JP5207137B2 - Bcl-xL transgenic animals and uses thereof - Google Patents

Description

  The present invention relates to Bcl-xL transgenic animals and uses thereof. Specifically, the present invention relates to a Bcl-xL expression system, a transgenic animal, and use thereof useful for elucidating bone mass and bone strength.

Osteoporosis is a disease caused by bone loss and causes fractures. Increasing with the aging society, it is estimated that about 10 million people in Japan alone and about 200 million people worldwide are affected. Since bone loss begins with cancellous bone rather than cortical bone, fragile fractures occur at sites where cancellous bone is distributed more. That is, it occurs frequently in the vertebral body, the femoral neck, the distal end of the radius, the humeral neck, and the like. Because of the fractures caused by osteoporosis, many elderly people are bedridden, and the activation of society is reduced, and medical expenses and nursing care expenses are expanded.
As therapeutic agents for osteoporosis, there are activated vitamin D3, estrogen, selective estrogen receptor modulator, vitamin K2, bisphosphonate, parathyroid hormone (only approved in the United States) and the like. Most of these therapeutic agents suppress the activation of osteoclasts, and few drugs act on osteoblasts to promote bone formation. When osteoclasts are suppressed, bone increases, but bone remodeling decreases and bone quality decreases, which may reduce bone strength. Among the therapeutic agents, bisphosphonates and parathyroid hormone have been observed to decrease the fracture frequency, but this is still insufficient and the development of new therapeutic agents is desired.
Bcl-xL belongs to the Bcl-2 family and is a molecule that suppresses apoptosis. In addition to Bcl-2 and Bcl-xL, the Bcl-2 family includes BCL-w, BFL-1, A1, MCL-1, BOO, BRAG-1, NR-13, CDN-1, CDN-2, CDN- 3, BHRF-1, LMW5-HL, CED-9, and the like are known. Bcl-xL is mainly present on the outer membrane of mitochondria and inhibits the general pathway of apoptosis by at least partially preventing the release of cytochrome c into the cytoplasm (Non-Patent Documents 1 to 3). However, the function of Bcl-xL in bone formation and bone maintenance is not clear.
Adams, J .; M.M. , And Cory, S .; Science, 281: p. 1322-1326, 1998. Gross, A.M. McDonnell, J .; M.M. , And Korsmeyer, S .; J. et al. Genes Dev. , 13: p. 1899-1911, 1999. Tsumotomoto Y. J. et al. Cell. Physiol. 195: p. 158-167, 2003.

An object of the present invention is to provide a model animal useful for elucidating a disease that causes a decrease in bone mass and bone strength, such as osteoporosis, and a disease that causes an increase in bone mass and bone strength, and use thereof.
The present inventors pay attention to Bcl-xL, which plays a major role as an apoptosis-inhibiting type in the Bcl family, and examines whether normal bone mass can be increased by inhibiting apoptosis of osteoblasts. A Bcl-xL transgenic animal that was strongly expressed in osteoblasts using an osteoblast-specific promoter was produced. As a result of analyzing and intensively studying the transgenic animals, the present inventors have found the formation and maintenance of normal bone and the dose dependency between Bcl-xL and have completed the present invention. That is, the present invention is as follows.
[1] A bone mass and bone strength increasing agent comprising a gene encoding Bcl-xL.
[2] The increasing agent according to [1], wherein the Bcl-xL-encoding gene is operably linked downstream of an osteoblast-specific promoter.
[3] The increasing agent according to [2], wherein the promoter is a type I collagen promoter.
[4] The increasing agent according to any one of [1] to [3], which is an expression vector.
[5] A transgenic animal into which an expression vector containing a gene encoding an osteoblast-specific promoter and Bcl-xL has been introduced.
[6] The animal according to [5], wherein the promoter is a type I collagen promoter.
[7] The animal according to the above [5] or [6], wherein the bone mass and bone strength are increased as compared to the wild type animal of the same species.
[8] The animal according to any one of [5] to [7], wherein the expression of the Bcl-xL in osteoblasts is increased about twice as much as that of a wild-type animal.
[9] The animal according to any one of [5] to [7], wherein the expression of the Bcl-xL in osteoblasts is increased by about 3 to 5 times as compared with a wild-type animal of the same species.
[10] The animal according to any one of [5] to [7], wherein the expression of the Bcl-xL in osteoblasts is increased by about 10 times compared to a wild-type animal of the same species.
[11] The animal according to any one of [5] to [10], wherein the animal is a laboratory animal or a domestic animal.
[12] The animal according to [11], wherein the experimental animal is a mouse.
[13] The above-mentioned animal is an adult, and the number of cancellous bone trabeculae, the decrease in the distance between cancellous bone trabeculae, the increase in connectivity of cancellous bone, and the structure of cancellous bone are compared to adult wild type animals. The animal according to any one of [5] to [12], wherein at least one parameter selected from a measurement parameter group consisting of a change in model index has a significant difference.
[14] The animal according to any one of [5] to [13], which is used as a model in a state where bone mass and bone strength are increased.
[15] The animal according to any one of [5] to [13], which is used as a model for prevention or treatment of osteoporosis.
[16] A progeny animal of the animal according to any one of [5] to [15].
[17] A bone disease model animal obtained by crossing the animal according to any one of [5] to [16] with another disease model of the same species.
[18] The model animal according to [17], wherein the bone disease is osteoporosis, a disease that causes increased apoptosis of osteoblasts, a disease that causes increased proliferation of osteoblasts, or a disease that causes early aging .
[19] A tissue or cell obtained from the animal according to any one of [5] to [16] or the model animal according to [17] or [18].
[20] Increase in bone mass and / or bone strength, characterized by using the animal according to any one of [5] to [16] or the model animal according to [17] or [18] Screening method of substances to be caused.
[21] The following steps:
(A) a step of administering a test substance to the animal according to any one of [5] to [16] or the model animal according to [17] or [18],
(B) examining the bone mass and bone strength in an animal to which the test substance is administered and comparing the bone mass and bone strength in an animal to which the test substance is not administered; and (c) based on the comparison result, A method for screening a substance that increases bone mass and / or bone strength, comprising a step of selecting a test substance that increases bone strength.
[22] The following steps:
(A) a step of administering a test substance to the animal according to any one of [5] to [16] or the model animal according to [17] or [18],
(B) examining the bone mass and bone strength in an animal administered with the test substance, comparing with the bone mass and bone strength in an animal not administered with the test substance, and (c) based on the comparison result of step (b) Selecting a test substance that reduces bone mass and / or bone strength;
A method for screening a substance that decreases bone mass and / or bone strength.
[23] The following steps:
(A) a step of administering a test substance to the animal according to any one of [5] to [16] or the model animal according to [17] or [18],
(B) examining the bone mass and bone strength in an animal administered with the test substance, and comparing the bone mass and bone strength in an animal not administered with the test substance;
(C) selecting a test substance that reduces bone mass and / or bone strength based on the comparison result of step (b),
(D) a step of administering to the animal used in step (a) the test substance selected in step (c) and an antagonist candidate substance for the test substance;
(E) examining the bone mass and bone strength in the animal administered the test substance and the antagonist candidate substance in step (d), and comparing the bone mass and bone strength in the animal administered with the test substance alone; and (f) A screening method for a substance that increases bone mass and / or bone strength, comprising selecting an antagonist candidate substance that enhances bone mass and / or bone strength based on the comparison result of step (e).
[24] The following steps:
(A) contacting osteoblasts with a test substance;
(B) examining the expression level of Bcl-xL in osteoblasts contacted with the test substance, comparing with the expression level in osteoblasts not contacted with the test substance, and (c) based on the comparison result, A method for screening a substance that increases bone mass and / or bone strength through increased expression of Bcl-xL, comprising a step of selecting a test substance that increases the expression of Bcl-xL.
[25] A bone mass and bone strength increasing agent comprising Bcl-xL.
According to the bone mass and bone strength increasing agent of the present invention, since it has a Bcl-xL gene or Bcl-xL as a main component, the bone mass and the bone strength when introduced into osteoblasts and expressed. It is possible to form or regenerate a bone with an increase in the number of bones, that is, a bone having a cancellous bone excellent in quality and quantity. According to the transgenic animal and disease model animal of the present invention, Bcl-xL is strongly expressed in osteoblasts, thereby serving as an ideal model for elucidating various physiological states in bone tissue. Since the tissue or cell of the present invention is obtained from the animal, it can be used for studying bone mass and bone strength in vitro as well as in vivo. According to the screening method of the present invention using the animal, it is possible to contribute to the development of a drug that promotes bone formation via Bcl-xL, and has excellent quality and strength of bone formed.

FIG. 1 shows the results of Northern blotting of Bcl-xL mRNA expression in three strains of Bcl-xL transgenic mice (K1, K2 and K3) and wild type mice (WT).
FIG. 2 is a radiograph of the femurs of 10 week old female transgenic mice (K1, K2, and K3) and wild type mice (WT).
FIG. 3 is an X-ray photograph of the whole body (FIG. 3A), femur (FIG. 3B), and vertebra (FIG. 3C) of a 10-week-old transgenic mouse (K1) and a wild-type mouse (WT).
FIG. 4 is a photograph of a 10-week-old, 7-month-old, and 1-year-old transgenic mouse and wild-type mouse.
FIG. 5 is a diagram showing a comparison of various histological images in cortical bone of transgenic mice (K1) and wild-type mice (WT) at various weeks of age. From the top, HE image of 10-week-old male femur distal part, TUNEL staining of 10-week-old male femoral cortical bone, 4-week-old cortical bone transmission electron microscope image (TEM), and wrinkles of 10-week-old male femoral cortical bone Silver stained image, secondary electron image of bone cells and bone cell processes in 10-week-old cortical bone by scanning electron microscope (SEM).
FIG. 6 shows Northern blotting of type I collagen (Type I), osteopontin (OP) and osteocalcin (OC) expression in osteoblasts of 5 week old transgenic mice (K1) and wild type mice (WT). Shows the results of the investigation.
FIG. 7 shows the results of examining in situ hybridization the expression of HE staining, type I collagen, osteopontin and osteocalcin in the femurs of 10-week-old transgenic mice (K1) and wild-type mice (WT). .
FIG. 8 shows the results of examining DNA replication in osteoblasts using bromodeoxyuridine (BrdU).
FIG. 9 shows the results of examining the proportion of apoptosis in osteoblasts using the TUNEL method.
FIG. 10 is a graph showing a bone morphometric study in the cancellous bone region of the femur distal part of wild-type (WT) and Bcl-xL transgenic mice (K1).
FIG. 11 is a graph (upper and middle) showing the analysis results of the femoral shaft cortical bone by pQCT and a pQCT image (lower) of the femoral distal cortical bone.
FIG. 12 is a graph showing the analysis results of femoral shaft cortical bone by μCT.
FIG. 13 is a graph showing the analysis results of the femoral distal cancellous bone by pQCT.
FIG. 14 is a graph showing the analysis results of the femoral distal cancellous bone by μCT.
FIG. 15 is a μCT image of femoral distal cancellous bone (upper) and second lumbar cancellous bone (lower) of transgenic mice (K1) and wild-type mice (WT).
FIG. 16 shows a femoral distal X-ray image of a male transgenic mouse (K1) and a wild-type mouse at 10 weeks of age, 7 months of age, and 1 year of age, and a cancellous bone image of the distal femoral region by μCT.
FIG. 17 is a graph showing the analysis results of the trabecular bone cancellous bone mass (BV / TV) by μCT of male transgenic mice (K1) and wild-type mice at 10 weeks, 7 months and 1 year of age. Yes (mean ± SD, *: p <0.05. 10-week-old WT: n = 6, K1: n = 9; 7-month-old WT: n = 4, K1: n = 6; 1-year-old WT: n = 3, K1: n = 8).
FIG. 18 shows the results of examining the mRNA expression in transgenic mice and wild-type mice by Northern blotting. Left: mRNA expression of Bcl-2 in two strains of Bcl-2 transgenic mice (N2, N3) and wild type mice (WT); right: two strains of Bcl-xL transgenic mice (K1, K2) and wild type Bcl-xL mRNA expression in mice (WT).
FIG. 19 shows X-ray images of a 10-week-old male femoral distal part of a transgenic mouse and a wild-type mouse. Left: X-ray images of two strains of Bcl-2 transgenic mice (N2, N3) and wild type mice (WT); Right: two strains of Bcl-xL transgenic mice (K1, K2) and wild type mice (WT) ) X-ray image.
FIG. 20 shows a graph in which the bone density of cancellous bone and cortical bone of transgenic mice and wild type mice by pQCT was analyzed, and a cortical bone cross-sectional image.
Upper left: graph showing analysis of bone density of cancellous bone and cortical bone of 10-week-old male, Bcl-2 transgenic mouse (N2) (black circle) and wild-type mouse (white). WT: n = 4, N2: n = 5.
Lower left: graph showing analysis of bone density of cancellous bone and cortical bone of 10-week-old female, Bcl-xL transgenic mouse (K1) (black circle) and wild-type mouse (white). WT: n = 5, K1: n = 5.
Upper right: Cortical bone cross-sectional images of 10-week-old male, Bcl-2 transgenic mouse (N2) and wild type mouse.
Lower right: Cortical bone cross-sectional images of 10-week-old female, Bcl-xL transgenic mouse (K1) and wild type mouse.
FIG. 21 shows images of the trabecular bone of the femur of the 10-week-old male wild-type mouse (WT), Bcl-2 transgenic mouse (N2), and Bcl-xL transgenic mouse (K1) using μCT. A lumbar cancellous bone image is shown.
FIG. 22 shows cancellous bone (bone mass, bone) of 10-week-old male wild-type mouse (WT), Bcl-2 transgenic mouse (N2), and Bcl-xL transgenic mouse (K1) using μCT. Analysis results of connectivity, SMI, trabecular number, trabecular width) and cortical bone (cortical bone width) are shown (mean ± SD, *: p <0.05, WT: n = 6, N2: n = 7) , K1: n = 9).
FIG. 23 shows bone morphometry in the secondary cancellous bone region of the femoral centrifuge of 10-week-old male, wild-type mouse (WT), Bcl-2 transgenic mouse (N2), and Bcl-xL transgenic mouse (K1). The results are shown (WT: n = 5, N2: n = 4, K1: n = 4).
FIG. 24 shows BrdU and TUNEL stained images in a 2-week-old femoral primary cancellous bone region of wild-type mice (WT), Bcl-2 transgenic mice (N2), and Bcl-xL transgenic mice (K1). The percentage of positive osteoblasts to total osteoblasts in the primary cancellous bone region is shown in each panel. Further, positive cells are indicated by black arrows in the TUNEL-stained photograph.
FIG. 25 shows a graph in which the proportion of BrdU and TUNEL positive osteoblasts was calculated for each of a wild type mouse (WT), a Bcl-2 transgenic mouse (N2), and a Bcl-xL transgenic mouse (K1) (BrdU). WT: n = 6, N2: n = 6, K1: n = 4, mean ± SD, *: p <0.05; TUNEL WT: n = 12, N2: n = 4, K1: n = 7, mean ± SD, *: p <0.05).
FIG. 26 is a diagram showing a comparison of various histological images in cortical bone of transgenic mice (N2, K1) and wild-type mice (WT) at various weeks of age. From the top, HE image of 10-week-old male femur distal part, TUNEL staining of 10-week-old male femoral cortical bone, 4-week-old cortical bone transmission electron microscope image (TEM), and wrinkles of 10-week-old male femoral cortical bone It is a silver-stained image and a secondary electron image of bone cells and bone cell processes in 10-week-old cortical bone by a scanning electron microscope (SEM).
FIG. 27 shows type I collagen in osteoblasts of 4-week-old wild-type mice (WT) and Bcl-2 transgenic mice (N2), 5-week-old wild-type mice and Bcl-xL transgenic mice (K1). The result of having investigated the expression of (Type I collagen), osteopontin (osteopontin), and osteocalcin (osteocalcin) by the northern blotting is shown.
FIG. 28 shows the results of examining in situ hybridization the expression of type I collagen, osteopontin and osteocalcin in the tibia of an 8-week-old wild type mouse (WT), the tibia of an 8-week-old N2 mouse, and the tibia of a 10-week-old K1 mouse. Indicates.

In the present invention, “gene” is used to include not only double-stranded DNA but also each single-stranded DNA such as sense strand and antisense strand constituting the DNA. The length is not particularly limited. In the present invention, unless otherwise specified, a gene (DNA) is a double-stranded DNA containing human genomic DNA and a single-stranded DNA containing DNA (positive strand) and a single-stranded DNA having a sequence complementary to the positive strand. (Complementary strands) and any of these fragments are included. The “gene” includes not only a “gene” represented by a specific base sequence (SEQ ID NO: 1), but also a protein having a biological function equivalent to the protein encoded by these (eg, a homologue (homolog) And “splicing variants”, variants and derivatives) are included. The “gene” encoding such a homologue, variant or derivative is specifically a base sequence that hybridizes under stringent conditions with a complementary sequence of the specific base sequence represented by SEQ ID NO: 1. “Genes” having Here, stringent conditions are taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques in Enzymology, Vol. 152, Academic Press, San Diego T.). Can be determined based on For example, as washing conditions after hybridization, the conditions of about “1 × SSC, 0.1% SDS, 37 ° C.” can be given. The complementary strand is preferably one that maintains a hybridized state with the target positive strand even when washed under such conditions. Although it is not particularly limited, the severer hybridization conditions are about “0.5 × SSC, 0.1% SDS, 42 ° C.”, and the more severe hybridization conditions are “0.1 × SSC, 0.1% SDS, 65 ° C.” The cleaning conditions can be raised.
For example, as a gene encoding a homologue of a human-derived protein, genes of other species such as mouse and rat corresponding to the human gene encoding the protein can be exemplified, and these genes (homologues) are homogene (http: / /Www.ncbi.nlm.nih.gov/HomoloGene/). Specifically, a specific human base sequence is matched by BLAST (Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993, http://www.ncbi.nlm.nih.gov/BLAST/) ( Acquire the accession number of the sequence (Score is the highest, E-value is 0 and Identity is 100%). The UniGene Cluster ID (the number indicated by Hs.) Obtained by inputting the accession number into UniGene (http://www.ncbi.nlm.nih.gov/UniGene/) is input into Homogene. From the list showing the correlation of gene homologues between the other species gene and the human gene obtained as a result, genes of other species such as mice and rats as homologues corresponding to the human gene indicated by a specific base sequence Can be selected.
In the present invention, the gene encoding Bcl-xL (hereinafter sometimes abbreviated as Bcl-xL gene) may be any animal-derived gene. For the production of transgenic animals, an exogenous Bcl-xL gene is used, but for the production of a human model, it is preferable to use a human Bcl-xL gene. For example, the human Bcl-xL gene is described in Boise et al. (Boise L. H. et al., 1993, Cell 74 (4), pp. 597-608) and Genbank Accession No. Z23115 and the like can be isolated by a method known per se. In addition, the mouse Bcl-xL gene is a Genbank Accession No. L35049 and the like can be isolated by a method known per se.
In the present invention, “Bcl-xL gene” or “Bcl-xL DNA” means a human Bcl-xL gene (DNA) represented by a specific base sequence (SEQ ID NO: 1), and homologues, variants and derivatives thereof. It is used with the meaning which includes the gene (DNA) which codes etc. Specifically, the human Bcl-xL gene described in SEQ ID NO: 1, and mouse homologs and rat homologs thereof are included. The gene or DNA does not ask for distinction of the functional region, and can include, for example, an expression control region, a coding region, an exon, or an intron.
In the present invention, the term “protein” or “peptide” includes not only “protein” or “peptide” represented by a specific amino acid sequence (SEQ ID NO: 2), but also that the biological functions thereof are equivalent. , Homologues thereof (homologs and splice variants), mutants, derivatives, matured forms and amino acid modified forms. Here, examples of homologs include proteins of other species such as mice and rats corresponding to human proteins, and these were identified by HomoLoGene (http://www.ncbi.nlm.nih.gov/HomoloGene/). It can be identified a priori from the base sequence of the gene. Such variants include naturally occurring allelic variants, non-naturally occurring variants, and variants having amino acid sequences modified by artificial deletions, substitutions, additions and insertions. . Examples of the mutant include those that are at least 70%, preferably 80%, more preferably 95%, and still more preferably 97% homologous to a protein or (poly) peptide without mutation. Such derivatives include those substituted at the amino terminus or carboxy terminus or side chain of the protein. The amino acid modifications include naturally occurring amino acid modifications and non-naturally occurring amino acid modifications, and specific examples include phosphorylated forms of amino acids.
When the term “Bcl-xL” is used in the present invention, human Bcl-xL represented by the specific amino acid sequence (SEQ ID NO: 2) and its homologues, mutants, derivatives, matures, and the like, unless otherwise specified by the SEQ ID NO: Used to include amino acid modifications.
The bone mass and bone strength increasing agent of the present invention contains a gene encoding Bcl-xL. The increasing agent exerts its action by being introduced into osteoblasts. Therefore, the increasing agent preferably contains a gene encoding the Bcl-xL operably linked downstream of an osteoblast-specific promoter.
The osteoblast-specific promoter, when a gene encoding Bcl-xL is operably linked downstream of the promoter, determines the transcription start site of the gene encoding Bcl-xL in an osteoblast-specific manner And a DNA having a base sequence that directly regulates its frequency. The promoter can be appropriately selected according to the animal from which the osteoblast is derived.
Examples of the osteoblast-specific promoter include a type I collagen promoter and an osteocalcin promoter, and the type I collagen promoter is preferable. As a type I collagen promoter, for example, in the case of a human-derived promoter, Jimenez, S .; A. , Varga, J .; Olsen, A .; Li, L .; , Diaz, A .; Herhal, J .; and Koch, J. et al. Functional analysis of human alpha 1 (I) procollagen gene promoter. Differential activity in collagen-production and non-production cells and response to transforming growth factor beta 1 J. Phys. Biol. Chem. 269 (17), 12684-12691 (1994), in the case of a mouse-derived promoter, Ravazzolo, R .; Karsenty, G .; and de Crombrugge, B.M. A fibroblast-specific factor binds to an upstream negative control element in the promoter of the mouse alpha 1 (I) collagen gene. Biol. Chem. 266 (12), 7382-7387 (1991), Rossert, J. et al. Eberspächer, H .; , And de Crombrugge, B.M. (1995). Separate cis-acting DNA elements of the mouse pro-a1 (I) collagen promoter direct expression of reporter gen. J. et al. Cell Biol. 129, 1421-1432. It is described in.
The increasing agent is preferably an expression vector in order for the promoter and the Bcl-xL gene to function in osteoblasts. As the expression vector, a known or commercially available one can be used, and a viral vector or a non-viral vector is exemplified. Examples of viral vectors include viral vectors such as retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, vaccinia viruses, polioviruses, and Sindbis viruses. The method of linking the promoter and gene to an expression vector can be performed by a method known per se.
The increasing agent may contain any component for introducing the promoter, the Bcl-xL gene, and an expression vector containing them into a desired cell. Although it does not specifically limit as an arbitrary component, A solvent, a buffering agent, a chelating agent, a preservative, etc. are mention | raise | lifted. In order to increase the efficiency of introduction into cells, the increasing agent may be in the form of a liposome preparation.
The total content of the promoter, the Bcl-xL gene and / or the expression vector containing them in the increasing agent is not particularly limited, but is usually 0.001% to 100% by weight.
Examples of the method for introducing the increasing agent into desired cells (preferably osteoblasts) include an in vivo method and an ex vivo method. In the in vivo method, the increasing agent can be administered into a vein, artery, subcutaneous, intra-articular, intramuscular or the like in an appropriate administration form such as an injection. In the case of the ex vivo method, the above-mentioned increasing agent is transfected into cells of the subject in vitro during transfection, infection or injection by a known method, and cultured for a certain period of time to express the Bcl-xL gene. After confirmation, a method of returning to the body of the same subject can be adopted.
The dosage of the active ingredient in these various forms of preparation can be appropriately adjusted depending on the degree of the disease to be treated, the age of the patient, the body weight, and the like. Usually, the total of the promoter and the Bcl-xL gene may be about 0.0001-100 mg, preferably about 0.001-10 mg per adult patient, once a few days to several months. In the case of a retroviral vector, the retroviral titer is about 1 × 10 5 per kg patient body weight per day. ³ pfu or 1 × 10 ¹⁵ It can be selected from the range of the amount of pfu. In the case of a cell into which the present increasing agent has been introduced by the ex vivo method, 1 × 10 ⁴ Cells / body to 1 × 10 ¹⁵ What is necessary is just to administer a cell / body grade.
The bone mass and bone strength increasing agent of the present invention may contain the Bcl-xL protein. The increasing agent of the present invention may contain any component as long as it does not inhibit the activity of the Bcl-xL protein. Although it does not specifically limit as an arbitrary component, SH group protecting agents, such as a solvent, a buffer agent, antiseptic | preservative, dithiothreitol (DTT), surfactant, etc. are mention | raise | lifted. Further, the content of the Bcl-xL protein in the increasing agent is not particularly limited, but is usually 0.001% by weight to 100% by weight.
The present invention provides a transgenic animal into which an expression vector containing an osteoblast-specific promoter and a gene encoding Bcl-xL has been introduced.
In the present invention, “transgenic” refers to introduction of an expression vector in which an exogenous Bcl-xL gene is bound downstream of an osteoblast-specific promoter into a chromosomal gene. The introduced animal and its progeny are referred to as transgenic animals.
The animal to be used is not particularly limited as long as it is an animal other than a human, but an animal having a developed skeleton is preferable. Suitable animals include experimental animals such as mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, monkeys, etc. and domestic animals such as cows, sheep, horses, pigs, etc., which are easy to use in genetic engineering. Therefore, a mouse is more preferable.
The transgenic animal of the present invention can be produced by a method known per se. Next, a method for producing a transgenic animal of the present invention will be described.
The transgenic animal of the present invention can be produced, for example, by a method including the following steps (a) to (c).
(A) preparing an expression vector in which a human Bcl-xL gene and an osteoblast-specific promoter are linked;
(B) introducing the expression vector into a fertilized egg and transplanting the transgenic fertilized egg into a foster mother;
(C) selecting a transgenic animal from offspring born from said animal;
(D) A step of establishing a line from the selected animal (founder).
In the step (a), an expression vector that uses the bone mass and bone density increasing agent of the present invention as an expression vector can be preferably used. The expression vector is preferably linearized with a restriction enzyme or the like and subjected to step (b).
In the step (b), the method of introducing the expression vector into a fertilized egg is, for example, by washing a female oviduct after mating, collecting the fertilized egg, and microinjecting the pronucleus derived from the sperm or egg. Examples thereof include a method of directly injecting the expression vector. This fertilized egg is transplanted into the oviduct of a foster parent who has been pseudopregnant, and the development continues in the womb.
In the step (c), as a method for selecting a transgenic animal from the offspring born of the animal transplanted in the step (b), for example, whether or not the injected expression vector is integrated into the chromosomal DNA is determined. A method of screening chromosomal DNA separated and extracted from the tail of the mouse by Southern blotting or PCR.
In the step (d), a method for establishing a strain with a uniform genetic background from the animal (founder) selected in the step (c) is an inbred such as C57BL / 6, FVB, etc. A method of backcrossing with a wild-type animal of the system is mentioned.
Progeny animals obtained by further mating the transgenic animals of the present invention thus obtained, and tissues or cells derived from these animals are also included in the present invention. Examples of the tissue include all tissues, and a long bone, skull, vertebra and the like are preferable. Examples of the cells include cells contained in the tissue, cells isolated from the tissue, and cell lines established from these cells. Specifically, osteoblasts and the like are preferable.
In the transgenic animal of the present invention, the expression of Bcl-xL is regulated. That is, Bcl-xL is overexpressed in osteoblasts compared to allogeneic wild type animals. As a result, the transgenic animal of the present invention has increased bone mass and bone strength compared to the allogeneic wild type animal. The present invention is the first to find a dose dependency between the expression level of Bcl-xL and the increase in bone mass and bone strength. The present invention provides the following transgenic animals.
The transgenic animals of the present invention can be roughly classified into animals (hereinafter also referred to as low-expressing animals) in which the expression of the Bcl-xL in osteoblasts is increased by about 2 times compared to allogeneic wild-type animals. The expression of Bcl-xL in cells is about 3 to 5 times higher than that of allogeneic wild type animals (hereinafter also referred to as medium expression animals) or the expression of Bcl-xL in osteoblasts is allogeneic wild It can be classified into animals (hereinafter also referred to as high-expressing animals) that have increased by about 10 times compared to type animals.
The transgenic animal of the present invention is suitable according to the purpose from among the low-expressing animal, medium-expressing animal and high-expressing animal in bone disease research or bone disease prevention or treatment drug screening using the animal. Animals can be selected and used. For example, in the case of mice, Bcl-xL necessary to bring bone mass and bone strength back to normal levels by mating with various levels of osteoporosis model mice and low expression, medium expression, and high expression Bcl-xL transgenic mice. You can guess the level. Moreover, in the effect determination using the wild-type animal of the substance obtained from the screening using the Bcl-xL promoter, which will be described later, by comparing with each of the low expression, intermediate expression, and high expression Bcl-xL transgenic mice. The correlation between the expression induction level of Bcl-xL and the bone mass / bone strength / bone microstructure can be compared. That is, which of the low expression, medium expression, and high expression Bcl-xL transgenic mice can result in an increase in bone and other negative effects (bone strength) Can be compared to determine whether or not there has been a decrease in bone microstructure, abnormal bone microstructure, low bone rotation, etc. Further, as described later, low-expressing mice are suitable for screening for Bcl-xL signal activators, and medium-expressing and high-expressing mice are suitable for screening for suppressors.
When the transgenic animal of the present invention is used for bone disease research including treatment and prevention of osteoporosis, it is preferable to use an adult. The reason is that the occurrence of the bone disease usually increases with aging. The term “adult” differs depending on the animal, but it means at least the time excluding the fetus and immediately after birth. In the case of mice, examples include 6 to 12 months of age, and the significant difference from wild type increases with age. Until death by aging (up to about 2 years). About 10 weeks old is preferably used.
When the transgenic animals of the present invention are adults, the number of cancellous bone trabeculae, decreased cancellous bone trabecular distance, increased cancellous bone connectivity, and cancellous bone It is preferable that at least one parameter selected from the measurement parameter group including a change in the structure model index has a significant difference. For example, as a result of micro CT (μCT) analysis of the cancellous bone of the femur in a 10-week-old male, wild-type mouse (n = 6), and transgenic mouse (n = 9), the following five parameters A significant difference can be recognized.
Bone mass: p = 0.012 <0.05
Connectivity: p = 0.004 <0.01
Structure model index (SMI): p = 0.177 <0.05
Number of trabeculae: p = 0.005 <0.01
Trabecular thickness: p = 0.048 <0.05
In particular, the connectivity and trabecular number parameters are significantly different.
The measurement parameter can be measured by micro CT (μCT), and details thereof are described in Examples.
Since the transgenic animal of the present invention has an increased amount and quality of bone as compared to the wild-type animal of the same species, it can be used as a model with increased bone mass and strength, or as a model for preventing or treating osteoporosis. It is preferably used.
When used as a model for bone disease research, a model animal obtained by mating the transgenic animal of the present invention with another disease model of the same kind may be used. The present invention provides such a model animal.
Examples of the other disease models include an osteoporosis model, an osteoblast proliferation enhancement model, an osteoblast apoptosis enhancement model, an early aging model, and various knockout animals that cause bone loss. For example, in the case of mice, CDK6 / CyclinD1 double transgenic mice, Klotho mice, Bcl-2 transgenic mice (eg, mice that express exogenous Bcl-2 specifically in osteoblasts) and the like can be suitably used.
The present invention provides a screening method (screening method I) for a substance that increases bone mass and / or bone strength, characterized by using the transgenic animal or the model animal. In this case, it is preferable to use the low expression animal.
[Screening Method I]
The screening method I of the present invention specifically includes the following steps:
(A) a step of administering a test substance to the transgenic animal or model animal of the present invention;
(B) examining the bone mass and bone strength in an animal to which the test substance is administered and comparing the bone mass and bone strength in an animal to which the test substance is not administered; and
(C) A step of selecting a test substance that enhances bone mass and / or bone strength based on the comparison result.
In the above (a), the test substance may be any known substance or new substance, for example, a nucleic acid, a carbohydrate, a lipid, a protein, a peptide, a low-molecular-weight organic compound, or a combinatorial chemistry technique. Examples include compound libraries, random peptide libraries prepared by solid phase synthesis and phage display methods, and natural components derived from microorganisms, animals and plants, marine organisms, and the like. Moreover, the mixture of 2 or more types of these compounds can also be provided as a sample.
Although the method for administering the test substance to the animal of the present invention is not particularly limited, it can be administered orally or parenterally. Examples of parenteral administration routes include systemic administration such as intravenous, intraarterial, and intramuscular, or local administration in the vicinity of a target cell in a joint.
The dosage of the test substance can be appropriately set according to the type of active ingredient, the size of the molecule, the administration route, the animal species to be administered, the drug acceptability of the administration subject, body weight, age, and the like.
Examples of the method for examining the bone mass and bone strength in the animal to which the test substance is administered in the step (b) include X-ray analysis, μCT, and pQCT.
In the step (b), the bone mass and bone strength in the animal not administered with the test substance are also examined simultaneously or separately, and the result of the administered animal is compared with the result of the non-administered animal. It is also desirable to administer a known bone mass increasing agent in the same manner as a positive control.
In the step (c), a test substance that increases bone mass and / or bone strength is selected based on the comparison result obtained in the step (b). The criterion for selection may be Bcl-xL transgenic mice not administered with the test substance.
The test substance thus selected has an action capable of activating the Bcl-xL signal transduction pathway in osteoblasts, and is a candidate for improving the quantity and / or quality of bone mass and / or bone density. It can be a medicine.
The therapeutic agent for bone disease that can be selected by the screening method of the present invention is not particularly limited, and is, for example, a therapeutic agent for osteoporosis, periodontal disease, severe fracture, bone defect, and alveolar bone resorption.
[Screening Method II]
Moreover, this invention provides the screening method (screening method II) of the substance which reduces the bone mass and bone strength through Bcl-xL including the following processes. In this case, it is preferable to use the medium expression animal or the high expression animal.
(A) a step of administering a test substance to the transgenic animal or model animal of the present invention;
(B) examining the bone mass and bone strength in an animal to which the test substance is administered and comparing the bone mass and bone strength in an animal to which the test substance is not administered; and
(C) A step of selecting a test substance that reduces bone mass and / or bone strength based on the comparison result of step (b).
In the step (a), the test substance is the same as in screening method I, and the administration method is the same as in screening method I.
Examples of the method for examining the bone mass and bone strength in the animal to which the test substance is administered in the step (b) include X-ray analysis, μCT, and pQCT.
In the step (b), the bone mass and bone strength in the animal not administered with the test substance are also examined simultaneously or separately, and the result of the administered animal is compared with the result of the non-administered animal.
In the step (c), a test substance that decreases the bone mass and / or bone strength is selected based on the comparison result obtained in the step (b). The criterion for selection may be a transgenic mouse that does not administer the test substance.
The test substance thus selected has an action capable of reducing the bone mass and / or bone density by suppressing the signal transduction pathway of Bcl-xL in osteoblasts. Can be a candidate drug to treat or prevent diseases with an abnormally increased density.
The therapeutic agent for bone disease that can be selected by the screening method of the present invention is not particularly limited, and is, for example, a therapeutic agent for bone marble disease.
[Screening Method III]
The present invention also provides a screening method (screening method III) for a substance that increases bone mass and bone strength, including the following steps. In this case, any of the low-expressing animals, medium-expressing animals, and high-expressing animals may be used depending on the purpose.
(A) a step of administering a test substance to the transgenic animal or model animal of the present invention;
(B) examining the bone mass and bone strength in an animal administered with the test substance, and comparing the bone mass and bone strength in an animal not administered with the test substance;
(C) selecting a test substance that reduces bone mass and / or bone strength based on the comparison result of step (b),
(D) a step of administering to the animal used in step (a) the test substance selected in step (c) and an antagonist candidate substance for the test substance;
(E) examining the bone mass and bone strength in the animal administered the test substance and the antagonist candidate substance in step (d), and comparing the bone mass and bone strength in the animal administered with the test substance alone;
(F) A step of selecting an antagonist candidate substance that enhances bone mass and / or bone strength based on the comparison result of step (e).
The steps (a) to (c) are the same as in the screening method II.
In the step (d), an antagonist candidate substance for the test substance selected in the step (c) can be appropriately selected from candidate substances to be screened in the step (a). As the antagonist candidate substance, when the selected test substance is a protein, a neutralizing antibody against the protein is preferably selected. When the selected test substance is a nucleic acid having a specific sequence, an antisense nucleic acid against the nucleic acid, two Strand RNA and the like are preferably selected.
In the step (d), the method of administering the test substance and the antagonist candidate substance is the same as in the step (b) of the screening method I. However, the timing of administration may be the same, and a certain interval is used. May be separate. Also, the administration route may be the same route or different routes.
Examples of the method for examining the bone mass and bone strength in the animal to which the test substance and the antagonist candidate substance are administered in the step (e) include X-ray analysis, μCT and pQCT.
In the step (e), the bone mass and bone strength in the animal to which only the test substance is administered are also examined simultaneously or separately, and the results of the antagonist candidate substance-administered animal and the non-administered animal are compared.
In the step (f), an antagonist candidate substance that enhances bone mass and / or bone strength is selected based on the comparison result of the step (e). The criteria for selection may be based on the transgenic mice not administered with the antagonist candidate substance.
The antagonist candidate substance thus selected has an action capable of suppressing the inhibitory action via the expression system of Bcl-xL in osteoblasts, and the bone mass and / or by suppressing the expression of Bcl-xL. Can be a candidate drug to treat or prevent diseases with reduced bone density.
The therapeutic agent for bone disease that can be selected by the screening method of the present invention is not particularly limited, and is, for example, a therapeutic agent for osteoporosis or steroid osteoporosis.
The present invention provides a screening method (screening method IV) for a substance that increases bone mass and / or bone strength through increased expression of Bcl-xL, characterized by using osteoblasts. In this case, the osteoblast is preferably a wild-type animal-derived cell.
[Screening Method IV]
The screening method IV of the present invention specifically includes the following steps:
(A) contacting osteoblasts with a test substance;
(B) examining the expression level of Bcl-xL in osteoblasts contacted with the test substance, and comparing the expression level with osteoblasts not contacted with the test substance;
(C) A step of selecting a test substance that increases the expression of Bcl-xL based on the comparison result.
In step (a), osteoblasts placed under conditions that maintain the expression of Bcl-xL can be used. As such a condition, it is preferable to place osteoblasts in an appropriate medium and to survive or culture in an incubator at about 25 to 40 ° C. Next, contact can be made by adding a test substance to the medium and continuing the incubation.
The addition amount of the test substance can be appropriately set depending on the type of active ingredient, solubility in a medium, cell sensitivity, and the like.
In the step (b), the expression level of Bcl-xL in osteoblasts contacted with the test substance can be examined by a method known per se. The expression level of endogenous Bcl-xL is measured by, for example, Western blotting using an antibody that recognizes Bcl-xL by extracting a protein from the cell; RT-PCR or Northern blotting by extracting RNA from the cell And a method of measuring Bcl-xL mRNA transcribed by the above; a method of examining Bcl-xL in situ by immunohistochemistry using an antibody recognizing Bcl-xL, and the like. The expression level of endogenous Bcl-xL can also be estimated by a reporter assay or the like in which an expression vector in which the promoter region of Bcl-xL and a reporter gene are linked is introduced into osteoblasts and the expression of the reporter gene is measured. Reporter assays are preferably used. As the reporter gene, a known gene can be used without limitation, and examples thereof include, but are not limited to, luciferase, GFP (green fluorescent protein), CAT (chloramphenicol acetyltransferase) and the like. The expression of the reporter gene can be measured by a method known per se according to the reporter gene used.
In the step (b), osteoblasts that are not brought into contact with the test substance are also examined at the same time or separately, and the results when they are brought into contact with the results when they are not brought into contact are compared. Further, as a positive control, for example, it is preferable to prepare osteoblasts that are similarly contacted with FGF, BMP-2, or the like.
In the step (c), a test substance that increases the expression of Bcl-xL is selected based on the comparison result obtained in the step (b). The criterion for selection may be based on an increase in Bcl-xL protein or mRNA. It is preferable to use the increase in the expression of the reporter gene as an indicator.
The test substance selected in this way is useful as an agent having an agonistic action of Bcl-xL in osteoblasts, and, for example, by conducting further screening and pharmacological tests such as screening method I, side effects can be obtained. It can also be a candidate drug for a therapeutic agent for bone diseases such as osteoporosis with a small amount.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited at all by these Examples.
[Example 1]
Production of Bcl-xL transgenic mice
Human Bcl-xL cDNA (provided by Dr. Enomoto, Osaka University) was cloned into the plasmid pUC-CAGGS / Xhol. The plasmid was digested with EcoR I, and the resulting fragment was subcloned into a plasmid pNASSβ containing the mouse type I collagen promoter to obtain a transgenic construct (for pCAGGS-hbcl-xL from Osaka University, Dr. Enomoto). , Oncogene 19: 5736, 2000). A DNA construct linearized with EcoR I and Sal I was microinjected into the pronucleus of a C57BL / 6Cr (Japan SLC) conjugate, which was transferred to a foster mother to create a Bcl-xL transgenic founder. Using the full length Bcl-xL cDNA as a probe, the genotype was examined by Southern blotting.
The transgenic founder was backcrossed with C57BL / 6Cr to produce a Bcl-xL transgenic mouse. When RNA was extracted from each tissue of the obtained transgenic mice by a conventional method and the expression of Bcl-xL was examined by a Northern blotting method, it was confirmed that it was expressed only in osteoblasts and dental ivory cells. It was.
[Example 2]
Establishment of strains with different expression levels from Bcl-xL transgenic mice
RNA was isolated from the femur and tibia of 3 strains of Bcl-xL transgenic mice (4 weeks old) obtained in Example 1 using the RNA extraction kit ISOGEN (NIPPON GENE). In accordance with a conventional method, the expression of Bcl-xL was examined by Northern blot. The results are shown in FIG.
From FIG. 1, it was found that the three lines of transgenic mice (K1, K2 and K3) had an increased expression level of Bcl-xL compared to the wild type mouse (WT). When the expression levels of wild type and transgenic mice were compared using a densitometer, it was revealed that K1 was about 10 times, K2 was about 3 times, and K3 was about 2 times higher. Hereinafter, K1 was classified as a strong expression line, K2 as a medium expression line, and K3 as a weak expression line, and the analysis was advanced.
[Example 3]
X-ray analysis of Bcl-xL transgenic mice
Nembutal (0.75 mg / 10 g body weight) was intraperitoneally injected into 10-week-old Col I-Bcl-XL transgenic mice and wild-type mice, followed by general anesthesia, and then micro-FX1000 (Fuji Film, Inc.). A X-ray image of the whole body image was taken. Further, the mouse was dissected, and the femur and vertebra were collected and fixed with 70% ethanol, and then X-ray photography was performed using micro-FX1000 (Fuji Film, Inc.).
[result]
Fig. 2: Femur X-ray images of 10-week-old female wild-type mouse and Col I-Bcl-XL transgenic mouse
Compared with the wild type, the Col I-Bcl-XL transgenic mouse has increased bone mass in the secondary cancellous bone region in proportion to its expression level, and even the weakest expression line (K3) is different from the wild type. The amount of bone in the secondary cancellous bone region is large compared.
FIG. 3 (A): 10-week-old male X-ray image (whole body) wild type (left) and Col I-Bcl-XL transgenic mouse strong expression line (right)
(B): 10-week-old female X-ray image (femur) wild type (left) and Col I-Bcl-XL transgenic mouse strong expression line (right)
(C): 10-week-old male X-ray image (vertebra) wild type (left) and Col I-Bcl-XL transgenic mouse strong expression line (right)
Compared to the wild type, Col I-Bcl-XL transgenic mice have increased secondary cancellous bone and vertebral secondary cancellous bone.
[Example 4]
Analysis of Bcl-xL transgenic mice
10-week-old, 7-month-old and 1-year-old Col I-Bcl-XL transgenic mice and wild-type mice were used. After general anesthesia with Nembutal (0.75 mg / 10 g body weight), perfusion fixation was performed with 4% paraformaldehyde. The femur was collected, fixed by osmotic fixation at 4 ° C. overnight with the same fixing solution, washed with 0.01 M PBS, and decalcified with a 10% EDTA solution for about 1 week. After the decalcification, a paraffin block was prepared according to a conventional method. The block was sliced at 3 μm and stained with hematoxylin-eosin (HE).
[result]
As shown in FIG. 4, in the Col I-Bcl-XL transgenic mouse, many secondary cancellous bones are observed in the distal femoral region and many secondary cancellous bones are also observed in the diaphysis. And most secondary cancellous bone has been absorbed in the 1-year-old wild-type mouse, but in the Col I-Bcl-XL transgenic mouse, many secondary cancellous bones are observed even at the age of 1.
Next, various tissue images in cortical bone were compared. Cortical bones of 4- and 10-week-old Bcl-XL transgenic mice (K1) and wild-type mice were stained with HE, TUNEL, transmission electron microscope (TEM), silver-stained, and scanning electron microscope (SEM). It was examined in detail by observation.
[result]
FIG. 5 shows, from the top, a HE image of a 10-week-old male femoral distal part, a TUNEL staining of a 10-week-old male femoral cortical bone, a 4-week-old cortical bone transmission electron microscope image (TEM), and a 10-week-old male femoral bone It is a silver-stained image of cortical bone, and secondary electron images of bone cells and bone cell processes in 10-week-old cortical bone by a scanning electron microscope (SEM). The bone cells of the transgenic mouse (K1) showed a young form (the cytoplasm was more abundant compared to the wild type) as observed by transmission electron microscopic images and silver staining. A number of bone tubules and bone cell processes that are completely the same as the wild type were observed from secondary electron images of silver stained and scanning electron microscopes (generally when bone cells are young, bone tubules and bone cell processes decrease) ).
[Example 5]
Northern blotting
Total RNA was extracted from femurs of 5-week-old Col I-Bcl-XL transgenic mice and wild type mice. The femur frozen with liquid nitrogen was homogenized in 1 ml of ISOGEN (NIPPON GENE) and left at room temperature for 5 minutes. Then, 200 μl of chloroform was added, and the mixture was inverted and left at room temperature for 3 minutes. After cooling at 4 ° C. and centrifuging at 15,000 × g for 15 minutes, the aqueous layer (upper layer) was extracted, 500 μl of isopropanol was added, and the mixture was allowed to stand at room temperature for 10 minutes. After cooling at 4 ° C. and centrifuging at 15,000 × g for 15 minutes, the supernatant was removed and suspended in DEPC water. After adding phenol and centrifuging at 15,000 × g for 5 minutes, the upper layer was extracted, sodium acetate and 100% ethanol were added, and the mixture was centrifuged at 15,000 × g for 20 minutes. After removing the supernatant, the RNA precipitate was suspended in DEPC water. 20 μg of total RNA was denatured with formamide, electrophoresed on a 1.0% agarose gel, and blotted on a nylon membrane. The membrane is made of pro-α1 (1) collagen, osteopontin, osteocalcin ³² Complementary strand binding was carried out with a cDNA probe labeled with P.
From FIG. 6, as a result of examining the expression of the differentiation marker gene of osteoblasts in the Col I-Bcl-XL transgenic mouse and the wild type mouse, the Col I-Bcl-XL transgenic mouse was compared with the wild type mouse. The expression levels of type I collagen, osteopontin and osteocalcin remained almost unchanged.
in situ hybridization
After deparaffinization, the sections were washed with 0.01 M PBS, prefixed with 4% PFA solution, washed with 0.1 M PB, and treated with 8 μg / ml protease K (Roche Diagnostics) at 37 ° C. for 15 minutes. Post-fixation was performed with 4% PFA solution. The acetylation process was performed for 10 minutes with 0.1M triethanolamine containing 0.25% acetic anhydride, washed with 0.1M PB, dehydrated with ethanol, and dried. Thereafter, hybridization was performed at 50 ° C. for 16 hours using a probe diluted to 1 ng / ml with a hybridization solution. After hybridization, the membrane was washed in 50% formamide solution containing 2 × standard sodium citrate solution (hereinafter abbreviated as SSC) at 52 ° C. for 30 minutes, and treated with 18 μg / ml RNase A (Roche Diagnostics) at 37 ° C. for 30 minutes. . Washing at 52 ° C. for 20 minutes was repeated twice with 2 × SSC, and washing was performed at 0.2 ° SSC for 20 minutes at 52 ° C. After blocking with Blocking Reagent (Roche Diagnostics) for 1 hour at room temperature, the mixture was reacted with an alkaline phosphatase-labeled anti-digoxigenin antibody (Roche Diagnostics) for 45 minutes at room temperature. After washing, a color reaction was carried out for about 0.5-2 hours using 4-nitroblue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Roche Diagnostics) as substrates. Was detected. After completion of the detection reaction, it was washed with water and sealed with a water-soluble mounting medium crystal mount (biomeda corp. Foster City, CA).
[result]
In order to examine the differentiation stage of osteoblasts in Col I-Bcl-XL transgenic mice, the tibia at the age of 10 weeks was used, type I collagen gene (Col (I)), which is a marker gene for osteoblast differentiation, osteopontin Expression patterns of the gene (OP) and the osteocalcin gene (OC) were examined by in situ hybridization. As shown in FIG. 7, in wild-type mice, type I collagen was expressed in osteoblasts at various stages of differentiation. Osteopontin was mainly expressed in immature osteoblasts, and osteoblasts mainly present in cancellous bone were stained. Osteocalcin was expressed mainly in mature osteoblasts present in the cortical bone endometrium. Since the Col I-Bcl-XL transgenic mice have no change in the expression regions and expression levels of type I collagen, osteopontin, and osteocalcin compared to the wild type mice, the bones in the Col I-Bcl-XL transgenic mice The blasts were shown to be normally differentiated.
[Example 6]
BrdU
BrdU (Nacalai Tesque, Kyoto, Japan) 100 μg / g was intraperitoneally administered to 2-week-old Col I-Bcl-XL transgenic mice and wild-type mice, and 1 hour later, whole body in Nembutal (0.75 mg / 10 g body weight) After anesthesia, physiological saline containing 5 units / ml heparin was injected into the left ventricle, and perfusion fixation was performed with 4% PFA. According to the above, a femur section was prepared, and BrdU staining was performed using a BrdU Staining Kit (Zymed Laboratories, South San Francisco, USA). That is, the section was deparaffinized with xylene and washed with ethanol. The sections were immersed in Peroxidase quenching solution for 10 minutes to inhibit endogenous peroxidase activity, and then washed with PBS. Proteolytic treatment with 0.0625% trypsin at 37 ° C for 5 minutes, after exposing DNA, adding Denaturing solution dropwise, incubating at room temperature for 20 minutes, denaturing, dropping Blocking solution, blocking for 10 minutes at room temperature It was. After treatment with biotinylated anti-BrdU Reagent for 60 minutes at room temperature and washing with PBS, Streptavidin-peroxidase Reagent was added dropwise and incubated at room temperature for 10 minutes, followed by DAB staining. After the color development reaction, hematoxylin staining was performed as a counterstaining.
As shown in FIG. 8, the Col I-Bcl-XL transgenic mice showed a significant increase in BrdU incorporation in osteoblasts in the primary cancellous bone region as compared to the wild-type mice.
TUNEL
A femoral slice was prepared from 2-week-old Col I-Bcl-XL transgenic mice and wild-type mice as described above, and subjected to terminal deoxynucleotidyltransferase-mediated dUTP-digoxigeninned nick labeling (TUNEL) staining. For TUNEL staining, an ApopTag® system (Intergen, NY, USA) was used. That is, the sections were deparaffinized with xylene, washed with ethanol, and then washed with phosphate buffered saline (PBS). Proteinase K (20 μg / ml) was added, incubated at room temperature for 15 minutes to expose the DNA, washed with distilled water, and then washed with 3% H ₂ O ₂ / In PBS for 5 minutes at room temperature, endogenous peroxidase activity was inhibited. After washing with PBS, Equilibration Buffer was added dropwise to the sections and incubated at room temperature for 10 minutes, TdT / digoxigenin-labeled dUTP was added, and the mixture was allowed to react at 37 ° C. for 1 hour. The sections were immersed in a stop / wash buffer for 30 minutes at room temperature, the reaction was stopped, washed with PBS, an Antidigigenin peroxidase-labeled antibody was added dropwise, and the mixture was incubated for 30 minutes at room temperature. After washing with PBS, color was developed with a diaminebenzoline (DAB) solution. After the color development reaction, as a counterstain, methylgreen staining and toluidine blue staining were performed to count TUNEL positive osteoblasts.
FIG. 9 shows that a significant decrease in apoptosis-positive cells was observed in osteoblasts in the primary cancellous bone region of the Col I-Bcl-XL transgenic mice compared to the wild-type mice (TUNEL-positive osteoblasts, (Indicated by arrows in the figure).
[Example 7]
Bone morphometry
Static bone morphometry was performed using 10-week-old male Col I-Bcl-XL transgenic mice and wild-type mice. In order to perform dynamic bone morphometry, calcein (0.16 mg / 10 g body weight) was injected into a 10-week-old mouse twice intraperitoneally at intervals of 4 days and sacrificed the next day. . The femur was removed, fixed with 70% ethanol, and embedded with glycol methacrylate. A longitudinal section having a thickness of 7 μm was prepared from the distal part of the femur, and after toluidine blue staining, bone morphology was measured using Bone Histomorphometric System, (System Supply, Nagano, Japan).
From FIG. 10, as a result of comparing a 10-week-old male Col I-Bcl-XL transgenic mouse with a wild-type mouse, only a bone mass (BV / TV) was found to be significantly different in static parameters. There was no significant difference in the parameters. Regarding the dynamic parameters such as bone mineralization rate, bone formation rate, bone mineralization surface / bone surface, Col I-Bcl-XL transgenic mice were not significantly different from wild type mice, but bone mineralization surface / There was a tendency for the two parameters of bone surface and bone formation rate to be higher than those of the wild type.
[Example 8]
Analysis of femoral shaft cortical bone by pQCT
After 10 weeks of age, male Col I-Bcl-XL transgenic mice and wild-type mice were intraperitoneally injected with Nembutal (0.75 mg / 10 g body weight) and then subjected to general anesthesia, and then the femur was collected and 70% EtOH It was fixed using. The collected femur was subjected to pQCT analysis using a pQCT bone densitometer for animal research XCT Research SA + (Stratec Medintechnik GmbH, Pforzheim, Germany). The cancellous bone parameters were measured continuously from the distal femur growth plate toward the diaphysis at 0.19 mm intervals, and the cortical bone parameters were measured at the femoral diaphysis. Measurement was performed with a slice thickness of 0.46 mm and a voxel size of 0.08 mm.
As shown in FIG. 11, the Col I-Bcl-XL transgenic mice showed no significant difference in all parameters of cortical bone mineral density, cortical bone average thickness, cortical bone density, and XSSI, compared to wild type mice. Moreover, even if the pQCT images of the femoral distal cortical bone were compared, the properties of the cortical bone of the transgenic mouse and the wild type mouse were not changed at all.
[Example 9]
Analysis of femoral shaft cortical bone by μCT
After 10 weeks old female Col I-Bcl-XL transgenic mice (K1, K2, K3) and wild type mice were intraperitoneally injected with Nembutal (0.75 mg / 10 g body weight), general anesthesia was performed. Femurs were collected and fixed with 70% EtOH. Volume data of one slice is acquired from the femoral shaft using a micro-CT (Scanco Medical) with a 50 kV, 0.1 mA X-ray beam with a beam width of 20 μm and a slice interval of 12 μm. did. Then, a three-dimensional image processing program (TRI / 3D-Bone, Ratoc System Engineering) was used to extract cortical bone images and analyze bone parameters.
From FIG. 12, cortical bone area and cortical bone with respect to the bone area of the femoral diaphysis cortical bone for three 10-week-old female Col I-Bcl-XL transgenic mice (K1, K2, K3) and wild type mice As a result of examining the width, the Col I-Bcl-XL transgenic mouse was not significantly different from the wild type. Furthermore, no significant difference was observed between the three Col I-Bcl-XL transgenic mouse lines (K1, K2, K3) having different expression levels.
[Example 10]
Analysis of cancellous bone in the distal femur by pQCT
Ten week-old female Col I-Bcl-XL transgenic mice and wild-type mice were intraperitoneally injected with Nembutal (0.75 mg / 10 g body weight) and then subjected to general anesthesia, and then the femur was collected and 70% ethanol was collected. It was fixed using. The collected femur was subjected to pQCT analysis using a pQCT bone density measuring apparatus XCT Research SA + (Stratec Meditechntech GmbH, Pforzheim, Germany) for animal research. The cancellous bone parameters were continuously measured from the distal femur growth plate toward the diaphysis at 0.19 mm intervals. Measurement was performed with a slice thickness of 0.46 mm and a voxel size of 0.08 mm.
From FIG. 13, it was confirmed that the bone density of the femoral distal cancellous bone was significantly higher in the Col I-Bcl-XL transgenic mouse than in the wild type mouse.
[Example 11]
Analysis of distal cancellous bone of the femur by μCT
After 10 weeks old female Col I-Bcl-XL transgenic mice (K1, K2, K3) and wild type mice were intraperitoneally injected with Nembutal (0.75 mg / 10 g body weight), general anesthesia was performed. The femur and second lumbar vertebra were harvested and fixed with 70% EtOH. Volume data of 200 slices is acquired from directly under the centrifuge growth plate with a beam width of 20 μm and a slice interval of 12 μm using a 50 kV, 0.1 mA X-ray beam from the collected femur using a micro CT (Scanco Medical). did. Then, the image extraction of the cancellous bone part and the analysis of the bone parameters were performed with a three-dimensional image processing program (TRI / 3D-Bone, Ratok System Engineering). Similarly, images of the cancellous bone part were extracted from the collected second lumbar spine using micro CT.
From FIG. 14, the Col I-Bcl-XL transgenic mice showed significant differences in the bone mass, the number of trabeculae, and the distance between trabeculae, as compared with the wild type mice in all three strains. Although there was no significant difference in trabecular width, the tendency of the Col I-Bcl-XL transgenic mice to be greater than that of the wild type was observed. Regarding the connectivity related to bone strength and SMI, Col I-Bcl-XL transgenic mice were significantly different from wild-type mice.
From FIG. 15, the transgenic mouse (K1) also had increased femoral cancellous bone and vertebral cancellous bone as compared to the wild-type mouse.
X-ray and μCT of 10-week-old, 7-month-old and 1-year-old male Col I-Bcl-XL transgenic mice (K1) and wild-type mice cancellous cancellous bone (BV / TV) The analysis results are shown in FIGS.
16 and 17, the transgenic mouse (K1) showed a significant increase in bone mass in all of 10-week-old, 7-month-old, and 1-year compared with the wild-type mouse. In wild-type mice, the amount of cancellous bone at the age of 10 weeks was (22 ± 8%), but decreased to half or less at the age of 7 months (9 ± 6%). In the transgenic mouse (K1), the bone mass was (34 ± 3%) at the age of 10 weeks, and the bone mass was (19 ± 4%) at the age of 7 months. It was smaller than the mouse.
[Comparative Example 1]
Generation of Bcl-2 transgenic mice and comparison with Bcl-xL transgenic mice
In Example 1, osteoblast-specific Bcl-2 transgenic mice were established by the same production method as in Example 1 except that human Bcl-2 cDNA was used instead of human Bcl-xL cDNA. Bcl-2 protein also has an anti-apoptotic effect, and was compared with Bcl-xL to elucidate its function and role in osteoblasts.
[Comparative Example 2]
Northern blot
In the same manner as in Example 5, gene expression in Bcl-2 transgenic mice was examined by Northern blot. The results are shown in FIG.
FIG. 18 left: shows the results of Bcl-2 mRNA expression in two strains of Bcl-2 transgenic mice (N2, N3) and wild type mice (WT). N2 was a line of a strong expression line, N3 was a line of a weak expression line, and a subsequent Ncl mouse with high expression was used for comparison with the Bcl-xL transgenic mouse.
FIG. 18 right: shows the results of examining Bcl-xL mRNA expression in two strains of Bcl-xL transgenic mice (K1, K2) and wild type mice (WT).
[Comparative Example 3]
X-ray analysis
In the same manner as described in Example 3, X-ray imaging was performed on the distal femur of a Bcl-2 transgenic mouse. The results are shown in FIG.
FIG. 19 left: X-ray images of the 10-week-old male femur centrifuge part of two strains of Bcl-2 transgenic mice (N2, N3) and wild type mice (WT) are shown. In both N2 and N3 mice, secondary cancellous bone was observed up to the diaphyseal region compared to wild-type mice, and the bone mass increased in the entire femur, but in the primary cancellous bone region just below the growth plate, An image of bone loss was shown.
FIG. 19 right: X-ray images of the 10-week-old male femur centrifuge part of two strains of Bcl-xL transgenic mice (K1, K2) and wild type mice (WT) are shown. Both the K1 and K2 mice had increased bone mass compared to the wild type mice. In addition, unlike the N2 and N3 mice, the bone mass was increased just below the growth plate.
[Comparative Example 4]
pQCT analysis
In the same manner as described in Examples 8 and 10, cancellous bone and cortical bone of Bcl-2 transgenic mice were analyzed by pQCT. The results are shown in FIG.
FIG. 20 upper left: shows a graph analyzing the bone density of cancellous bone and cortical bone of 10-week-old male, Bcl-2 transgenic mouse (N2) (black circle) and wild-type mouse (white).
FIG. 20 bottom left: shows a graph analyzing the bone density of cancellous bone and cortical bone of 10-week-old female, Bcl-xL transgenic mouse (K1) (black circle) and wild-type mouse (white).
FIG. 20 (upper right): shows cortical bone cross-sectional images of 10-week-old male, Bcl-2 transgenic mouse (N2) and wild-type mouse.
FIG. 20, lower right panel: shows cortical bone cross-sectional images of 10-week-old female, Bcl-xL transgenic mouse (K1) and wild-type mouse.
From the bone mineral density graph, N2 mice have no change in cancellous bone density compared to wild-type mice, but cortical bone density is significantly lower. Many low yellow sites (the bone density increased in the order of red-yellow-blue-white) were observed. K1 mice had significantly increased cancellous bone density compared to wild-type mice, and cortical bone density did not change.
[Comparative Example 5]
10-week-old male mouse cancellous bone image using μCT
In the same manner as described in Examples 9 and 11, the cancellous bone of the femur of 10-week-old male wild-type mouse, Bcl-2 transgenic mouse (N2), and Bcl-xL transgenic mouse (K1) The second lumbar cancellous bone was analyzed by μCT. The results are shown in FIG.
From FIG. 21, the N2 mouse showed a thin trabecular bone with less bone mass in both the femoral distal part and the second lumbar vertebra compared with the wild type mouse. The K1 mouse showed an image in which the bone mass increased in both the femoral distal part and the second lumbar vertebra compared with the wild type mouse.
[Comparative Example 6]
Analysis of 10-week-old male femoral distal cancellous bone and cortical bone using μCT
In the same manner as described in Example 11, the femoral distal cancellous bone (bone mass) of 10-week-old male wild-type mice, Bcl-2 transgenic mice (N2) and Bcl-xL transgenic mice (K1) , Connectivity, SMI, trabecular number, trabecular width) and cortical bone (cortical bone width) were analyzed by μCT. The results are shown in FIG.
FIG. 22 shows that K1 mice have significantly increased cancellous bone mass, connectivity, trabecular number, and trabecular width, while N2 mice have significantly more cancellous bone mass compared to wild-type mice. The phenotype was quite the opposite, with poor connectivity.
[Comparative Example 7]
Morphometry in the secondary cancellous bone region of the 10-week-old male femur
Similar to the method described in Example 7, the secondary cancellous bone region of the femur of 10-week-old male, wild-type mouse, Bcl-2 transgenic mouse (N2), Bcl-xL transgenic mouse (K1) Bone morphology was measured. The results are shown in FIG.
FIG. 23 shows that although N2 mice have significantly increased osteoblast surface (Ob.S / BS) and osteoblast number (N.Ob / BPm) compared to wild-type mice, Bone mass (BV / TV) was significantly reduced. The bone formation rate (BFR / BS) and calcification rate (MAR) were not different from the wild type. That is, it was considered that the function per osteoblast cell was reduced in the N2 mouse compared to the wild type mouse. N2 mice had a large number of bone cells in cortical bone. In contrast, in the K1 mouse, bone mass (BV / TV), calcified surface (MS / BS) and bone formation rate (BFR / BS) increased, and osteoblasts maintained normal functions. Unlike N2 mice, the bone cell density was normal.
[Comparative Example 8]
2-week-old femoral primary cancellous bone area BrdU and TUNEL stained images
In the same manner as described in Example 6, in the 2-week-old femoral primary cancellous bone region of wild-type mice (WT), Bcl-2 transgenic mice (N2) and Bcl-xL transgenic mice (K1), BrdU and TUNEL staining was performed. The results are shown in FIG.
From FIG. 24, the ratio (%) of positive osteoblasts to total osteoblasts in the primary cancellous bone region is shown in each panel. Further, positive cells are indicated by black arrows in the TUNEL-stained photograph.
The ratio of BrdU and TUNEL positive osteoblasts shown in FIG. 24 was calculated for each mouse and shown in the graph. The results are shown in FIG. FIG. 25 shows that the percentage of positive osteoblasts in BrdU staining of N2 and K1 mice is significantly higher than that of wild-type mice, indicating that proliferation of osteoblasts in the primary cancellous bone region is enhanced. It was. In TUNEL staining, K1 mice were significantly lower than wild-type mice, indicating that apoptosis was suppressed. On the other hand, no significant inhibition of apoptosis was observed in N2 mice. However, even in N2 mice, the proliferation of osteoblasts is promoted, and thus apoptosis is considered to be suppressed to some extent. However, the degree is mild compared to K1 mice.
[Comparative Example 9]
Comparison of various histological features in cortical bone
In the same manner as described in Example 4, the cortical bones of various-week-old transgenic mice (N2, K1) and wild-type mice were examined in detail. The results are shown in FIG. From the upper part of FIG. 26, HE image of 10-week-old male femoral distal part, TUNEL staining of 10-week-old male femoral cortical bone, 4-week-old cortical bone transmission electron microscope image (TEM), 10-week-old male femoral cortex It is a silver-stained image of bone and a secondary electron image of bone cells and bone cell processes in 10-week-old cortical bone by scanning electron microscope (SEM).
Cortical bone observation showed that the bone cells of K1 mice showed a young form as seen in transmission electron microscopic images and silver halide staining. Many bone tubules were found that were completely the same.
However, in the cortical bone of the N2 mouse, most bone cells are dead (HE), and the remaining bone cells and the dead bone cavities are TUNEL positive, so the bone cells of the N2 mouse die by apoptosis. Condensed nuclei were also observed in the transmission electron microscope image. When observing bone tubules and bone cell processes using silver halide staining and scanning electron microscopy, an image with very few bone tubules and bone cell processes compared to wild-type mice is observed. Reduction of bone cell processes was thought to be the cause of bone cell apoptosis.
[Comparative Example 10]
Northern blotting
In the same manner as described in Example 5, osteoblasts of 4-week-old wild-type mice and Bcl-2 transgenic mice (N2), 5-week-old wild-type mice and Bcl-xL transgenic mice (K1) Expression of type I collagen, osteopontin and osteocalcin in cells was examined by Northern blotting. The results are shown in FIG.
From FIG. 27, the expression level was the same in the K1 mouse and the wild type, but in the N2 mouse, the expression of osteopontin was stronger and the expression of osteocalcin was lower than in the wild type mouse.
[Comparative Example 11]
in situ hybridization
In the same manner as described in Example 5, expression of type I collagen, osteopontin and osteocalcin in the tibia of 8-week-old wild-type mice, tibia of 8-week-old N2 mice, and tibia of 10-week-old K1 mice was in situ high. Investigated by hybridization. The results are shown in FIG.
From FIG. 28, the expression pattern of these genes in the K1 mouse directly under the growth plate and in the periosteum was not different from that in the wild type mouse. There was a strong expression and a decrease in osteocalcin expression.

According to the present invention, a transgenic animal suitable for elucidation of bone diseases and development of therapeutic agents therefor is provided. The screening method using the animal can contribute to the development of a drug that acts specifically on the Bcl-xL expression system and has few side effects.
This application is based on Japanese Patent Application No. 2006-138287 filed in Japan (filing date: May 17, 2006), the contents of which are incorporated in full herein.
[Sequence Listing]

Claims (23)

  A bone mass and bone strength increasing agent comprising a gene in which a gene encoding Bcl-xL is operably linked downstream of an osteoblast-specific promoter.   The increasing agent according to claim 1, wherein the promoter is a type I collagen promoter.   The increasing agent according to claim 1 or 2, which is an expression vector.   A transgenic non-human animal into which an expression vector containing a gene encoding an osteoblast-specific promoter and Bcl-xL is introduced.   The non-human animal according to claim 4, wherein the promoter is a type I collagen promoter.   The non-human animal according to claim 4 or 5, wherein the bone mass and the bone strength are increased as compared to a wild-type animal of the same species.   The non-human animal according to any one of claims 4 to 6, wherein the expression of the Bcl-xL in osteoblasts is increased by a factor of about 2 compared to that of allogeneic wild type animals.   The non-human animal according to any one of claims 4 to 6, wherein the expression of the Bcl-xL in osteoblasts is increased 3 to 5 times as compared with the allogeneic wild type animal.   The non-human animal according to any one of claims 4 to 6, wherein the expression of the Bcl-xL in osteoblasts is increased by about 10 times as compared with that of a wild-type animal.   The non-human animal according to any one of claims 4 to 9, wherein the animal is a laboratory animal or a domestic animal.   The non-human animal according to claim 10, wherein the experimental animal is a mouse.   The animal is an adult, and compared to adult wild-type animals, the number of cancellous trabeculae, the distance between cancellous trabeculae, the increased connectivity of cancellous bone, and the structure model index of cancellous bone The non-human animal according to any one of claims 4 to 11, wherein at least one parameter selected from a measurement parameter group consisting of a change has a significant difference.   The non-human animal according to any one of claims 4 to 12, which is used as a model in a state in which bone mass and bone strength are increased.   The non-human animal according to any one of claims 4 to 12, which is used as a model for prevention or treatment of osteoporosis. A progeny animal of the non-human animal according to any one of claims 4 to 14 , wherein an expression vector containing an osteoblast specific promoter and a Bcl-xL gene is introduced .   A bone into which an expression vector containing an osteoblast-specific promoter and a Bcl-xl gene, which is obtained by crossing the non-human animal according to any one of claims 4 to 15 with another disease model of the same species, is introduced. Disease model animal.   The model animal according to claim 16, wherein the bone disease is osteoporosis, a disease that causes increased apoptosis of osteoblasts, a disease that causes increased proliferation of osteoblasts, or a disease that causes aging at an early stage.   A tissue or cell obtained from the non-human animal according to any one of claims 4 to 15 or the model animal according to claim 16 or 17.   A screening method for a substance that increases bone mass and / or bone strength, comprising using the non-human animal according to any one of claims 4 to 15 or the model animal according to claim 16 or 17. The following process:
(A) administering a test substance to the non-human animal according to any one of claims 4 to 15 or the model animal according to claim 16 or 17,
(B) examining the bone mass and bone strength in an animal to which the test substance is administered and comparing the bone mass and bone strength in an animal to which the test substance is not administered; and (c) based on the comparison result, A method for screening a substance that increases bone mass and / or bone strength, comprising a step of selecting a test substance that increases bone strength. The following process:
(A) administering a test substance to the non-human animal according to any one of claims 4 to 15 or the model animal according to claim 16 or 17,
(B) examining the bone mass and bone strength in an animal administered with the test substance, comparing with the bone mass and bone strength in an animal not administered with the test substance, and (c) based on the comparison result of step (b) A method for screening a substance that decreases bone mass and / or bone strength, comprising a step of selecting a test substance that decreases bone mass and / or bone strength. The following process:
(A) administering a test substance to the non-human animal according to any one of claims 4 to 15 or the model animal according to claim 16 or 17,
(B) examining the bone mass and bone strength in an animal administered with the test substance, and comparing the bone mass and bone strength in an animal not administered with the test substance;
(C) selecting a test substance that reduces bone mass and / or bone strength based on the comparison result of step (b),
(D) a step of administering to the animal used in step (a) the test substance selected in step (c) and an antagonist candidate substance for the test substance;
(E) examining the bone mass and bone strength in the animal administered the test substance and the antagonist candidate substance in step (d), and comparing the bone mass and bone strength in the animal administered with the test substance alone; and (f) A screening method for a substance that increases bone mass and / or bone strength, comprising selecting an antagonist candidate substance that enhances bone mass and / or bone strength based on the comparison result of step (e). The following process:
(A) contacting osteoblasts with a test substance;
(B) examining the expression level of Bcl-xL in osteoblasts contacted with the test substance, comparing with the expression level in osteoblasts not contacted with the test substance, and (c) based on the comparison result, A method for screening a substance that increases bone mass and / or bone strength through increased expression of Bcl-xL, comprising a step of selecting a test substance that increases the expression of Bcl-xL .