CN112680507B - Application of CDC20 gene in diagnosis or treatment of bone metabolism related diseases - Google Patents
Application of CDC20 gene in diagnosis or treatment of bone metabolism related diseases Download PDFInfo
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- CN112680507B CN112680507B CN202110034868.3A CN202110034868A CN112680507B CN 112680507 B CN112680507 B CN 112680507B CN 202110034868 A CN202110034868 A CN 202110034868A CN 112680507 B CN112680507 B CN 112680507B
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Abstract
The invention relates to application of CDC20 gene in diagnosis or treatment of bone metabolism related diseases, and particularly discloses application of CDC20 gene or CDC20 protein as a drug target in screening and/or preparing a drug for preventing and/or treating bone metabolism diseases, and application of an inhibitor and an agonist of CDC20 in preparing a drug for preventing and/or treating bone metabolism diseases.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to application of CDC20 gene in diagnosis or treatment of bone metabolism related diseases.
Background
Osteoporosis is a systemic metabolic bone disease characterized by a damaged microstructure of bone, low bone mass, increased bone fragility, and susceptibility to bone fracture, and is clinically manifested by pain, spinal deformity, and brittle fracture. According to statistics, about 2 hundred million people all over the world currently suffer from osteoporosis, the incidence rate of the osteoporosis jumps over the sixth common disease and frequently-occurring disease of the world, and the osteoporosis is classified as one of three old diseases by the world health organization. In China, 9000 ten thousand patients with osteoporosis account for about 7 percent of the total population, and the total disease rate of osteoporosis is 15.7 percent in people over 50 years old. With the rapid development of economy, the change of life style and the continuous aggravation of the aging problem of population in China, the occurrence and development of serious diseases become important obstacles for further improving the life quality of people, and the problems of medical expenses, related social economy and the like caused by the serious burdens are brought to individuals, families and countries.
Ubiquitination refers to a process in which ubiquitin molecules classify proteins in cells under the action of ubiquitin activating enzyme, binding enzyme, degrading enzyme, etc., and target protein molecules are selected from the proteins, and are specifically modified. Ubiquitination is closely related to the onset of various diseases such as tumors, cardiovascular diseases, neurodegenerative diseases and the like. Therefore, as a result of significant research in the field of biochemistry in recent years, it has become an important target for research and development of new drugs. It has been shown that Bortezomib (Bzd, the first proteasome inhibitor approved for clinical use) injected into osteoporotic mice significantly increases bone mineral density and trabecular bone volume and alleviates bone loss symptoms, and has been found to promote osteogenic differentiation of mesenchymal stem cells in mice.
The current studies on the role of the ubiquitin-proteasome system in bone homeostasis are mostly concerned with ubiquitin ligases. Mitogen regulator 20(CDC20) is a regulator of cell cycle checkpoint, is an activator of the APC/C complex of ubiquitin ligase, and plays an important role in the G1 and M phases of mitosis. Many studies report the biological functions of CDC20 in brain development, cilia disintegration and regulation of apoptosis, and CDC20 is associated with a poor prognosis in a variety of cancers, lung, bladder, colon, breast and oral squamous cell carcinoma, but the role of CDC20 in bone metabolism-related diseases is unclear.
The development and enhancement of basic research, clinical transformation research and new drug development closely related to CDC20 are significant and urgent. The invention relates to a skeletal conditional knockout mouse (Sp 7-Cre; CDC20) prepared by gene knockout technologyf/f) It was found that mice developed severe osteoporosis and bone marrow mesenchymal cellsThe osteogenic capacity is reduced.
Aiming at the new discovery of CDC20 in regulation and control of mouse bone formation, the novel application of CDC20 in osteoporosis diseases is verified, a novel target point is provided for development of anti-osteoporosis drugs, and a foundation is laid for development of CDC20 agonists or antagonists for treatment of bone metabolism related diseases.
Disclosure of Invention
One aspect of the present invention provides the use of a CDC20 gene or CDC20 protein in the screening of a drug target for the treatment or prevention of a bone metabolic disease.
One aspect of the present invention provides a use of CDC20 gene or CDC20 protein as a therapeutic target for preventing and/or treating a bone metabolic disease, or as a diagnostic target for a bone metabolic disease.
In the technical scheme of the invention, the bone metabolism disease is related to bone growth or bone abnormality; such as osteoporosis, osteopetrosis, Paget's disease (Paget), dysplasia of bone fibers, and osteogenesis.
Another aspect of the present invention provides a use of an inhibitor of CDC20 gene or CDC20 protein in the preparation of a medicament for preventing or treating a disease associated with abnormal increase in bone growth or bone mass or a disease associated with increased osteogenic differentiation activity of mesenchymal stem cells of bone marrow and/or decreased osteoclastic differentiation activity of bone marrow mononuclear cells.
Preferably, the disease associated with abnormal increase in bone growth or bone mass is osteopetrosis.
Wherein the inhibitor of CDC20 gene or CDC20 protein is selected from CDC20 antagonist, CDC20sh RNA, CDC20siRNA, etc.
Another aspect of the present invention provides a use of an agonist of CDC20 gene or CDC20 protein in the preparation of a medicament for preventing or treating a disease associated with abnormal decrease in bone growth or bone quality, or a disease associated with decreased osteogenic differentiation activity of mesenchymal stem cells and/or increased osteoclastic differentiation activity of bone marrow mononuclear cells.
Preferably, the disease associated with abnormal reduction of bone growth or bone mass is osteoporosis, Paget's disease (Paget), osteogenesis imperfecta, bone fiber dysplasia, or the like.
Another aspect of the present invention provides an animal model for mimicking a disease associated with abnormal reduction of bone growth or bone mass, wherein only CDC20 gene of animal skeletal system is knocked out.
One aspect of the present invention provides a method for the preparation of an animal model for mimicking a disease associated with abnormal reduction of bone growth or bone mass, wherein Sp7-Cre conditional knockout CDC20 mice are used.
CDC20 gene described in the present invention is a gene known in the art and is a unique sequence, GeneID 108995 at NCBI.
The invention firstly proves the relation between CDC20 gene and bone metabolism related diseases, and provides basis for the prevention, diagnosis and treatment of diseases. Specifically, an agonist of CDC20 gene or CDC20 protein may be used in the preparation of a medicament for preventing or treating diseases associated with abnormal decrease in bone growth or bone mass, such as osteoporosis, Paget's disease (Paget), osteogenesis imperfecta, bone fiber dysplasia, etc., or for the preparation of a medicament for treating diseases associated with decreased osteogenic differentiation activity of mesenchymal stem cells and/or increased osteoclastic differentiation activity of bone marrow mononuclear cells. Inhibitors of CDC20 gene or CDC20 protein for the preparation of a medicament for the prophylaxis or treatment of diseases associated with abnormal increase in bone growth or bone mass, such as osteopetrosis. Or can be used for preparing medicines for treating related diseases caused by the enhancement of osteogenic differentiation activity of the mesenchymal stem cells and/or the reduction of osteoclastic differentiation activity of the mononuclear cells.
Drawings
FIG. 1 is a Micro-CT scan of 6-week male control mice (CDC20)f/f) And 6-week-old androgen conditioned knockout mice (Sp 7-Cre; CDC20f/f) Femur drawing and H&E staining pattern.
FIG. 2 is a Micro-CT analysis of 6-week male control mice (CDC20)f/f) And 6-week-old androgen conditioned knockout mice (Sp 7-Cre; CDC20f/f) The bone parameter index map of the femur.
FIG. 3 is a Micro-CT scan of 6 week female control mice (CDC20)f/f) And 6-week female skeletal conditional knockdown mice (Sp 7-Cre; CDC20f/f) Femur drawing and H&E staining pattern.
FIG. 4 is Micro-CT analysis of 6 week female control mice (CDC20)f/f) And 6-week female skeletal conditional knockdown mice (Sp 7-Cre; CDC20f/f) The bone parameter index map of the femur.
Detailed Description
Example 1Micro-CT scanning of 6-week male control mice (CDC20)f/f) And 6-week-old androgen conditioned knockout mice (Sp 7-Cre; CDC20f/f) Femur and H&E dyeing
Collection of mouse femoral samples
CDC20 skeletal conditional gene knockout 6-week male mice (Sp 7-Cre; CDC20) with C57BL/6J backgroundf/f) And control 6-week male mice (CDC20)f/f) Purchased from baiosaiguratin biotechnology limited. The mice were kept in a constant temperature, humidity pathogen free (SPF) environment with a 12h light and dark cycle, and were allowed to eat and drink water autonomously at all times. Welfare and experimental procedures for mice strictly follow the rules of the experimental animal practice and the ethical regulations of the animal committee of the oral hospital, Beijing university. After the mice are killed at the neck-broken positions, right femur formalin is fixed and used for CT analysis, and left femur formalin is fixed and then decalcified and paraffin-embedded.
Bone section H & E (hematoxylin-easin) staining
(1) Paraffin sections were cut and routinely dewaxed to water.
(2) Hematoxylin is added dropwise to cover the specimen and the staining is carried out for 3 min.
(3) The hematoxylin is washed clean by tap water.
(4) The slices were immersed in a bluing solution for 60s and washed clean with tap water.
(5) The slices were immersed in 95% ethanol for 30s, and then stained with eosin dropwise for 1 min.
(6) Washing with tap water.
(7) Dehydrating, transparentizing, sealing and taking a picture.
Example 2Micro-CT analysis of 6-week Male control mice (CDC20)f/f) And 6-week-old androgen conditioned knockout mice (Sp 7-Cre; CDC20f/f) The bone parameter index of the femur
Mouse femur CT scan analysis
Micro CT was used on the mouse thigh of example 1Bone scanning was performed, femur position was fixed using a foam plate, instrument scanning parameters were set to 220 μ Α current, 60kV voltage, and 1500ms exposure time. Data analysis was performed on the femoral metaphyseal trabecular bone. After 50 layers are continuously discarded from the disappearance position of the femur distal metaphysis growth plate, 400 layers are selected as a trabecular bone reconstruction area for analysis, reconstruction is carried out by using self-contained software, and the threshold value is 80. The main analytical indexes include bone density (BMI), bone volume integral number (BV/TV), trabecular number (Tb.N), trabecular resolution (Tb.Sp),*P<0.05,**P<0.01,***P<0.001. statistical analysis of the collected data was performed using GraphPad Prism software, all data presented as mean ± standard deviation, with 4-8 samples per group.
Example 3Micro-CT scanning 6 week female control mice (CDC20)f/f) And 6-week female skeletal conditional knockdown mice (Sp 7-Cre; CDC20f/f) Femur and H&E dyeing
Experimental method As in example 1, the experimental mice were replaced with CDC20 bone-conditioned gene knockout 6-week female mice (Sp 7-Cre; CDC20) with C57BL/6J backgroundf/f) And control 6-week female mice (CDC20)f/f) Purchased from baiosaitan gene biotechnology limited.
Example 4Micro-CT analysis of 6 week female control mice (CDC20)f/f) And 6-week female skeletal conditional knockdown mice (Sp 7-Cre; CDC20f/f) The bone parameter index of the femur
Experimental methods As in example 2, the experimental mice were replaced with CDC20 skeletal conditional gene knockout 6-week female mice (Sp 7-Cre; CDC20) with C57BL/6J backgroundf/f) And control 6-week female mice (CDC20)f/f) Purchased from baiosaitan gene biotechnology limited.
Female mice have overall lower bone parameters than male mice and are therefore grouped individually. The occurrence of severe bone loss in knockout of CDC20 in the skeletal system was established in both males and females, a common phenomenon.
FIG. 1 shows Micro-CT scanning of 6-week male control mice (CDC20)f/f) And 6-week-old androgen conditioned knockout mice (Sp 7-Cre; CDC20f/f) Femur drawing (D) and (H)&E staining pattern, FIG. 3 is Micro-CT scan 6 week female control mice (CDC20)f/f) And 6-week female skeletal conditional knockdown mice (Sp 7-Cre; CDC20f/f) Femur drawing and H&E staining pattern. As shown in the femoral diagrams in fig. 1 and 3, the distribution of trabecular cancellous bone under the femoral and femoral distal growth plate regions is shown by 3D reconstruction of Micro-CT scan data: skeletal conditioned knockout CDC20 mice exhibited significant trabecular reduction in the cancellous bone region distal to the femur, including both transverse and longitudinal sections of the femur, with similar results compared to wild-type mice.
The results of analyzing bone parameter data by software are shown in fig. 2 and 4, the bone density (BMD), bone volume fraction (BV/TV) and number (tb.n) of bone trabeculae of mice knockout CDC20 mice are all significantly reduced, the separation degree (tb.sp) of bone trabeculae is significantly increased in the distal femur bone conditional gene of mice, which is consistent with the 3D reconstruction graph, and further prove that serious bone loss of mice knockout CDC20 mice due to the bone conditional gene of mice is first reported in vivo about bone formation of mice mediated by CDC 20.
In order to research the structural change in the femur of a mouse, the femur of the mouse is decalcified, then the femur is sliced in paraffin, H & E staining can clearly observe the structural change of the trabecular bone of the mouse, in order to enhance the experimental reliability, the mouse is divided into a female mouse and a male mouse, 4-8 samples in each group are respectively taken for analysis, and the H & E staining graphs in figures 1 and 3 show that the number of the trabecular bone in the distal cancellous bone area of the femur of the mouse subjected to the gene knockout CDC20 under the skeletal condition is obviously reduced, and the bone separation degree is obviously increased. The same results were obtained in the female and male groups, indicating that knockout of CDC20 in the skeletal system results in bone loss.
Claims (2)
1. A method for producing an animal model for simulating bone loss, which comprises knocking out CDC20 gene of the skeletal system of an animal.
2. The method of claim 1, wherein the CDC20 gene knockout of the animal skeletal system is performed using Sp7-Cre conditional knockout CDC 20.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2013204098A1 (en) * | 2005-11-14 | 2013-05-02 | Amgen Inc. | Rankl antibody-PTH/PTHrP chimeric molecules |
| CN107114311A (en) * | 2017-04-26 | 2017-09-01 | 西北工业大学 | The structure for the mouse model that MACF1 genes are knocked out in Gegenbaur's cell conditional and application |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2013204098A1 (en) * | 2005-11-14 | 2013-05-02 | Amgen Inc. | Rankl antibody-PTH/PTHrP chimeric molecules |
| CN107114311A (en) * | 2017-04-26 | 2017-09-01 | 西北工业大学 | The structure for the mouse model that MACF1 genes are knocked out in Gegenbaur's cell conditional and application |
Non-Patent Citations (2)
| Title |
|---|
| CDC20 is critical for meiosis I and fertility of female mice;Fang Jin等;《Plos Genetics》;20100930;第6卷(第9期);e1001147第1-16页 * |
| 白细胞介素-1基因多态性与类风湿关节炎病情活动及骨代谢的相关性;张晓等;《中华风湿病学杂志》;20010430;第5卷(第2期);第87-90页 * |
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