A method for constructing and applying an animal model of osteoporosis based on the Mmp14 gene.

Abstract

This invention provides a method and application for constructing an animal model of osteoporosis based on the Mmp14 gene, belonging to the technical field of osteoporosis animal model construction. This invention utilizes the Ctsk-Cre system to target osteoclasts and their precursor cells to achieve conditional knockout of the Mmp14 gene, thereby constructing an animal model with the Mmp14 genotype. flox / flox A double-positive / Ctsk-Cre osteoporosis animal model. This osteoporosis animal model provides direct evidence for studying the function of Mmp14 in bone homeostasis regulation and provides an experimental platform for developing early diagnostic biomarkers based on Mmp14 gene expression levels. Furthermore, this osteoporosis animal model exhibits pathological features highly consistent with human diseases, including decreased bone mineral density and trabecular bone structure destruction, and can be applied to the study of bone metabolism mechanisms, screening of drugs for the prevention / treatment of osteoporosis, or the development of diagnostic tools.

Description

Technical Field

[0001] This invention relates to the field of osteoporosis animal model construction technology, specifically to a method and application for constructing an osteoporosis animal model based on the Mmp14 gene. Background Technology

[0002] Osteoporosis (OP) is a metabolic bone disease characterized by decreased bone mineral density, bone microarchitectural deterioration, and increased bone fragility. Its high incidence and disability rate impose a heavy socioeconomic burden and severely threaten patients' quality of life. Current clinical drugs generally suffer from limitations in efficacy, safety risks, and relapse rates. Therefore, elucidating its molecular pathological mechanisms, developing early diagnostic biomarkers, and targeted intervention strategies are of great significance. Human osteoporosis is mainly classified into postmenopausal, disuse, and glucocorticoid-induced types. Currently, animal model construction mainly relies on traditional methods such as surgical intervention (e.g., oophorectomy), mechanical load deprivation (e.g., tail suspension), or drug induction (e.g., glucocorticoid injection). However, these methods are difficult to accurately simulate disease progression due to surgical trauma stress, unclear sympathetic nerve regulation mechanisms, or ambiguous osteogenic inhibition mechanisms. More efficient and stable modeling strategies are urgently needed.

[0003] Genetic engineering modeling, through gene editing technology, provides a highly specific and pathologically controllable solution for studying the mechanisms of osteoporosis by targeting gene expression. Among these, membrane type 1 matrix metalloproteinase (MT1-MMP / Mmp14), as a membrane-anchored endopeptidase in the MMP family, plays a central role in bone homeostasis by hydrolyzing extracellular matrix components and non-matrix proteins to regulate osteoclast-related signaling pathways related to bone remodeling. Cathepsin Ctsk, as a marker protease of osteoclasts, dominates matrix degradation during bone resorption and is expressed in mesenchymal progenitors in the Ranvier cartilage groove and osteoblasts in the periosteum, suggesting its potential for cross-cellular synergistic regulation of bone metabolism. Although Mmp14 has been shown to participate in bone development and repair, its functional role in osteoporosis has not been reported, and there is a lack of disease models and therapeutic strategies targeting this gene. Summary of the Invention

[0004] To address the shortcomings of the existing technologies, this invention provides a method and application for constructing an animal model of osteoporosis based on the Mmp14 gene. This invention utilizes the Mmp14 gene... floxAnimals were systematically hybridized with Ctsk-Cre animals to obtain an osteoporosis animal model. This animal model can stably simulate the pathological phenotype of osteoporosis, including but not limited to decreased bone density and destruction of trabecular bone structure, providing a standardized platform for elucidating the disease mechanism of osteoporosis, drug screening, and the development of diagnostic and therapeutic tools.

[0005] To achieve the above objectives, the specific technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention provides a method for constructing an animal model of osteoporosis, comprising the following steps:

[0007] Mmp14 flox Animals were crossbred with Ctsk-Cre animals to obtain F1 generation animals;

[0008] In F1 generation animals, simultaneously satisfying Mmp14 flox Animals with positive expression and Ctsk-Cre positive expression compared to Mmp14 flox Animals are crossbred to obtain F2 generation animals;

[0009] Select animals from F2 generation that simultaneously satisfy Mmp14 flox Positive expression of both Ctsk-Cre and Mmp14 genotype. flox / flox ;Ctsk-Cre + / - These animals are known as osteoporosis model animals.

[0010] Furthermore, the Mmp14 flox The animal is a conditionally repressed Mmp14 gene expression variant with loxP sites flanking the exon region of the Mmp14 gene. flox / flox The Ctsk-Cre animal is a homozygous animal whose genotype is Ctsk-Cre and whose promoter region of the Ctsk gene integrates the Cre recombinase expression element. + / - A hybrid animal.

[0011] Furthermore, the F1 generation animals simultaneously satisfy Mmp14 flox The genotype of animals that positively express Ctsk-Cre and those that positively express Ctsk-Cre is Mmp14. flox / + ;Ctsk-Cre + / - .

[0012] Furthermore, the genotypes of the F2 generation animals were detected using the detection primers shown in SEQ ID NO. 1-4 to screen for osteoporosis model animals. Specifically, Mmp14 was detected by PCR amplification using the primer pairs shown in SEQ ID NO. 1-2. floxGenotype; The Ctsk-Cre genotype was detected by PCR amplification using the primer pairs shown in SEQ ID NO.3~4.

[0013] Furthermore, the animals include rodents.

[0014] Furthermore, the animal is a rodent, and in one example of the present invention, the animal is a C57BL / 6 strain mouse.

[0015] Secondly, the present invention provides reagents for assisting in the selection and breeding of osteoporosis model animals, used for screening model animals in the construction method, wherein the reagents include the detection primers shown in SEQ ID NO.1~4.

[0016] Furthermore, the reagents also include water, dNTPs, Taq DNA polymerase, and PCR buffer.

[0017] Thirdly, the present invention provides the application of osteoporosis model animals obtained by the construction method in the study of bone metabolism mechanisms, screening of drugs for the prevention / treatment of osteoporosis, or development of diagnostic tools.

[0018] Compared with the prior art, the advantages of the present invention are:

[0019] (1) This invention is the first to successfully construct an osteoporosis animal model with a clear pathological phenotype and high clinical consistency by specifically inactivating Mmp14 in osteoclasts and their precursor cells through Ctsk-Cre-mediated conditional knockout technology. The animal model of this invention avoids the non-specific interference of traditional induction methods, accurately reveals the regulatory role of Mmp14 in bone metabolism imbalance, and provides an ideal experimental platform for the development of anti-osteoporosis drugs and personalized treatment strategies.

[0020] (2) The present invention has selected an animal model with a stable osteoporosis pathological phenotype based on the Mmp14 gene. The animal model and its offspring can be used to study the role mechanism of the Mmp14 gene in osteoporosis. Based on this model, early diagnostic kits and instruments can be developed, targeted prevention / treatment drugs can be screened, and efficacy can be evaluated. Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating the strategy for constructing the osteoporosis mouse model in Example 1.

[0022] Figure 2 Mmp14, the osteoporosis model mouse in Example 2 flox / flox Ctsk-Cre + / - PCR gene identification results;

[0023] Figure 3The results of phenotypic detection of osteoporosis model mice in Example 3 are shown in Figure A, which is a picture of 12-week-old osteoporosis model mice; Figure B is the statistical results of body length of 12-week-old osteoporosis model mice; and Figure C is the statistical results of body weight of 12-week-old osteoporosis model mice.

[0024] Figure 4 Figure 4 shows the X-ray examination results of the long bones and the histological analysis results of the femur of the osteoporosis model mice in Example 4. Among them, Figure A shows the X-ray examination results of the long bones of the 12-week-old osteoporosis model mice; Figure B shows the HE staining results of the femur of the 12-week-old osteoporosis model mice; and Figure C shows the data analysis results of the trabecular bone volume of the 12-week-old osteoporosis model mice. Detailed Implementation

[0025] To enable those skilled in the art to clearly and completely understand the technical solution of the present invention, the present invention will be further described in detail below with reference to embodiments. Obviously, the embodiments described herein are only for explaining the present invention and are not intended to limit the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0026] It should be noted that in this invention, "measurement", "determination", "detection" or "examination" refers to evaluating the presence, absence, quantity or amount (which may be an effective amount) of a given substance or object-derived sample (including derivatives of qualitative or quantitative concentration levels of such substance) in a clinical setting, or otherwise assessing the value or classification of non-analytical clinical parameters or clinical determinants of an object.

[0027] In the context of this invention, "sample" refers to a biological sample isolated from an object, such as, but not limited to, rat tails, tissue biopsies, cytological samples, etc.

[0028] The term "treatment" as used in this invention means to slow down, interrupt, prevent, control, stop, alleviate, or reverse the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

[0029] In the context of this invention, all data meet statistical requirements (statistical significance). "Statistical significance" means that the change is greater than that of a single, accidental occurrence (which could be a "false positive"). Statistical significance can be determined using any method known in the art. Commonly used measures of significance include the p-value, which represents the probability that at least the limit value is obtained at a given data point, assuming that the data point is a single, accidental result. A p-value of 0.05 or less is generally considered to indicate a significant difference in the results.

[0030] It should be noted that, in the following embodiments, unless otherwise specified, the techniques or conditions described in the literature in this field, or in accordance with the product instructions, shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0031] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments.

[0032] Example 1: Construction of an osteoporosis mouse model

[0033] like Figure 1 As shown in the paper "MT1-MMP-Dependent Control of Skeletal Stem Cell Commitment via a β1-Integrin / YAP / TAZ Signaling Axis", a conditional knockout of the Mmp14 fragment was inserted into the Mmp14 gene to obtain the genotype Mmp14. flox / flox homozygous mice.

[0034] Based on the paper "Estrogen Prevents Bone Loss via Estrogen Receptor α and Induction of Fas Ligand in Osteoclasts", a Cre recombinase expression element was integrated into the promoter region of the Ctsk gene to obtain the genotype Ctsk-Cre. + / - A heterozygous mouse.

[0035] Genotype Mmp14 flox / flox homozygous mice and mice with genotype Ctsk-Cre + / - Hybrid mice were crossed to obtain F1 generation mice. F1 generation mice included the following two genotypes: Mmp14 flox / + Ctsk-Cre + / - and Mmp14 flox / + .

[0036] F1 generation mice with genotype Mmp14 flox / + Ctsk-Cre + / - Mice with genotype Mmp14 flox / flox Homozygous mice were crossed to obtain F2 generation mice. F2 generation mice included the following four genotypes: Mmp14 flox / + Ctsk-Cre + / - Mmp14 flox / flox Ctsk-Cre + / - Mmp14 flox / + and Mmp14 flox / floxAmong them, Mmp14 flox Double positive for Ctsk-Cre (genotype Mmp14) flox / flox Ctsk-Cre + / - The mice in question are osteoporotic mice, i.e. model mice.

[0037] Distinguishing Mmp14 in F2 generation mice flox / + Or Mmp14 flox / flox The primers used for genotyping detection are shown in Table 1, distinguishing Ctsk-Cre in F2 generation mice. + / - and Ctsk-Cre - / - The detection primers used for the (wild-type) genotype are shown in Table 2.

[0038] Table 1: Distinguishing Mmp14 flox / + Mmp14 flox / flox Detection primers used for genotyping

[0039]

[0040] Table 2: Differentiating Ctsk-Cre + / - Primers used for detecting genotype and wild type

[0041]

[0042] In this embodiment, the Mmp14 construction is used. flox / flox Mice and Ctsk-Cre + / - The mouse technique is a mature technology and will not be described in detail here; meanwhile, this invention relates to Mmp14. flox / flox Mice and Ctsk-Cre + / - The method of constructing mice is not strictly limited. In other embodiments not shown, other construction methods known to those skilled in the art can also be used to construct Mmp14. flox / flox Mice and Ctsk-Cre + / - The mice were used for construction.

[0043] Example 2: Mouse Genotyping Detection

[0044] To obtain Mmp14 flox In Ctsk-Cre double-positive osteoporosis model mice, the Mmp14 genotype in Example 1 was... flox / + Ctsk-Cre + / - F1 generation mice and genotype Mmp14 flox / flox The F2 generation mice produced by hybridization of homozygous mice were tail-cropped, and genomic DNA was extracted and verified by PCR. The target band should include Mmp14. flox / floxThe presence of two bands, Ctsk-Cre and a double band, indicates an osteoporosis model mouse. Please refer to the test results for details. Figure 2 Mmp14 flox / flox PCR product size: 300bp; Ctsk-cre: PCR product size: 992bp.

[0045] In this embodiment, the PCR reaction system and reaction conditions are shown in Tables 3, 4 and 5, respectively. The four detection primers in Table 3 refer to the two detection primers in Table 1 and the two detection primers in Table 2.

[0046] Table 3: PCR reaction system

[0047]

[0048] Table 4: Mmp14 flox / flox PCR reaction conditions

[0049]

[0050] Table 5: Ctsk-cre PCR reaction conditions

[0051]

[0052] Example 3: Morphological and weight detection of model mice

[0053] The 12-week-old osteoporosis model mice selected in Example 2 were photographed for comparison, and their weight and body length were recorded. The results are as follows: Figure 3 As shown in the figure. Compared with the control group mice, the osteoporosis model mice had significantly smaller body size, shorter body length, and significantly lower body weight. These results indicate that the osteoporosis model mice screened in Example 2 exhibit a distinct osteoporosis phenotype. Furthermore, the changes in body length of the osteoporosis model mice in this example share many similarities with previously reported human osteoporosis, suggesting that the model mice constructed in this invention are a suitable model for osteoporosis.

[0054] Example 4: Histological analysis of the femur in model mice

[0055] Further X-ray examination was performed on the long bones of 12-week-old osteoporosis model mice. Histological analysis was conducted on the femurs of 12-week-old osteoporosis model mice. The femurs were paraffin-embedded and sectioned for observation. The results are as follows: Figure 4As shown, compared with the control group mice, the trabecular bone volume of the model mice was significantly reduced. These results indicate that the osteoporosis model mice constructed in this invention have a stable osteoporosis pathological phenotype. This mouse model and its progeny can be used to study the role of the Mmp14 gene in osteoporosis, and based on this model, early diagnostic kits and instruments can be developed, targeted prevention / treatment drugs can be screened, and efficacy can be evaluated.

[0056] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for constructing an animal model of osteoporosis, characterized in that, Includes the following steps: Mmp14 flox Animals were crossbred with Ctsk-Cre animals to obtain F1 generation animals; In F1 generation animals, simultaneously satisfying Mmp14 flox Animals with positive expression and Ctsk-Cre positive expression compared to Mmp14 flox Animals are crossbred to obtain F2 generation animals; Select animals from F2 generation that simultaneously satisfy Mmp14 flox Positive expression of both Ctsk-Cre and Mmp14 genotype. flox / flox ;Ctsk-Cre + / - The animals in question are osteoporosis model animals; the osteoporosis model animals have a stable osteoporosis pathological phenotype. The Mmp14 flox The animal is a conditionally repressed Mmp14 gene with loxP sites flanking the intron region of the Mmp14 gene. flox / flox The Ctsk-Cre animal is a homozygous animal whose genotype is Ctsk-Cre and whose promoter region of the Ctsk gene integrates the Cre recombinase expression element. + / - Hybrid animals; The animal in question is a mouse.

2. The method for constructing an animal model of osteoporosis according to claim 1, characterized in that, The F1 generation animals simultaneously satisfying Mmp14 flox The genotype of animals that positively express Ctsk-Cre and those that positively express Ctsk-Cre is Mmp14. flox / + ;Ctsk-Cre + / - .

3. The method for constructing an animal model of osteoporosis according to claim 1, characterized in that, The genotypes of F2 generation animals were detected using the detection primers shown in SEQ ID NO. 1~4, and osteoporosis model animals were screened out.

4. The application of osteoporosis model animals obtained by the construction method according to any one of claims 1-3 in the study of bone metabolism mechanisms, screening of drugs for the prevention or treatment of osteoporosis, or development of osteoporosis diagnostic tools.

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