Use of a substance that increases the expression level of ras-related protein 2 or a biological carrier containing a gene encoding ras-related protein 2 in the preparation of a drug for treating osteoporosis

Abstract

The application discloses application of a substance for improving expression level of RAS-related protein 2 or a biological carrier containing a RAS-related protein 2 coding gene in preparation of a medicine for treating osteoporosis, and relates to the technical field of biological medicine. The application provides application of the substance for improving expression level of RAS-related protein 2 in preparation of the medicine for treating osteoporosis. It is found by the application that the expression level of RAS-related protein 2 in bone tissue of an old mouse is significantly reduced, and the absence of the RAS-related protein 2 coding gene can cause the osteogenic differentiation ability of mouse bone marrow mesenchymal stem cells to be impaired, and cause osteoporosis of the mouse. Therefore, the substance for improving expression level of RAS-related protein 2 can realize treatment of osteoporosis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of substances that enhance the expression level of RAS-associated protein 2 or biological vectors containing the gene encoding RAS-associated protein 2 in the preparation of drugs for treating osteoporosis. Background Technology

[0002] Bone homeostasis is maintained by a delicate balance between osteoblast-mediated bone formation and osteoclast-driven bone resorption. Disruption of this balance can lead to skeletal diseases such as osteoporosis and osteopenia. The core of bone formation is the differentiation of bone marrow mesenchymal stem cells (BMSCs) into functional osteoblasts, a process regulated by multiple signaling pathways, including the bone morphogenetic protein (BMP) / SMAD pathway (which is a core signal transduction molecule in the transforming growth factor-β signaling pathway).

[0003] Bone morphogenetic protein 2 (BMP2) is the largest subclass in the transforming growth factor-β (TGF-β) superfamily, transmitting signals via transmembrane type II serine / threonine kinase receptors (BMPR2) and type I serine / threonine kinase receptors (BMPR1). Clinically, recombinant bone morphogenetic protein 2 (BMP2) is a bone-inducing drug with bone anabolic effects; however, it requires supraphysiological doses and often causes adverse reactions such as inflammation, edema, nerve compression, and heterotopic ossification. Therefore, there is an urgent need to develop drugs for the treatment of osteoporosis, providing more options for its treatment.

[0004] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0005] Based on the shortcomings of the prior art, the purpose of this invention is to provide the application of substances that enhance the expression level of RAS-associated protein 2 or biological vectors containing the RAS-associated protein 2 encoding gene in the preparation of drugs for treating osteoporosis, with the aim of developing drugs for treating osteoporosis and providing more options for the treatment of osteoporosis.

[0006] The technical solution of the present invention is as follows: In a first aspect, the invention provides the use of a substance that enhances the expression level of RAS-associated protein 2 in the preparation of a medicament for treating osteoporosis.

[0007] In a second aspect, the invention provides the use of a biological vector containing a gene encoding RAS-associated protein 2 in the preparation of a medicament for treating osteoporosis.

[0008] RAS-associated protein 2 (RAS-A2) belongs to the RAS family of guanosine triphosphatases (GTPases) and participates in immune homeostasis by regulating antigen receptors on B cells and T cells. However, this invention found that compared to young mice, RAS-A2 and its encoding gene mRNA are reduced in the bone tissue of aged mice, indicating that a decrease in RAS-A2 or its encoding gene is an important pathological feature of age-related osteoporosis. The deletion of the RAS-A2 encoding gene leads to impaired osteogenic differentiation of mouse bone marrow mesenchymal stem cells, causing osteoporosis. Furthermore, RAS-A2 and its encoding gene regulate bone formation in osteogenic progenitor cells through a cell-autonomous mechanism. RAS-A2 enhances the BMP signaling pathway by inhibiting SMMURF1 (SMAD-specific E3 ubiquitin ligase 1)-mediated ubiquitination and degradation of BMPR2 protein, providing a new strategy for enhancing cellular sensitivity to BMP2 protein. Further research in this invention revealed that lentivirus-mediated overexpression of the RAS-A2 encoding gene can enhance the osteogenic differentiation and bone regeneration capacity of human bone marrow mesenchymal stem cells. Injecting an adeno-associated virus expression vector carrying the RAS-associated protein 2 (RAS-2) encoding gene into the bone of aged osteoporotic mice significantly increased bone density, improved bone microstructure, and promoted bone formation, effectively treating osteoporosis. Therefore, this invention provides a novel therapeutic target for osteoporosis. Substances that enhance RAS-2 expression levels, substances that enhance the expression levels of the RAS-2 encoding gene, or biological vectors containing the RAS-2 encoding gene can be used to treat osteoporosis, offering more treatment options.

[0009] Optionally, the gene encoding RAS-associated protein 2 can be human or mouse-derived.

[0010] Optionally, the nucleotide sequence of the human RAS-associated protein 2 encoding gene is shown in SEQ ID NO: 1.

[0011] Optionally, the nucleotide sequence of the mouse-derived RAS-associated protein 2 encoding gene is shown in SEQ ID NO: 2.

[0012] Optionally, the biological vector is a viral vector or a cell.

[0013] Optionally, the viral vector is an adenovirus vector, an adeno-associated virus vector, or a lentivirus vector; The cells in question are human bone marrow mesenchymal stem cells.

[0014] In this embodiment, the nucleotide sequence of the biological vector containing the RAS-associated protein 2 encoding gene is shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0015] Optionally, the dosage form of the drug includes injections, tablets, or capsules.

[0016] Optionally, the medicament may further include a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable additive.

[0017] Optionally, the pharmaceutically acceptable carrier includes at least one of a flow aid, diluent, wetting agent, suspending agent, solvent, and emulsifier; the pharmaceutically acceptable adjuvant includes at least one of a preservative, colorant, flavoring agent, stabilizer, and isotonic agent.

[0018] Beneficial Effects: RAS-associated protein 2 (RAS-A2) belongs to the RAS family of small GTPases and participates in immune homeostasis by regulating antigen receptors on B cells and T cells. However, this invention found that compared to young mice, the mRNA levels of RAS-A2 and its encoding gene are reduced in the bone tissue of aged mice, indicating that a decrease in RAS-A2 or its encoding gene is an important pathological feature of senile osteoporosis. The deletion of the RAS-A2 encoding gene leads to impaired osteogenic differentiation of mouse bone marrow mesenchymal stem cells, causing osteoporosis. Furthermore, RAS-A2 and its encoding gene regulate bone formation in osteogenic progenitor cells through a cell-autonomous mechanism. RAS-A2 enhances the BMP signaling pathway by inhibiting SMURF1-mediated ubiquitination and degradation of BMPR2 protein, providing a new strategy for enhancing cellular sensitivity to BMP2 protein. Further research in this invention revealed that lentivirus-mediated overexpression of the RAS-A2 encoding gene can enhance the osteogenic differentiation and bone regeneration capacity of human bone marrow mesenchymal stem cells. Injecting an adeno-associated virus expression vector carrying the RAS-associated protein 2 (RAS-2) encoding gene into the bone of aged osteoporotic mice significantly increased bone density, improved bone microstructure, and promoted bone formation, effectively treating osteoporosis. Therefore, this invention provides a novel therapeutic target for osteoporosis. Substances that enhance RAS-2 expression levels, substances that enhance the expression levels of the RAS-2 encoding gene, or biological vectors containing the RAS-2 encoding gene can be used to treat osteoporosis, offering more treatment options. Attached Figure Description

[0019] Figure 1 The figures show the results of the study on Rras2 levels in bone tissues of young and old wild-type mice in Example 1. In Figure A, the results show the expression levels of Rras2 mRNA in bone tissues of young and old wild-type mice, and in Figure B, the results show the expression levels of RRAS2, BMPR2, P16, and P21 proteins in bone tissues of young and old wild-type mice.

[0020] Figure 2 This is a diagram showing the results of an osteoblast-specific Rras2 knockout study in Example 2 that induced osteoporosis in mice. In this diagram, A represents Rras2. f / fThree-dimensional reconstructed images of the distal femur of mice and RPKO mice using microcomputed tomography (MCT), B represents Rras2. f / f Figure 1 shows the quantitative bone mineral density (BMD) results of the femur in mice and RPKO mice. C represents Rras2. f / f Figure 1 shows the quantitative results of femoral bone volume fraction in mice and RPKO mice, where D represents Rras2. f / f Quantitative results of trabecular bone number in the femur of mice and RPKO mice, E represents Rras2. f / f Quantitative results of trabecular bone thickness in the femur of mice and RPKO mice, F represents Rras2. f / f Quantitative results of trabecular separation in the femur of mice and RPKO mice, G represents Rras2. f / f Quantitative results of cortical bone thickness in the femur of mice and RPKO mice, H represents Rras2. f / f Hematoxylin-eosin staining images of the femurs of mice and RPKO mice, I represents Rras2. f / f Figure 1 shows the quantitative results of osteoblast counts in the femur of mice and RPKO mice. J represents Rras2. f / f Calcein-xylenol orange double-labeled fluorescence images of trabecular and cortical bone in mice and RPKO mice, where K represents Rras2. f / f Quantitative results of bone formation rate in trabecular and cortical bone of mice and RPKO mice, L represents Rras2. f / f Figure 1 shows the mRNA levels of osteogenic marker genes after osteogenic induction in bone marrow mesenchymal stem cells of mice and RPKO mice. M represents Rras2. f / f Alkaline phosphatase staining images of bone marrow mesenchymal stem cells after osteogenic induction in mice and RPKO mice, where N represents Rras2. f / f Alizarin red staining of bone marrow mesenchymal stem cells after osteogenic induction in mice and RPKO mice.

[0021] Figure 3 This is a diagram showing the results of the molecular mechanism study on Rras2-driven osteoogenesis in Example 3, where A represents Rras2. f / f Volcano plot of differentially expressed RNA sequences of bone marrow mesenchymal stem cells in mice and RPKO mice 2 days after osteogenic induction. B represents Rras2. f / f Figure 1 shows the results of differential gene enrichment analysis of the KEGG pathway 2 days after osteogenic induction of bone marrow mesenchymal stem cells in mice and RPKO mice. C represents Rras2. f / f Heatmap of BMP signaling pathway-related gene expression 2 days after osteogenic induction of bone marrow mesenchymal stem cells in mice and RPKO mice. D is the result of the Id1-BRE luciferase reporter assay.

[0022] Figure 4The figures show the results of a study on the inhibition of BMPR2 protein ubiquitination and degradation mediated by SMURF1 protein by RRAS2 protein. In figure A, immunoprecipitation-mass spectrometry analysis identifies interacting proteins of RRAS2; figure B shows the results of immunoprecipitation verification of the interaction between RRAS2 and BMPR2 proteins in human bone marrow mesenchymal stem cells; and figure C shows the results of RRAS2 protein inhibition. + / + Mice and Rras2 - / - The graph shows the expression level of BMPR2 protein during osteogenic differentiation of mouse bone marrow mesenchymal stem cells. D represents Rras2. + / + Mice and Rras2 - / - The graph shows the Bmpr2 mRNA level during osteogenic differentiation of mouse bone marrow mesenchymal stem cells. E represents Rras2. - / - The graph shows the BMPR2 protein levels in mouse bone marrow mesenchymal stem cells after treatment with MG132 or chloroquine. F represents Rras2. + / + Mice and Rras2 - / - The graph shows the results of BMPR2 protein ubiquitination levels in mouse bone marrow mesenchymal stem cells. G represents the results of immunoprecipitation-immunoblotting detection of BMPR2 protein ubiquitination levels in control cells and RRAS2-overexpressing human bone marrow mesenchymal stem cells. H represents the results of immunoprecipitation detection of the effect of Smurf1 knockdown on BMPR2 protein ubiquitination. I represents the results of GST pull-down assay detection of the effect of RRAS2 protein on the interaction between SMURF1 and BMPR2 proteins. J represents the results of immunoprecipitation-immunoblotting detection of the effect of RRAS2 overexpression on BMPR2 protein ubiquitination in human bone marrow mesenchymal stem cells. K represents the results of immunoblotting detection of the effect of SMURF1 overexpression and RRAS2 overexpression on BMPR2 protein levels in human bone marrow mesenchymal stem cells.

[0023] Figure 5Figure 5 shows the results of the study on the promotion of osteogenic differentiation and bone regeneration of human bone marrow mesenchymal stem cells by lentivirus-mediated human RRAS2 overexpression in Example 5. A is a fluorescence micrograph of human bone marrow mesenchymal stem cells infected with Lenti-RRAS2-OE-RFP lentivirus; B is the RRAS2 mRNA level in the RRAS2 overexpression group and the control group; C is the RRAS2 protein level in the RRAS2 overexpression group and the control group; D is the alkaline phosphatase staining of the RRAS2 overexpression group and the control group; E is the phosphatase activity after alkaline phosphatase staining of the RRAS2 overexpression group and the control group; and F is the Von phosphatase activity of the RRAS2 overexpression group and the control group. Kossa staining images: G shows the mineralization rate of RRAS2-overexpressing cells and control cells; H shows the hematoxylin-eosin staining image 8 weeks after subcutaneous transplantation of 2,3Col-GFP-labeled RRAS2-overexpressing cells and control cells into SCID mice; I shows the fluorescence image 8 weeks after subcutaneous transplantation of 2,3Col-GFP-labeled RRAS2-overexpressing cells and control cells into SCID mice; J shows the bone volume fraction 8 weeks after subcutaneous transplantation of 2,3Col-GFP-labeled RRAS2-overexpressing cells and control cells into SCID mice; K shows the number of GFP-positive cells 8 weeks after subcutaneous transplantation of 2,3Col-GFP-labeled RRAS2-overexpressing cells and control cells into SCID mice; L shows the micro-computed tomography scan 8 weeks after transplantation of RRAS2-overexpressing cells and control cells into the SCID mouse cranial defect model; M shows the bone volume fraction 8 weeks after transplantation of RRAS2-overexpressing cells and control cells into the SCID mouse cranial defect model.

[0024] Figure 6The figures shown in Example 6 illustrate the treatment results of osteoporosis in aged mice. A represents the Rras2 mRNA level in bone tissue of mice in the AAV-Rras2 treatment group (2 weeks) and the AAV-control treatment group (2 weeks); B represents the RRAS2 protein level in bone tissue of mice in the AAV-Rras2 treatment group (2 weeks) and the AAV-control treatment group (2 weeks); C represents a fluorescence micrograph of bone tissue sections from mice in the AAV-Rras2 treatment group (2 weeks); D represents a computed tomographic microscopy image of the femur from mice in the AAV-Rras2 treatment group (6 weeks) and the AAV-control treatment group (6 weeks); E represents the quantitative results of bone volume fraction in the femur from mice in the AAV-Rras2 treatment group (6 weeks) and the AAV-control treatment group (6 weeks); F represents the quantitative results of trabecular bone number in the femur from mice in the AAV-Rras2 treatment group (6 weeks) and the AAV-control treatment group (6 weeks); G represents the quantitative results of trabecular bone number in the femur from mice in the AAV-Rras2 treatment group (6 weeks) and the AAV-control treatment group (6 weeks); and G represents the quantitative results of trabecular bone number in the femur from mice in the AAV-Rras2 treatment group (6 weeks) and the AAV-control treatment group (6 weeks). The graph shows the quantitative results of trabecular bone thickness in the femur of mice in the AAV-Rras2 treatment group (6 weeks), H is the quantitative results of trabecular bone separation in the femur of mice in the AAV-Rras2 treatment group (6 weeks) and the AAV-control treatment group (6 weeks), I is the calcein-xylenol orange double-labeled fluorescence image of mice in the AAV-Rras2 treatment group (6 weeks) and the AAV-control treatment group (6 weeks), J is the quantitative results of bone formation rate of mice in the AAV-Rras2 treatment group (6 weeks) and the AAV-control treatment group (6 weeks), K is the alkaline phosphatase staining image of mice in the AAV-Rras2 treatment group (6 weeks) and the AAV-control treatment group (6 weeks), and L is the percentage of alkaline phosphatase positive area of ​​mice in the AAV-Rras2 treatment group (6 weeks) and the AAV-control treatment group (6 weeks).

[0025] In the picture, This indicates that P < 0.05. This indicates that P < 0.01. P < 0.001 indicates that all three values ​​are considered statistically significant. ns indicates no significant difference. Detailed Implementation

[0026] This invention provides the application of substances that enhance the expression level of RAS-associated protein 2 or biological vectors containing the RAS-associated protein 2 encoding gene in the preparation of drugs for treating osteoporosis. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0028] The present invention will be further described below through specific embodiments.

[0029] In the following examples, RAS-associated protein 2 is referred to as "RRAS2 protein"; the human RAS-associated protein 2 encoding gene is referred to as "RRAS2"; and the mouse RAS-associated protein 2 encoding gene is referred to as "Rras2".

[0030] In the following examples, all experimental procedures were approved by the Institutional Animal Care and Use Committee of Shenzhen University. All mice were maintained in a C57BL / 6J background and housed under specific pathogen-free conditions. Rras2 + / - The mice were generated by Shanghai Southern Model Organisms Technology Development Co., Ltd. using CRISPR / Cas9 technology, and Rras2 was obtained through self-crossing. + / + Mice and Rras2 - / - Mice. Rras2 f / f The mice (with loxP sites flanking exons 2 and 3 of the Rras2 gene) were generated by Cyagen (Guangzhou) Biotechnology Co., Ltd.

[0031] In the following examples, test data are presented as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism. One-way ANOVA and Tukey post-hoc tests were used for comparisons among multiple groups, and two-tailed Student's t-tests were used for comparisons between two groups.

[0032] The experimental methods used in the following embodiments are as follows: Co-immunoprecipitation (Co-IP): Cells were lysed for 15 minutes in IP lysis buffer (containing 20 mM Tris (pH 7.8), 150 mM NaCl, 0.2% ethylphenyl polyethylene glycol (NP-40), and 10% glycerol) supplemented with a protease inhibitor mixture (Roche Complete). The lysates were clarified by centrifugation and then incubated overnight at 4°C with specific antibodies or control IgG conjugated to Protein A / G agarose beads (Invitrogen, USA). After washing five times with IP lysis buffer, the immunoprecipitate was eluted by boiling in loading buffer for 6 minutes and analyzed by Western blot.

[0033] Western blot: Tissue or cells were added to RIPA lysis buffer (containing protease inhibitors), homogenized on ice, and lysed at 4°C for 30 minutes. Centrifuged at 12000 rpm for 10 minutes, and the supernatant was collected. Protein concentration was determined using the quinoline carboxylic acid (BCA) method. 20 μg of protein was subjected to SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% skim milk for 1 hour and incubated overnight at 4°C with primary antibody. Incubation with secondary antibody at room temperature for 1 hour was followed by enhanced chemiluminescence (ECL) imaging. Separation was achieved by SDS-PAGE, and immunoblotting analysis was performed. The immunoreaction bands were detected and quantified using ECL and Image J.

[0034] The primary antibodies used were as follows: rabbit anti-RRAS2 (Affinity Biosciences, DF9840), mouse anti-BMPR2 (Invitrogen, 3F6F8), rabbit anti-ubiquitin (Proteintech, 10201-2-AP), rabbit anti-Myc tag (Proteintech, 16286-1-AP), rabbit anti-Flag (Proteintech, 20543-1-AP), mouse anti-GAPDH (Beyotime, AF2819), mouse anti-GST (Beyotime, AF2888), mouse anti-Smurf1 (Santa Cruz, sc-100616), mouse anti-P16 (Santa Cruz, sc-1661), and mouse anti-P21 (Santa Cruz, sc-6246).

[0035] Example 1: Study on Rras2 levels in bone tissue of young and aged mice To investigate the role of Rras2 in age-related osteoporosis, the expression of Rras2 in the bones of young (3-month-old) and old (23-month-old) wild-type mice was examined. Specifically, the expression level of Rras2 mRNA in the bone tissue of young (3-month-old) and old (23-month-old) wild-type mice (males and females) was detected by qPCR (real-time quantitative polymerase chain reaction). The expression levels of RRAS2, BMPR2, P16 (cyclin-dependent kinase inhibitor 2A), and P21 (cyclin-dependent kinase inhibitor 1A) proteins in the bone tissue of young (3-month-old) and old (23-month-old) wild-type mice were detected by Western blotting.

[0036] The results are as follows Figure 1As shown in the figure (where GAPDH is glyceraldehyde-3-phosphate dehydrogenase), compared with young mice, the levels of Rras2 mRNA and RRAS2 protein in the bones of aged mice were significantly decreased, and the level of BMPR2 protein was also significantly reduced, while the levels of aging markers P16 and P21 proteins were significantly increased. These results indicate that the decrease in Rras2 levels during aging is associated with osteoporosis.

[0037] Example 2: Study on osteoporosis induced by osteoblast-specific Rras2 knockout in mice To clarify the causal relationship between Rras2 and the occurrence of osteoporosis, Rras2... f / f Mice were mated with Prrx1-Cre mice to obtain Rras2. f / f Prrx1-Cre osteoblast lineage-specific Rras2 knockout mice (designated RPKO, 3 months old), littermate Rras2 f / f Mice (3 months old) served as the control group.

[0038] The femurs of two groups of mice were dissected and fixed in 4% paraformaldehyde (PFA) solution at 4°C for 36 hours. Micro-computed tomography (μCT) imaging was performed using a SkyScan 1176 system (Brook, Kartuizersweg, Belgium) at a resolution of 9 μm for quantitative assessment. Trabecular bone parameters, including bone mineral density, bone volume fraction, trabecular number, trabecular thickness, and trabecular separation, were quantitatively analyzed in the region 0.45–0.90 mm distal to the femoral growth plate (50 slices). Cortical bone thickness at 2.88 mm below the growth plate was measured using CTAn (a specialized image analysis tool for quantitative analysis of bone tissue microstructure). Results are as follows: Figure 2 A to Figure 2 As shown in G. Where, with Rras2 f / f Compared to mice, RPKO mice, regardless of sex, showed significantly reduced bone mass (e.g. Figure 2 (As shown in A). Quantitative analysis results indicate that, compared to Rras2 f / f Bone mineral density (e.g., in mice, RPKO mice) Figure 2 (as shown in B), bone volume fraction (e.g.) Figure 2 (as shown in C), number of trabeculae (as shown in C) Figure 2 As shown in D), trabecular bone thickness (as shown in D). Figure 2 (as shown in E) and cortical bone thickness (as shown in E) Figure 2 The values ​​shown in G in the figure all decreased, while the trabecular separation increased (as shown in the figure). Figure 2 (as shown by F in the diagram).

[0039] The femurs of both groups of mice were fixed in 10% EDTA (ethylenediaminetetraacetic acid) solution and decalcified for 3 days. Paraffin-embedded bone sections (5 μm) were then prepared and stained with hematoxylin and eosin. The representative region of interest (ROI) was defined as the trabecular bone region located 500 μm below the growth plate. The results of hematoxylin and eosin staining and quantitative analysis are as follows: Figure 2 H and Figure 2 As shown in Figure I, the results indicate that, compared to Rras2 f / f Compared to mice, RPKO mice showed a significant reduction in the number of osteoblasts per unit bone circumference.

[0040] Dynamic bone formation was analyzed in two groups of mice by intraperitoneal injection of calcein (10 mg / kg) and xylenol orange (90 mg / kg) 7 days later. Femurs were fixed in 4% PFA, and undecalcified sections were prepared. Fluorescent labels were imaged using an Olympus BX63 fluorescence imaging system, and quantification was performed using ImageJ. Results of calcein-xylenol orange dual labeling and quantitative analysis of trabecular and cortical bone are shown below. Figure 2 J and Figure 2 As shown in K, the results indicate that, compared to Rras2 f / f Compared to mice, RPKO mice had a lower rate of bone formation, indicating impaired bone formation.

[0041] Separate Rras2 f / f Bone marrow mesenchymal stem cells from mice and RPKO mice were cultured in osteogenic induction medium containing 100 nM dexamethasone, 10 mM sodium β-glycerophosphate, and 50 μg / mL ascorbic acid.

[0042] qPCR analysis was performed on day 3 of culture, and the results are as follows: Figure 2 As shown in L, it can be seen that, compared with Rras2 f / f Compared with mice, the expression of key osteogenic marker genes (Alp, Runx2, Sp7) in bone marrow mesenchymal stem cells of RPKO mice was significantly downregulated.

[0043] Alkaline phosphatase staining was performed on day 7 of culture to assess early osteogenic differentiation, and the results were as follows: Figure 2 As shown in M, it can be seen that compared to Rras2 f / f In mice, the osteogenic differentiation capacity of bone marrow mesenchymal stem cells in RPKO mice was significantly reduced.

[0044] Alizarin red staining was performed on day 14 of culture to assess mineralization matrix formation, and the results were as follows: Figure 2 As shown in N, the mineralization capacity of bone marrow mesenchymal stem cells in RPKO mice is significantly reduced.

[0045] These results indicate that the absence of Rras2 leads to impaired osteogenic differentiation of mouse bone marrow mesenchymal stem cells, resulting in osteoporosis in mice.

[0046] Example 3: Study on the molecular mechanism of Rras2-driven osteogenic formation From Rras2 f / f Bone marrow mesenchymal stem cells isolated from mice and RPKO mice (specific information for both mouse types is the same as in Example 2) were cultured for 2 days in osteogenic induction medium containing 100 nM dexamethasone, 10 mM sodium β-glycerophosphate, and 50 μg / mL ascorbic acid. RNA was extracted and sequenced, and the results are as follows: Figure 3 As shown in A, the results indicate that 68 genes were significantly upregulated and 145 genes were significantly downregulated in the bone marrow mesenchymal stem cells of RPKO mice.

[0047] Differentially expressed genes were analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG), and the results are as follows: Figure 3 As shown in B, the results indicate that Rras2 deficiency significantly affects gene expression associated with the TGF-β / BMP (transforming growth factor-β / bone morphogenetic protein) signaling pathway.

[0048] A heatmap of gene expression related to the BMP signaling pathway is shown below. Figure 3 As shown in C, it can be seen that compared to Rras2 f / f In mice, downstream target genes of BMP2 protein (such as Sp7, Id1, Id3, Smad6 and Dlx3) were significantly downregulated in bone marrow mesenchymal stem cells of RPKO mice.

[0049] The Id1 gene promoter contains the most sensitive BMP response element. To detect whether Rras2 affects the transcriptional activity of BMP2 signaling, the Id1-BRE-luciferase reporter plasmid was used to perform the Id1-BRE luciferase reporter assay. Specifically, HEK293T cells were seeded in 24-well plates and the following nucleic acids were co-transfected using Lipofectamine 3000 transfection reagent (Invitrogen, USA): (1) 1 μg of Id1-BRE-luciferase reporter plasmid (Id1-BRE-Luc, PPL, Nanjing, China); (2) 0.5 μg of CMV-β-galactosidase internal control plasmid; (3) small interfering RNA targeting human RRAS2 (denoted as si-RRAS2) or negative control small interfering RNA (denoted as si-control) at a final concentration of 50 nM. Forty-eight hours post-transfection, the old culture medium was discarded, and fresh culture medium containing recombinant human BMP2 protein (final concentration 100 ng / mL) or an equal volume of solvent (phosphate buffer containing 0.1% bovine serum albumin, pH 7.4) was added as a control. After culturing for another 6 hours, the culture medium was aspirated, cells were lysed using cell lysis buffer, and luciferase activity was measured according to the instructions of a commercial kit (Beyotime, China). Simultaneously, β-galactosidase activity was measured using the same cell lysis buffer, and the luciferase readings were normalized to the β-galactosidase activity values ​​to correct for transfection efficiency. Results are as follows: Figure 3 As shown in D, it can be seen that in HEK293T cells, the loss of RRAS2 significantly inhibits its response to BMP2 protein.

[0050] Example 4: Study on the inhibition of SMURF1-mediated ubiquitination and degradation of BMPR2 protein by RRAS2 protein To determine how RRAS2 protein promotes the BMP2 signaling pathway, this embodiment identified proteins interacting with RRAS2 protein using immunoprecipitation combined with mass spectrometry. The results showed that BMPR2 protein was significantly enriched in the RRAS2 protein transcribed product, suggesting a potential interaction between RRAS2 and BMPR2 proteins (e.g., ...). Figure 4 (As shown in A in the diagram).

[0051] The interaction between RRAS2 and BMPR2 proteins was confirmed in human bone marrow mesenchymal stem cells using co-immunoprecipitation (Co-IP). Figure 4 (As shown in B in the diagram).

[0052] Rras2 was detected by immunoblotting and qPCR. + / + Mouse bone marrow mesenchymal stem cells and Rras2 - / - The levels of BMPR2 protein and Bmpr2 mRNA during osteogenic differentiation of mouse bone marrow mesenchymal stem cells were as follows: Figure 4 C and Figure 4 As shown in D, it can be seen that during osteogenic differentiation, Rras2... + / + Compared to mouse bone marrow mesenchymal stem cells, Rras2 - / - The level of BMPR2 protein in mouse bone marrow mesenchymal stem cells was significantly reduced, while its mRNA level remained almost unchanged. This indicates that the loss of RRAS2 protein mainly affects the stability of BMPR2 protein.

[0053] Rras2 was detected by immunoblotting. - / - The levels of BMPR2 protein in mouse bone marrow mesenchymal stem cells after treatment with MG132 (a proteasome inhibitor) or chloroquine (CQ, a lysosomal inhibitor) were as follows: Figure 4 As shown in E, it can be seen that for Rras2 - / - In mouse bone marrow mesenchymal stem cells, the lysosomal inhibitor chloroquine can increase the level of BMPR2 protein, but the proteasome inhibitor MG132 has no such effect.

[0054] Detection of Rras2 by immunoprecipitation (IP)-immunoblotting (IB) + / + Mouse bone marrow mesenchymal stem cells and Rras2 - / - The ubiquitination level of BMPR2 protein in mouse bone marrow mesenchymal stem cells was as follows: Figure 4 As shown in F, it can be seen that, compared with Rras2 + / + Compared to mouse bone marrow mesenchymal stem cells, Rras2 - / - The ubiquitination level of BMPR2 protein is increased in mouse bone marrow mesenchymal stem cells.

[0055] The ubiquitination level of BMPR2 protein in control cells (prepared using the method described in Example 5, which is the same as the method for preparing control cells) and RRAS2-overexpressing human bone marrow mesenchymal stem cells (prepared using the method described in Example 5, which is the same as the method for preparing RRAS2-overexpressing cells) was detected by immunoprecipitation (IP)-Western immunoblotting (IB). The results are as follows: Figure 4 As shown in G, RRAS2 overexpression reduces the ubiquitination level of BMPR2 protein.

[0056] SMURF1 is the main E3 ubiquitin ligase mediating BMPR2 protein ubiquitination. The effect of knockdown of Smurf1 (the shRNA targeting Smurf1, sh-Smurf1, was purchased and obtained) on BMPR2 protein ubiquitination in mouse bone marrow mesenchymal stem cells was investigated using immunoprecipitation. The results are as follows: Figure 4 As shown in H, knockdown of Smurf1 in mouse bone marrow mesenchymal stem cells enhances the ubiquitination of BMPR2 protein at K63 and K48, indicating that Smurf1 protein mediates BMPR2 protein degradation.

[0057] The effect of RRAS2 protein on the interaction between SMUFR1 and BMPR2 proteins was investigated using a GST pull-down assay. Specifically, GST-SMURF1 and GST-RRAS2 proteins (where GST represents glutathione S-transferase) were expressed in BL21 cells and purified using glutathione agarose 4B beads (GE Healthcare). The purified GST-RRAS2 protein was cleaved by PreScission protease to remove the GST tag, yielding tag-free RRAS2 protein. Flag-BMPR2-KD (i.e., the BMPR2 kinase domain containing the flag tag) was expressed in HEK293T cells and purified using anti-Flag M2 affinity gels (Sigma-Aldrich). All proteins were incubated overnight at 4°C in GST binding buffer. After washing the beads four times with GST binding buffer, they were boiled and analyzed by Western blotting. Results are as follows: Figure 4 As shown in Figure I, the results indicate that RRAS2 protein overexpression significantly reduces the interaction between SMUFR1 and BMPR2 proteins.

[0058] The GST binding buffer contains 20 mM Tris (pH 7.8), 150 mM NaCl, 0.2% NP-40 and 10% glycerol, and is supplemented with a protease inhibitor mixture (Roche Complete).

[0059] Immunoprecipitation-Western blotting was used to detect the effect of RRAS2 overexpression on BMPR2 protein ubiquitination in human bone marrow mesenchymal stem cells (the preparation method is described in Example 5 for the preparation of RRAS2 overexpression group cells). The results are as follows: Figure 4 As shown in J, RRAS2 overexpression reduces the ubiquitination level of BMPR2 protein in human bone marrow mesenchymal stem cells.

[0060] Immunoblotting was used to detect the effects of SMURF1 overexpression and RRAS2 overexpression on BMPR2 protein levels in human bone marrow mesenchymal stem cells (the preparation method is described in Example 5 for the preparation of RRAS2-overexpressing cells). The results are as follows: Figure 4 As shown in Figure K, RRAS2 overexpression reduced the ubiquitination level of BMPR2 protein in human bone marrow mesenchymal stem cells and alleviated the SMURF1-mediated decrease in BMPR2 protein levels. These results indicate that RRAS2 protein stabilizes BMPR2 protein by inhibiting SMURF1-mediated ubiquitination degradation.

[0061] Example 5: Study on the effect of lentivirus-mediated human RRAS2 overexpression on osteogenic differentiation and bone regeneration of human bone marrow mesenchymal stem cells. Human RRAS2 cDNA was cloned into a lentiviral expression vector carrying the red fluorescent protein (RFP) gene to construct a recombinant lentivirus overexpressing RRAS2, named Lenti-RRAS2-OE-RFP lentivirus (its nucleotide sequence is shown in SEQ ID NO: 3). Specifically, the recombinant lentiviral expression plasmid, packaging plasmid psPAX2, and envelope plasmid pMD2.G were mixed at a mass ratio of 4:3:1 and transfected into HEK293T cells using Lipofectamine 2000 transfection reagent (Invitrogen). Cell supernatants were collected at 48 and 72 hours post-transfection, filtered through a 0.45 μm filter membrane, and concentrated by ultracentrifugation at 50,000 × g for 2 hours at 4°C. The viral pellet was resuspended in PBS to obtain Lenti-RRAS2-OE-RFP lentivirus.

[0062] Meanwhile, the same method was used, with the only difference that an empty vector without RRAS2 cDNA (expressing only RFP) was used to obtain Lenti-RFP lentivirus as a control.

[0063] RRAS2 overexpression group: Human bone marrow mesenchymal stem cells were infected with Lenti-RRAS2-OE-RFP lentivirus to obtain human bone marrow mesenchymal stem cells that overexpress human RRAS2, namely RRAS2 overexpression group cells.

[0064] Control group: Human bone marrow mesenchymal stem cells were infected with Lenti-RFP lentivirus to obtain control group cells.

[0065] The expression of RFP in human bone marrow mesenchymal stem cells after infection with Lenti-RRAS2-OE-RFP lentivirus was observed using fluorescence microscopy (i.e., the expression of RFP in human bone marrow mesenchymal stem cells overexpressing human RRAS2). The results are as follows: Figure 5 As shown in A, the infected cells express RFP.

[0066] The expression levels of RRAS2 mRNA and RRAS2 protein in RRAS2-overexpressing cells (i.e., human bone marrow mesenchymal stem cells overexpressing human RRAS2) and control cells were detected by qPCR and immunoblotting, respectively. The results are as follows: Figure 5 B and Figure 5 As shown in C, the RRAS2 overexpression group cells successfully overexpressed RRAS2 mRNA and RRAS2 protein.

[0067] Alkaline phosphatase staining and quantification were performed on cells in the RRAS2 overexpression group and the control group. The results are as follows: Figure 5 D and Figure 5As shown in E, the RRAS2 overexpression group cells have a stronger osteogenic differentiation capacity compared to the control group cells.

[0068] Von Kossa staining and quantitative analysis were performed on RRAS2 overexpression group cells and control group cells, and the results are as follows: Figure 5 F and Figure 5 As shown in G, the RRAS2 overexpression group cells have a stronger mineralization capacity compared to the control group cells.

[0069] To evaluate the in vivo osteogenic potential of RRAS2-overexpressing human bone marrow mesenchymal stem cells, an ectopic bone formation experiment was conducted. Specifically, 40 mg of hydroxyapatite / tricalcium phosphate (HA / TCP) scaffolds were respectively coupled with 2×10 6 One human bone marrow mesenchymal stem cell overexpressing human RRAS2 (i.e., RRAS2-overexpressing cells) and 2×10 6 Cells from control groups were co-cultured. Simultaneously, cells were labeled with GFP (2.3ColGFP) driven by the 2.3-kb Col1a1 promoter for in vivo tracking. Scaffolds were implanted subcutaneously into the backs of 8-week-old severely ill combined immunodeficiency (SCID) mice, and BMP2 protein was administered locally via subcutaneous injection. Specimens were harvested 6 weeks after implantation for histological processing, including hematoxylin-eosin staining to detect 2.3Col-GFP activity and histomorphometry analysis of ectopic bone volume in hematoxylin-eosin stained sections. Results are as follows: Figure 5 H in Figure 5 I in Figure 5 J and Figure 5 As shown in K, it can be seen that human bone marrow mesenchymal stem cells overexpressing human RRAS2 (i.e., RRAS2-overexpressing cells) have significantly enhanced ectopic osteogenic capacity.

[0070] To further evaluate the osteogenic differentiation capacity of human bone marrow mesenchymal stem cells overexpressing human RRAS2 (i.e., RRAS2-overexpressing cells), a SCID mouse skull defect model was established. The aforementioned RRAS2-overexpressing cells and control cells were then locally transplanted. After 8 weeks, micro-computed tomography and quantitative analysis of bone volume fraction were performed. The results are as follows: Figure 5 L and Figure 5 As shown in M, compared with the transplanted control group cells, the bone volume fraction of the defect site was significantly increased after transplantation of human bone marrow mesenchymal stem cells overexpressing human RRAS2, indicating enhanced bone regeneration capacity.

[0071] The construction process of the SCID mouse skull defect model is as follows: A linear scalp incision was made in 7-week-old male nude mice, and a full-thickness skin flap was dissected to expose the skull. The periosteum was completely removed, and a 4 mm diameter defect was created along the sagittal suture using a trephine, with thorough irrigation during the process. The skull fragment was carefully removed, avoiding damage to the dura mater or brain.

[0072] Cell transplantation process: Human bone marrow mesenchymal stem cells (5th generation, 1×10⁶ cells / year) overexpressing human RRAS2 were transplanted separately. 6 Gelatin sponge (1 × 10⁶ cells) and control group cells (1 × 10⁶ cells) 6 One gelatin sponge was placed at the defect site in the SCID mouse skull defect model, ensuring it was in contact with the bone edge. The incision was closed using Vetbond tissue adhesive (3M).

[0073] The above results indicate that lentivirus-mediated RRAS2 overexpression can enhance osteogenic differentiation and bone regeneration capacity of human bone marrow mesenchymal stem cells.

[0074] Example 6: Treatment of osteoporosis in aged mice AAV9 serotypes with bone affinity were selected, and mouse Rras2 cDNA was cloned into an adeno-associated virus expression vector carrying the green fluorescent protein (GFP) gene to construct recombinant rAAV9-Rras2-GFP virus (its nucleotide sequence is shown in SEQ ID NO: 4). Specifically, the adeno-associated virus expression plasmid, helper plasmid pHelper, and packaging plasmid pAAV-RC were mixed at a mass ratio of 1:2:1 (helper plasmid pHelper and packaging plasmid pAAV-RC are two commonly used helper plasmids in adeno-associated virus packaging systems), and AAV-293 cells were transfected using Lipofectamine 2000 transfection reagent (Invitrogen). Cells were collected 72 hours after transfection, subjected to three freeze-thaw cycles, centrifuged at 2000×g for 10 minutes, and the supernatant was collected. Nuclease was added to the supernatant, and the mixture was incubated at 37°C for 1 hour to remove the nucleic acid outside the virus. The mixture was then centrifuged at 600×g for 10 minutes, and the supernatant was collected. The virus was purified using an adeno-associated virus purification kit. The purified virus liquid was added to an ultrafiltration tube and centrifuged at 1400×g for 30 minutes to obtain the purified virus, which was the recombinant rAAV9-Rras2-GFP virus.

[0075] The only difference in using the above method is that an empty vector without RRAS2 cDNA (expressing only GFP) is used to obtain the control group virus, i.e., rAAV9-GFP virus.

[0076] AAV-Rras2 treatment group (2 weeks): 23-month-old mice (aged mice) were anesthetized with isoflurane. A skin incision of approximately 0.5 cm was made at the distal femur using fine forceps and surgical scissors. Subcutaneous fat and muscle were dissected to expose the femur. Under stereomicroscopic guidance, a 0.2 mm diameter hole was drilled at the distal femur using a fine metal drill. Recombinant rAAV9-Rras2-GFP virus was slowly injected into the bone marrow cavity through a microcapillary pipette. The surgical site was sealed with Vetbond tissue adhesive. Two weeks after injection, all mice were sacrificed, and bone tissue was collected.

[0077] AAV-Control Treatment Group (2 weeks): rAAV9-GFP virus was injected using the same method as a control.

[0078] Bone tissues from mice in the AAV-Rras2 treatment group (2 weeks) and the AAV-control treatment group (2 weeks) were collected for qPCR and Western blot analysis. The results are as follows: Figure 6 A and Figure 6 As shown in B, the levels of Rras2 mRNA and RRAS2 protein in the bone tissue of aged mice were significantly increased in the AAV-Rras2 treatment group (2 weeks).

[0079] Bone tissue sections from mice in the AAV-Rras2 treatment group (2 weeks) were observed under a fluorescence microscope. The results are as follows: Figure 6 As shown in C, the overexpressed signal is mainly located on the bone surface, demonstrating the virus's good specificity.

[0080] To further investigate whether Rras2 can serve as a therapeutic target for age-related osteoporosis, 23-month-old male mice were treated with recombinant rAAV9-Rras2-GFP virus and rAAV9-GFP virus, using the same injection method as above. Samples were taken 6 weeks after intraosseous injection and recorded as AAV-Rras2 treatment group (6 weeks) and AAV-control treatment group (6 weeks), respectively.

[0081] The results of femoral micro-computed tomography analysis are as follows: Figure 6 As shown in Figure D, bone mass was significantly increased in mice treated with AAV-Rras2 (6 weeks). Quantitative results for bone volume fraction, trabecular bone number, trabecular bone thickness, and trabecular bone separation are shown in Figures D. Figure 6 E to Figure 6 As shown in Figure H, compared to the AAV-control group (6 weeks), the AAV-Rras2 treatment group (6 weeks) showed significantly increased femoral bone volume fraction, trabecular bone number, and trabecular bone thickness, while trabecular bone separation decreased. The results of calcein-xylenol orange double labeling and quantitative analysis are shown below. Figure 6 I and Figure 6As shown in Figure J, compared to the AAV-control group (6 weeks), the bone formation rate of the femur in the AAV-Rras2 treatment group (6 weeks) was significantly increased, indicating improved bone formation. Furthermore, the results of alkaline phosphatase staining and quantitative analysis are shown in Figure J. Figure 6 K and Figure 6 As shown in L, compared to the AAV-control group (6 weeks), the AAV-Rras2 treatment group (6 weeks) showed increased bone formation in mice. In conclusion, Rras2 gene therapy can alleviate osteoporosis by promoting bone formation.

[0082] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. Application of substances that enhance the expression level of RAS-associated protein 2 in the preparation of drugs for the treatment of osteoporosis.

2. Application of biological vectors containing the RAS-associated protein 2 encoding gene in the preparation of drugs for the treatment of osteoporosis.

3. The application of the biological vector containing the RAS-associated protein 2 encoding gene according to claim 2 in the preparation of a drug for treating osteoporosis, characterized in that, The gene encoding RAS-associated protein 2 can be human or mouse-derived.

4. The application of the biological vector containing the RAS-associated protein 2 encoding gene according to claim 3 in the preparation of a drug for treating osteoporosis, characterized in that, The nucleotide sequence of the gene encoding human RAS-associated protein 2 is shown in SEQ ID NO:

1.

5. The application of the biological vector containing the RAS-associated protein 2 encoding gene according to claim 3 in the preparation of a drug for treating osteoporosis, characterized in that, The nucleotide sequence of the gene encoding the mouse-derived RAS-associated protein 2 is shown in SEQ ID NO:

2.

6. The application of the biological vector containing the RAS-associated protein 2 encoding gene according to claim 2 in the preparation of a drug for treating osteoporosis, characterized in that, The biological vector is a viral vector or a cell.

7. The application of the biological vector containing the RAS-associated protein 2 encoding gene according to claim 6 in the preparation of a drug for treating osteoporosis, characterized in that, The viral vector is an adenovirus vector, an adeno-associated virus vector, or a lentivirus vector; The cells in question are human bone marrow mesenchymal stem cells.

8. The application of the biological vector containing the RAS-associated protein 2 encoding gene according to claim 2 in the preparation of a drug for treating osteoporosis, characterized in that, The dosage forms of the drug include injections, tablets, or capsules.

9. The application of the biological vector containing the RAS-associated protein 2 encoding gene according to claim 2 in the preparation of a drug for treating osteoporosis, characterized in that, The drug also includes pharmaceutically acceptable carriers and / or pharmaceutically acceptable additives.

10. The use of the biological vector containing the RAS-associated protein 2 encoding gene according to claim 9 in the preparation of a drug for treating osteoporosis, characterized in that, The pharmaceutically acceptable carrier includes at least one of the following: a gliding agent, a diluent, a wetting agent, a suspending agent, a solvent, and an emulsifier; the pharmaceutically acceptable adjuvant includes at least one of the following: a preservative, a coloring agent, a flavoring agent, a stabilizer, and an isotonic agent.

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