequence number 6: 5'-GUUUTGGGUGACUGGUGUA-3 '

  “RNA having a base sequence in which one or two bases are substituted, inserted, deleted or added from any of these base sequences” means one base sequence selected from SEQ ID NOs: 2 to 6 and these bases RNA having a base sequence in which one or two bases are substituted, inserted, deleted or added from any base sequence of the base sequence complementary to the sequence. Any one kind of substitution, insertion, deletion or addition may occur, or two or more may occur.

  For example, International Publication WO2005-018675 Pamphlet and WO2005 / 074988 Pamphlet disclose siRNA for a gene encoding synoviolin and siRNA screening and evaluation methods for a gene encoding synoviolin. Also in the present invention, siRNA against a gene encoding synoviolin can be evaluated by appropriately using the method disclosed in this publication.

In one embodiment of the present invention, the Synoviolin inhibitor has a base sequence represented by SEQ ID NO: 7 or a base sequence in which one or two bases are substituted, inserted, deleted or added from the base sequence represented by SEQ ID NO: 7. Synoviolin decoy nucleic acid. The fact that the nucleic acid having the base sequence represented by SEQ ID NO: 7 is a synoviolin decoy nucleic acid is described in, for example, Tsuchimochi K, et al. , Mol Cell Biol. 2005; 25 (16): 7344-56. As shown with examples.
SEQ ID NO: 7: 5′-AUGGUGACGUGUGCUAGA-3 ′

  Synoviolin decoy nucleic acids and methods for confirming them are known as disclosed in, for example, International Publication WO 2005-093067 and International Publication WO 2005-074988.

  In one embodiment of the present invention, the Synoviolin inhibitor is a Synoviolin antisense nucleic acid. Methods for screening for Synoviolin antisense nucleic acid and Synoviolin antisense nucleic acid are disclosed, for example, in JP-A-2009-155204, Table No. 2006-137514, and Table No. 2005-074988. Synoviolin antisense nucleic acid means a nucleic acid having a sequence complementary to the synoviolin gene and capable of inhibiting the expression of the synoviolin gene by hybridizing to the gene. An antisense nucleic acid can be prepared by a synthetic chemical method or the like by a nucleic acid compound complementary to a partial base sequence of a gene encoding synoviolin. In order to evaluate whether the nucleic acid compound efficiently inhibits synoviolin production, a screening test using the gene expression level as an index may be performed. As the antisense nucleic acid compound, for example, the expression of Synoviolin can be suppressed to at least 50% or less as compared with the control.

  As a method for inhibiting the expression of a specific endogenous gene, a method using an antisense technique is well known to those skilled in the art. There are a number of factors that cause the antisense nucleic acid to inhibit the expression of the target gene as follows. Inhibition of transcription initiation by triplex formation, transcription inhibition by hybridization with a site where an open loop structure is locally created by RNA polymerase, transcription inhibition by hybridization with RNA that is undergoing synthesis, intron and exon Splicing inhibition by hybridization at the junction with nuclease, inhibition of splicing by hybridization with spliceosome formation site, inhibition of transition from nucleus to cytoplasm by hybridization with mRNA, hybridization with capping site and poly (A) addition site Inhibition of splicing, translation initiation inhibition by hybridization with a translation initiation factor binding site, translation inhibition by hybridization with a ribosome binding site near the initiation codon, hybridization with mRNA translation region and polysome binding site Outgrowth inhibitory peptide chain by de formation, and gene expression inhibition by hybrid formation at sites of interaction between nucleic acids and proteins, and the like. In this way, antisense nucleic acids inhibit the expression of target genes by inhibiting various processes such as transcription, splicing or translation (Hirashima and Inoue, Shinsei Kagaku Kougaku 2 Nucleic acid IV gene replication and expression, Japan biochemical Academic Society, Tokyo Kagaku Dojin, 1993, 319-347.).

  The antisense nucleic acid used in the present invention may inhibit the expression and / or function of the Synoviolin gene by any of the actions described above. In one embodiment, if an antisense sequence complementary to the untranslated region near the 5 'end of the Synoviolin gene mRNA is designed, it is considered effective for inhibiting gene translation. In addition, a sequence complementary to the coding region or the 3 ′ untranslated region can also be used. Thus, not only the translation region of the Synoviolin gene but also the nucleic acid containing the antisense sequence of the sequence of the untranslated region is included in the antisense nucleic acid used in the present invention. The antisense nucleic acid used is linked downstream of a suitable promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 ′ side. The nucleic acid thus prepared can be transformed into a desired animal (cell) by using a known method. The sequence of the antisense nucleic acid is preferably a sequence complementary to the endogenous synoviolin gene or a part thereof possessed by the animal (cell) to be transformed. However, as long as the gene expression can be effectively suppressed, May not be complementary. The transcribed RNA preferably has a complementarity of 90% or more, most preferably 95% or more, with respect to the transcription product of the target gene. In order to effectively inhibit the expression of a target gene (Shinobi folding) using antisense diffusion, the length of the antisense nucleic acid is preferably at least 15 bases and less than 25 bases. The nucleic acid is not necessarily limited to this length, and may be, for example, 100 bases or more, or 500 bases or more.

  Inhibition of the expression of the synoviolin gene can also be carried out using a ribozyme or a DNA encoding a ribozyme. A ribozyme refers to an RNA molecule having catalytic activity. Although ribozymes have various activities, research focusing on ribozymes as enzymes that cleave RNA has made it possible to design ribozymes that cleave RNA in a site-specific manner. Some ribozymes have a size of 400 nucleotides or more, such as group I intron type and M1 RNA contained in RNAse P, but some have an active domain of about 40 nucleotides called hammerhead type or hairpin type. (Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzyme, 1990, 35, 2191.).

  For example, the self-cleaving domain of the hammerhead ribozyme cleaves 3 ′ of C15 in the sequence G13U14C15, and base pairing between U14 and A9 is important for its activity. Instead of C15, A15 or U15 Has also been shown to be cleaved (KOizumi, M, et al., FEBS Lett, 1988, 228, 228.). By designing a ribozyme whose substrate binding site is complementary to the RNA sequence in the vicinity of the target site, a restriction enzyme-like RNA-cleaving ribozyme that recognizes the sequence UC, UU or UA in the target RNA can be created (KOizumi, M. et al., FEBS Lett, 1988, 239, 285., Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzymes, 1990, 35, 2191., Koizumi, M. et al., Nucl Acids, 1989, 17, 7059. ).

  Hairpin ribozymes are also useful for the purposes of the present invention. This ribozyme is found, for example, in the minus strand of satellite RNA of tobacco ring spot virus (Buzyan, JM., Nature, 1986, 323, 349.). It has been shown that target-specific RNA-cleaving ribozymes can also be produced from hairpin ribozymes (Kikuchi, Y. & Sasaki, N., Nucl Acids Res, 1991, 19, 6751., Hiroshi Kikuchi, Chemistry and Biology, 1992, 30, 112.). Thus, the expression of the gene can be inhibited by specifically cleaving the synoviolin gene transcript in the present invention using a ribozyme.

  Inhibition of the expression of the endogenous gene can also be performed by RNA interference (RNA interference, hereinafter abbreviated as “RNAi”) using double-stranded RNA having the same or similar sequence as the target gene sequence.

  The agent of the present invention described above can be administered either orally or parenterally. In the case of parenteral administration, pulmonary dosage forms (for example, those using a nephriser etc.), nasal dosage forms, transdermal dosage forms (for example, ointments, creams), injection dosage forms and the like can be mentioned. In the case of an injection type, it can be administered systemically or locally by intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like.

  The administration method is appropriately selected according to the patient's age and symptoms. The effective dose is 0.1 μg to 100 mg, preferably 1 to 10 μg per kg body weight per time. However, the therapeutic agent is not limited to these doses. When nucleic acids such as siRNA are mixed, an example of the dosage of the nucleic acid is 0.01 to 10 μg / ml, preferably 0.1 to 1 μg / ml.

  The agent of the present invention can be formulated according to a conventional method and may contain a pharmaceutically acceptable carrier or additive. Such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, carboxymethyl. Sodium starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, pharmaceutical Examples of acceptable surfactants include additives.

  The above additives are selected from the above alone or in appropriate combination depending on the dosage form of the therapeutic agent of the present invention. For example, when used as an injectable preparation, a purified ER stress inducer is dissolved in a solvent (eg, physiological saline, buffer solution, glucose solution, etc.), and Tween 80, Tween 20, gelatin, human serum albumin, etc. are added thereto. Can be used. Alternatively, it may be lyophilized to obtain a dosage form that dissolves before use. As the excipient for lyophilization, for example, sugar alcohols and saccharides such as mannitol and glucose can be used.

  The present invention produces a knockout mouse of a synoviolin gene, thereby inhibiting the expression of synoviolin or inhibiting the activity of synoviolin, thereby being effective in producing an osteoporosis model animal that inhibits the activity of osteoblasts. I found it. Synoviolin gene knockout mice mean that the synoviolin gene region has been artificially altered to inhibit its normal expression, and as a result, synoviolin is not functioning normally in the body. To do.

  Synoviolin gene can be partially or completely modified or deleted. Here, “all” refers to the region from the 5 ′ end of exon 1 to the 3 ′ end of the last exon of Synoviolin genomic DNA. Further, “part” means a part of this region and a length necessary for inhibiting normal expression of the synoviolin gene. Furthermore, “modification” means that the nucleotide sequence of the target region in genomic DNA is altered to another nucleotide sequence by replacing, deleting, inserting, and / or transposing single or multiple nucleotides. .

  A knockout animal in which a part or all of the Synoviolin gene is modified or deleted can be produced by a known method. For example, it can be prepared using a gene targeting method as described in the Examples below. In this method, normal expression of Synoviolin can be inhibited by substituting a region containing at least the start codon of exon 1 of Synoviolin gene with another base sequence by homologous recombination. Moreover, the knockout animal used for the screening method of the present invention includes not only the knockout animal produced by the same method but also its offspring.

  The animal that is the subject of the knockout animal of the present invention is a non-human animal and is not particularly limited. For example, mammals such as cows, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats and the like are exemplified. Of these, rabbits, dogs, cats, guinea pigs, hamsters, mice, and rats are preferred for use as experimental animals, of which rodents are more preferred, and many inbred strains have been produced. Considering techniques such as in vitro fertilization, mice and rats are particularly preferable.

  To construct a targeting vector, a part of the synoviolin gene of the target animal is isolated. For example, when a knockout mouse is prepared, a synoviolin gene is screened from a mouse genomic DNA library. Using the obtained genomic DNA clone, a targeting vector for homologous recombination is constructed. The genomic DNA clone need not be the full length of the gene, but only the region necessary for disrupting the synoviolin gene and suppressing the expression of synoviolin. A targeting vector can be prepared by a known method. For example, using a commercially available plasmid as a backbone, each fragment such as the genomic DNA clone, a marker for positive selection, and a marker for negative selection is appropriately linked. Can be produced.

  The targeting vector prepared by the above method is introduced into a cell having an individual forming ability (differentiating totipotency) such as a fertilized egg, early embryo, or embryonic stem cell (ES cell) by electroporation, etc. Cells that have undergone homologous recombination are selected. Sorting can be done using drugs by a positive-negative selection method. After selection, the cells in which the desired homologous recombination has occurred are confirmed by Southern blotting or PCR. The cells finally confirmed to have the desired homologous recombination are introduced into 8-cell embryos or blastocysts (blast cysts) collected from the oviduct or uterus during pregnancy.

  The 8-cell embryo or blastocyst is transplanted to a temporary parent according to a conventional method. By crossing a germline chimeric animal (preferably male) born from a foster parent with a wild type animal (preferably female) having a wild type synv1 gene homozygously, the first generation (F1) is obtained on the homologous chromosome. A heterozygote can be obtained in which one of the synv1 genes is disrupted by homologous recombination or can be deleted under certain conditions or in a site-specific manner. Furthermore, by mating these heterozygotes, as a second generation (F2), both synv1 genes on the homologous chromosome are disrupted, or can be deleted under certain conditions or site-specifically. That is, the synv1 knockout animal of the present invention can be obtained. Homozygotes can be identified by cutting a part of the body (for example, the tail), extracting the DNA, and examining the genotype by Southern blotting or PCR.

  The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples.

<Production Example 1: Production of Synoviolin Knockout Mouse>
FIG. 39 shows a design diagram of the gene targeting vector used for the preparation of the systemic conditional knockout (KO) mouse of this example (synovioline knockout mouse (syvn1 cKO mouse)). In the figure, in order from the top, a normal synoviolin gene (Wild allele), a targeting vector for producing Synoviolin knockout mice (Targeting VeCTor), an allele that has undergone homologous recombination (targeted allele), and an allele introduced with a loxP sequence ( The structure of an allele from which (Flox Allele) and loxP-Exon2 to 14-loxP have been removed is schematically shown.

  The 14.8 kb gene region, which is the region upstream of exon 1 to the downstream of exon 16 of the mouse synoviolin gene, was used for the construction of the targeting vector. A Neo resistance gene sandwiched between FRT sequences was inserted between exon 1 and exon 2. In addition, a loxP sequence was introduced upstream of exon 2 and downstream of exon 14.

  The above targeting vector was introduced into ES cells. Clones having the allele in which the desired homologous recombination occurred were selected by confirming the removal of the loxP-exon-loxP sequence by Cre treatment and the removal of FRT-neomycin-FRT by FLP treatment by the length of the PCR product. .

  A chimeric mouse was obtained by introducing the ES cell clone in which homologous recombination occurred into a mouse embryo according to a known method (for example, EMBO J 16: 1850-1857). Furthermore, this chimeric mouse was crossed with a wild type C57BL / 6 mouse to obtain a mouse from which the neomycin sequence was removed. Moreover, since the loxP sequence between the exon and the Longarm incorporated in the targeting vector may be deleted during homologous recombination, the presence was confirmed by PCR.

The obtained neomycin-removed mice were crossed with CAG-Cre-ER mice to obtain CAG-Cre-ER; syvn1 ^{flox / flox} mice.

(Example 1: Analysis of osteoblast-specific conditional KO mice by pQCT method)
In order to investigate the possibility of a correlation between synoviolin and bone mass, bone density was measured using osteoblast-specific conditional KO mice. Male and male KO mice were used for the measurement, and 0.02 mg / g body weight, 1 mg / g body weight 01-01- 2 mg / g tetracycline as a bone labeling agent (fluorescent Ca chelator) for morphometry. The femur was excised from each mouse by subcutaneous administration with a label schedule of 01-01 (one day after tetracycline administration and calcein administration, and another day after bone collection). The femur was similarly removed from male and female control mice. Using each extracted femur as a measurement sample, XCT Research SA + (Stratec Medizintechnik GmbH, Pforzheim, Germany) was used to measure the metaphysis of 1.2 mm from the distal growth plate cartilage and about ½ of the diaphysis of the bone length. For two slices, the bone density was measured by the pQCT method (peripheral quantitative computed tomography method). The results are shown in FIG.

  Total bone density (FIG. 1a), cancellous bone density (FIG. 1b) and cortical bone density (FIG. 1c) were decreased in KO mice compared to control mice in both sexes. Cortical bone thickness (FIG. 1d) was also decreased in KO mice compared to control mice in both sexes. Moreover, it was recognized that the cortical epicardial perimeter (FIG. 2a) and the cortical endosteal perimeter (FIG. 2b) were lower in KO mice than in control mice. Furthermore, the X-axis bone strength index (strength in the three-point bending direction) (Fig. 2c) and the polar coordinate bone strength index (strength in the torsional bending direction) (Fig. 2d) also decreased in the KO mice compared to the control mice. It was recognized that

  From these results, it was clarified that the bone of KO mice is being coarsened.

(Example 2: Analysis of osteoblast-specific conditional KO mice by two-dimensional micro CT)
In order to investigate the possibility of a correlation between Synoviolin and bone mass, bone microstructure was quantified using osteoblast-specific conditional KO mice.

  First, KO mice and control mice were treated in the same procedure as described in Example 1, and the femur was extracted from each mouse. Quantification of bone microstructure by micro CT method (computed tomography method) using Scan Xmate-L090 (Comscan Techno Co., Ltd.) with an area of 0.507 mm below to 2.609 mm as the measurement sample. Went. As analysis software, TRI / 3D-BON (Ratok System Engineering Co., Ltd.) was used. Further, the conditions of voltage 75 kV, current 100 μA, magnification factor 9.660 times, resolution 10.352 μm / pixel, and slice thickness 10.352 μm were used. The results are shown in FIGS.

  The ratio of bone mass per tissue mass (BD / TV) (Fig. 3a), trabecular width (Tb.Th) (Fig. 3b) and trabecular number (Tb.N) (Fig. 3c) were determined in both male and female control mice. In comparison, it decreased in KO mice and increased trabecular gap (Tb.Sp) (FIG. 3d). From these facts, it was revealed that the bone of the KO mouse is being coarsened.

  In addition, Marro Star volume (V * m.space) (FIG. 4a), which is the average volume of the bone marrow cavity in a range that can be seen without being blocked by the trabecular bone from all points in the bone marrow cavity, is a control mouse in both males and females. Increased in KO mice. Conversely, the trabecular star volume (V * tr) (FIG. 4b), which is the average of the volume from the point in the trabecular bone to the end of the bone mass in all directions, is higher in both males and females than in control mice. It was decreasing at. In addition, the number of Nd per tissue volume (Fig. 4c) when the connection point of trabecular bone is defined as a nodule (Nd) and the free end that is not connected with other bone mass is defined as the end (Fig. 4c) is the control mouse. Decreased in KO mice. Furthermore, the total length of skeletal lines per tissue volume (Total strut length) (FIG. 4d) was also decreased in KO mice compared to control mice in both males and females. FIG. 5 shows an image actually taken by micro CT.

  From these results, it became clear that the bone of KO mice is being coarsened.

Example 3 Bone Morphology Measurement Using Osteoblast Specific Conditional KO Mouse Knee Joint Preserved Slice
In order to investigate the effect of Synoviolin KO on bone morphology in detail, we prepared mouse femur and tibia specimens and measured bone morphology. Samples for bone morphometry were prepared as follows.

Male and male KO mice were used, and 2 mg / mL tetracycline was administered subcutaneously on the label schedule of 0.02 mg / g body weight, 1 mg / g body weight 01-01-01-01 as a bone marker (after tetracycline administration) 1 day later, calcein was administered, and another day later, bone was collected), and the tibia and femur were removed from each mouse.
Fix with 70% ethanol. Unnecessary soft tissues were removed and cut (trimmed) with a technical micromotor (Hypertech II, TYPE: # 93706J), and then immersed in Villanueva Bone Stain solution for 6 to 7 days to perform block staining.

  In order to prepare a sliced specimen, the block-stained sample was dehydrated stepwise with 70% ethanol to 100% ethanol and replaced with acetone, acetone / methyl methacrylate monomer mixture, methyl methacrylate monomer, and then methyl methacrylate resin ( Embedded in MMA resin). This was polymerized over about 10 days while raising the temperature from 30 ° C to 43 ° C. After the polymerization, unnecessary portions were cut with a diamond saw and formed into a block, and were cut into thin pieces with a thickness of 5 μm using a microtome (rotary microtome, Leica RM2255). Sections were encapsulated with a photo-curing encapsulant and made into thin slices.

  For measurement of bone morphology, a Histometry RT digitizer (System Supply Co., Ltd.) was used as the bone morphology measurement system, and a CSS-840 cancellous bone measurement version was used as the measurement software. Measurements were made on the items of articular cartilage width measurement, cancellous bone morphology measurement, growth rate measurement, hypertrophic chondrocyte measurement, number of osteoclast cells, and subarticular cartilage bone width measurement.

  In addition, the knee joint-preserved sliced specimen was observed using a microscope. For the observation, an epifluorescence polarization microscope OLYMPUS BX-53 was used.

  Knee-preserving sliced specimens were prepared according to the above procedure, and the articular cartilage width, subchondral bone width, and joint gap of the extracted femur and tibia were measured. The results are shown in FIGS. Further, FIG. 9 shows an image obtained by observing these knee joint-preserving sliced specimens with a microscope.

  In males, the articular cartilage width outside the tibia and the articular cartilage width inside the tibia were found to be narrower in KO mice than in control mice (FIGS. 6a and 6b). On the other hand, the width of the articular cartilage inside the tibia was wider in females than in control mice (FIG. 6b). In both males and females, there was no difference in the cartilage width outside the femur and the cartilage width inside the femur between the control mouse and the KO mouse (FIGS. 6c and 6d).

  In both sexes, the width of subchondral bone was found to be decreased in KO mice compared to control mice at all measured sites (FIG. 7). In females, the subchondral bone was marked, and ectopic calcification was observed (Fig. 9).

  In males, the inner joint gap was found to be narrower in KO mice than in control mice (FIG. 8b). In both males and females, there was no difference in the outer joint gap between control mice and KO mice (FIG. 8a).

  Next, we measured the growth rate, growth cartilage width, hypertrophic chondrocyte count, and number of chondrocytes using the knee-preserved slices of the isolated femur and tibia of the mouse. The results are shown in FIGS.

  In both sexes, the rate of long axis bone formation was increased in KO mice compared to control mice (FIG. 10a). In females, the growth plate cartilage width was found to be wider (FIG. 10b). From the microscopic images, KO mice have a wider range of labeling for the tetracycline-derived fluorescence (yellow) administered the first time and the calcein-derived fluorescence (green) administered the second time compared to the control mice. , It can be seen that the long bone formation rate has increased (FIG. 11). In addition, among the cells constituting the growth plate cartilage in both males and females, terminally differentiated hypertrophic chondrocytes were decreased in KO mice compared to control mice (FIGS. 10c and 10d). Furthermore, in female KO mice, hypertrophic chondrocytes were heterogeneous, and the regular arrangement from growth plate cartilage to primary cancellous bone seen in control mice was lost (FIG. 12). In females, it was revealed that the number of osteoclast cells was decreased in KO mice compared to control mice (FIG. 13).

(Example 4: Bone morphometry using an osteoblast-specific conditional KO mouse using a femoral shaft polished specimen)
In the same procedure as described in Example 3, a block-stained sample was prepared. In order to prepare a polished specimen, unnecessary resin was cut from the sample after block staining using a diamond saw, and then the cut surface was molded using a polishing sheet. Furthermore, the block was cut | disconnected with the micro cutter and the sticking surface was grind | polished using the abrasive sheet. Adhering the molded block of the suction plate with a photo-curing encapsulant, rough polishing using a speed wrap (Malto ML-521-d) (adsorption plate + about 25 μm), and finishing polishing while checking the state of the specimen with a microscope (Adsorption plate + about 15 μm). Thereafter, the section was sealed using a light encapsulant to prepare a polished specimen.

  For measuring bone morphology, a Histometry RT digitizer (System Supply Co., Ltd.) was used as the bone morphology measuring system, and a CSS-840 cortical bone measurement version was used as the measurement software.

  We also observed the femoral shaft polished specimen using a microscope. For the observation, an epifluorescence polarization microscope OLYMPUS BX-53 was used.

  Morphological measurement was performed using a polished mouse femoral shaft specimen prepared according to the above procedure. The results are shown in FIGS.

  In both males and females, the total column (FIG. 14a), bone marrow volume (FIG. 14b), and cortical bone width (FIG. 14c) were decreased in KO mice compared to control mice. In addition, when comparing the ratios of the resorption surface (ES), osteoid surface (OS), and rest surface (QS) on the cortical epicardial surface and cortical endocardial surface, the cortex was higher in KO mice than in control mice in both males and females. The absorptive surface decreased in both the epicardial surface and the cortical endosteal surface (FIGS. 15a and 15b). Furthermore, in KO mice, an increase in the porous area was observed (FIG. 15c).

  Next, the bone mineralization rate (FIG. 16a), the bone formation rate (FIG. 16b), and the ratio of the mineralized surface per bone surface (FIG. 16c) were compared. In males, both the cortical epicardial surface (Ps) and the cortical endosteal surface (Es) increased in both KO mice compared to the control mice. On the other hand, in the female, the cortical epicardial surface (Ps) showed an increase in all items in the same manner as the male, but in the cortical endosteal surface (Es), all items decreased.

  Next, FIG. 17 shows the results of microscopic observation of the femoral shaft polished specimen.

  It was observed that the cross section that was elliptical in the control mouse was circular in the KO mouse and the diameter was small. In the KO mouse, the cortical intimal surface was darkly stained with Villanueva Bone staining, which suggests that bone remodeling was not performed normally and old bone remained. Furthermore, in KO mice, bone cells were enlarged and bone tubules were thick and interrupted.

(Example 5: cancellous bone morphometry using an osteoblast-specific conditional KO mouse femoral shaft polished specimen)
In the same procedure as in Example 3, using a mouse-extracted femur, a knee joint-preserving sliced specimen was prepared, and cancellous bone morphology was measured. The results are shown in FIGS.

  Comparing the proportions of resorption surface (ES), osteoid surface (OS), and resting surface (QS) on the secondary cancellous bone surface, the proportion of resorption surface (ES) in KO mice is slightly higher in both males and females than in control mice. However, the proportion of the osteoid surface decreased (FIG. 18a). In both males and females, the number of osteoclasts increased in KO mice (FIG. 18b) and the osteoid width decreased (FIG. 18c). In female KO mice, the number of osteoblasts was reduced (FIG. 18d).

  Next, the bone mineralization rate (Fig. 19a) and the bone formation rate (Figs. 19b and 19c) on the secondary cancellous bone surface were compared. In males, there was no difference between control mice and KO mice in terms of bone mineralization rate and bone formation rate. On the other hand, in females, both bone mineralization rate and bone formation rate were lower in KO mice than in control mice. In addition, the calcified surface per bone surface (FIG. 19d) increased in males compared to control mice but decreased in females.

  Next, the bone mass and trabecular width on the secondary cancellous bone surface were measured. The results are shown in FIG.

  When the bone mass on the secondary cancellous bone surface was compared, both males and females had decreased in KO mice compared to control mice (FIG. 20a). When the trabecular width was compared, no difference was observed between control mice and KO mice in males, but thinning was observed in KO mice in females (FIG. 20b).

  Next, FIG. 21 shows the results of microscopic observation of the knee joint-preserved sliced specimen.

  On the secondary cancellous bone surface of KO mice, a decrease in osteoids was observed. In addition, the regular arrangement of osteoblasts found in control mice was lost.

(Example 6: Analysis of systemic conditional KO mice by pQCT method)
In order to examine the possibility of a correlation between Synoviolin and bone mass, bone mineral density was measured using systemic conditional KO mice. Male and male KO mice were used for the measurement, and 2 mg / mL tetracycline was used as a bone labeling agent for morphometry, and the label schedule was 0.02 mg / g body weight, 1 mg / g body weight 01-01-01-01. Was administered subcutaneously (calcein was administered 1 day after tetracycline administration, and bone was collected after another day), and the femur was removed from each mouse. The femur was similarly removed from male and female control mice. Using each extracted femur as a measurement sample, XCT Research SA + (Stratec Medizintechnik GmbH, Pforzheim, Germany) was used to measure the metaphyseal part of 1.2 mm from the distal growth plate cartilage and about ½ of the diaphysis of the bone length. For two slices, the bone density was measured by the pQCT method (peripheral quantitative computed tomography method). The results are shown in FIG.

  Total bone density (FIG. 22a) and cortical bone density (FIG. 22c) were decreased in KO mice compared to control mice in both sexes. Further, the cancellous bone density (FIG. 22b) was lower in males than in control mice. Cortical bone thickness (FIG. 22d) was also decreased in KO mice compared to control mice in both males and females. In addition, the cortical periosteal perimeter (FIG. 23a) and the cortical periosteal perimeter (FIG. 23b) were also lower in KO mice than in control mice. Furthermore, the X-axis bone strength index (strength in the three-point bending direction) (Fig. 23c) and the polar coordinate bone strength index (strength in the torsional bending direction) (Fig. 23d) are also lower in the KO mice than in the control mice. It was.

(Example 7: Analysis of systemic conditional KO mice by two-dimensional micro CT)
In order to investigate the possibility of a correlation between Synoviolin and bone mass, the bone microstructure was quantified using systemic conditional KO mice. First, KO mice and control mice were treated in the same procedure as described in Example 1, and the femur was extracted from each mouse. Quantification of bone microstructure by micro CT method (computed tomography method) using Scan Xmate-L090 (Comscan Techno Co., Ltd.) with an area of 0.507 mm below to 2.609 mm as the measurement sample. Went. As analysis software, TRI / 3D-BON (Ratok System Engineering Co., Ltd.) was used. Further, the conditions of voltage 75 kV, current 100 μA, magnification factor 9.660 times, resolution 10.352 μm / pixel, and slice thickness 10.352 μm were used. The results are shown in FIGS.

  The ratio of bone mass per tissue mass (BD / TV) (FIG. 24a), trabecular width (Tb.Th) (FIG. 24b) and trabecular number (Tb.N) (FIG. 24c) were determined in both control mice. In comparison, it decreased in KO mice and increased trabecular gap (Tb.Sp) (FIG. 24d). From these results, it was clarified that the bone of KO mice is being coarsened.

  In addition, Marro Star volume (V * m.space) (FIG. 25a), which is the average volume of the bone marrow cavity in a range that can be seen without being blocked by the trabecular bone in all directions from the point in the bone marrow cavity, is a control mouse. Increased in KO mice. Conversely, the trabecular star volume (V * tr) (FIG. 25b), which is the average of the volume from the point in the trabecular bone to the end of the bone mass in all directions, is KO mice compared to control mice in both males and females. It was decreasing at. In addition, when the connection point of trabecular bone is defined as a nodule (Nd) and the free end that is not connected to other bone mass is defined as the end, the Nd number per tissue mass (FIG. 25c) is the control mouse for both males and females. Decreased in KO mice. Furthermore, the total length of skeletal lines per tissue mass (Total strut length) (FIG. 25d) was also decreased in KO mice compared to control mice in both males and females. FIG. 26 shows an image actually taken by micro CT.

  From these results, it became clear that the bones of KO mice are becoming coarse.

(Example 8: Bone morphometry using a knee joint-preserved slice of a generalized conditional KO mouse)
In order to investigate the effect of Synoviolin KO on bone morphology in detail, we prepared mouse femur and tibia specimens and measured bone morphology. Samples for bone morphometry were prepared in the same procedure as described in Example 3, and bone morphometry and microscopic observation were performed in the same manner.

  The width of the articular cartilage was measured using the prepared knee-preserved slices of the femur and tibia. The results are shown in FIG. Further, FIG. 29 shows an image obtained by observing these knee joint-preserving sliced specimens with a microscope.

  In males, the width of articular cartilage outside the tibia and the width of articular cartilage outside the femur were found to be narrower in KO mice than in control mice (FIGS. 27a and 27c). In males, the width of the cartilage inside the tibia was wide (FIG. 27b). There was no difference in the cartilage width inside the femur in both sexes (FIG. 27d).

  Next, we measured the growth rate, growth cartilage width, hypertrophic chondrocyte count, and number of chondrocytes using the knee-preserved slices of the isolated femur and tibia of the mouse. The results are shown in FIGS.

  In both males and females, the long bone formation rate was increased in KO mice compared to control mice (FIG. 28a), and the growth plate cartilage width was shortened (FIG. 28b). Furthermore, among the cells constituting the growth plate cartilage in both sexes, the terminally differentiated hypertrophic chondrocytes were significantly reduced in the KO mice compared to the control mice (FIGS. 28c and 28d).

  Further, as a result of microscopic observation, in the KO mouse, the width of the growth plate cartilage was short and the number of hypertrophic chondrocytes was decreased (FIG. 29). In addition, deposition of the labeling agent was observed directly under the growth plate cartilage, and the appearance of endochondral ossification did not proceed normally (delay of the long axis bone growth rate). The number of osteoclast cells in both males and females was increased in KO mice compared to control mice (FIG. 30).

(Example 9: Measurement of bone morphology using a ground specimen of a femoral shaft of systemic conditional KO mice)
Using the femoral bone of the mouse, we prepared a ground specimen of the femoral shaft and measured the morphology. The results are shown in FIGS.

  In both males and females, no difference was observed between the control mice and the KO mice in the whole column (FIG. 31a). On the other hand, the amount of bone marrow was slightly increased in KO mice compared to control mice in both sexes (FIG. 31b). Cortical bone width was slightly decreased in KO mice compared to control mice (FIG. 31c).

  From these facts, bone remodeling from the bone marrow cavity may not function normally in the bones of KO mice, similar to the results obtained with pQCT.

  In addition, when comparing the ratios of the resorption surface (ES), osteoid surface (OS), and rest surface (QS) on the cortical epicardial surface and cortical endocardial surface, the cortex was higher in KO mice than in control mice in both males and females. The osteoid surface decreased on both the epicardial surface and the cortical endosteal surface (FIGS. 32a and 32b). In addition, the resorption surface increased on the cortical endosteal surface. Furthermore, an increase in the porous area was observed in KO mice compared to control mice (FIG. 32c).

  Next, the bone mineralization rate (FIG. 33a), the bone formation rate (FIG. 33b), and the ratio of the mineralized surface per bone surface (FIG. 33c) were compared. In both male and female KO mice, both the cortical epicardial surface (Ps) and the cortical endosteal surface (Es) decreased in both items compared to the control mice.

  Next, FIG. 34 shows the result of microscopic observation of the femoral shaft polished specimen.

  The control mouse had an elliptical cross section and the KO mouse had a circular shape. In KO mice, bone cells were enlarged and bone tubules were thick and interrupted.

(Example 10: cancellous bone morphometry using a whole body conditional KO mouse femoral shaft polished specimen)
Using a femoral bone from a mouse, we prepared a knee joint-preserved slice and measured the morphology of the cancellous bone. The results are shown in FIGS.

  Comparing the proportions of the resorption surface (ES), osteoid surface (OS), and resting surface (QS) on the secondary cancellous bone surface, the proportions of the resorbing surface and resting surface increased in KO mice compared to control mice in both males and females. , The proportion of osteoid surface decreased (Fig. 35a). In males, the number of osteoclasts increased in KO mice compared to control mice (FIG. 35b). Furthermore, the width of osteoids and the number of osteoblasts were decreased in KO mice compared to control mice in both sexes (FIGS. 35c and 35d).

  Next, the bone mineralization rate on the secondary cancellous bone surface (FIG. 36a), the bone formation rate (FIGS. 36b and 36c), and the ratio of the mineralized surface per bone surface (FIG. 36d) were compared. In both males and females, all items were lower in KO mice than in control mice.

  Next, the bone mass and trabecular width on the secondary cancellous bone surface were measured. In both males and females, the bone mass (FIG. 37a) and trabecular width (FIG. 37b) on the secondary cancellous bone surface were lower in KO mice than in control mice.

  Next, FIG. 38 shows the results of microscopic observation of the knee joint-preserved sliced specimen.

  On the secondary cancellous bone surface of KO mice, a decrease in osteoids was observed. In addition, the regular arrangement of osteoblasts found in control mice was lost. In addition, osteoclasts were multinucleated and enlarged, and bone resorption was actively observed. Bone cell hypertrophy was also observed. Furthermore, venous sinuses were observed in KO mice, suggesting that the individual was moribund.

  Since the present invention relates to a method for creating a model animal for osteoporosis, the present invention can be used, for example, in the pharmaceutical industry and the experimental equipment industry.

SEQ ID NO: 2: Synthetic RNA
Sequence number 3: Synthetic RNA
Sequence number 4: Synthetic RNA
Sequence number 5: Synthetic RNA
Sequence number 6: Synthetic RNA
Sequence number 7: Synthetic RNA

Claims (6)

  A method for producing a non-human osteoporosis model animal, comprising a step of inhibiting the expression or function of Synoviolin protein in a non-human mammal. The method of claim 1, comprising:
The step of inhibiting the expression or function of synoviolin protein in the non-human mammal comprises the step of knocking out or knocking down the synoviolin gene,
Method. The method of claim 1, comprising:
The non-human osteoporosis model animal is a non-human osteoporosis model animal in which osteoblast activity is inhibited,
Method. A method for evaluating the therapeutic effect of a test substance on osteoporosis,
Producing a non-human osteoporosis model animal according to the method of claim 1;
Administering the test substance to the non-human osteoporosis model animal;
Analyzing the non-human osteoporosis model animal to which the test substance has been administered,
Method. A carrier and a synoviolin inhibitor that inhibits the expression or function of synoviolin protein in a non-human mammal as an active ingredient,
The synoviolin inhibitor is
Synoviolin siRNA, Synoviolin decoy nucleic acid or Synoviolin antisense nucleic acid;
One base sequence selected from SEQ ID NOs: 2 to 6, a base sequence complementary to these base sequences, or a base in which 1 or 2 bases are substituted, inserted, deleted or added from any of these base sequences Synoviolin siRNA having the sequence; and a base sequence represented by SEQ ID NO: 7 or a synoviolin decoy having a base sequence in which one or two bases are substituted, inserted, deleted or added from the base sequence represented by SEQ ID NO: 7 Including any one or more of nucleic acids,
A composition for inhibiting the activity of osteoblasts. A composition for preparing an osteoporosis model animal, comprising the osteoblast activity inhibiting composition according to claim 5.

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