Bone marrow mesenchymal stem cell specific gene knockout mouse model and construction method

Abstract

The invention belongs to the technical field of medical treatment, and discloses a BMSC (bone mesenchymal stem cell) specific HIF-2alpha gene knockout mouse model based on a Cre-LxoP + technology and a construction method of the BMSC specific HIF-2alpha gene knockout mouse model. The method comprises the following steps: carrying out an in-vivo experiment on a mouse (Hif-2 [alpha] fl / fl mouse), and using a wild type (WT) same nest mouse (Hif-2 [alpha] fl / fl) with matched age and gender as a contrast; hif-2 [alpha] fl / fl and Prx1-Cre are selected from the group consisting of Prx1-Cre Hif-2 alpha fl / fl mice are subjected to ovariectomy, dexamethasone (DEX) treatment or semi-lethal radiation, and the mice with the same age (6-8 weeks), gender and genotype are randomly grouped for subsequent experiments (researchers are not covered); the method comprises the following steps: preparing Prx1-Cre; no gender-related difference has been observed in Hif-2 [alpha] fl / fl mice. According to the invention, a mouse with BMSC-specific HIF-2alpha knockout is generated and is used for evaluating the physiological function of HIF-2alpha in an early osteoblast lineage.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a bone marrow mesenchymal stem cell-specific HIF-2α gene knockout mouse model based on Cre-LxoP+ technology and a construction method thereof. Background Art

[0002] When discussing bone marrow mesenchymal stem cells (BMSCs) and related research fields, the technical problems existing in the existing technologies are mainly concentrated in the following aspects:

[0003] 1. Insufficient understanding of the regulatory mechanisms of cell differentiation

[0004] Complexity of signaling pathways and transcription factors: The differentiation of BMSCs is regulated by multiple signaling pathways and transcription factors, and these regulatory mechanisms are complex and have not yet been fully elucidated. Although current studies have revealed the role of some key signaling pathways (such as the HIF-2α signaling pathway) and transcription factors in the differentiation of BMSCs, there are still a large number of unknown factors to be explored.

[0005] 2. Insufficient research on the effects of hypoxia on BMSCs

[0006] Direct measurement of hypoxic environment is difficult: Although it is known that oxygen tension in the bone marrow is low, it is still challenging to directly measure and accurately reflect the oxygen tension of the microenvironment in which BMSCs are located in living animals. Although the study by Spencer et al. provides a method to measure local oxygen tension in the bone marrow, how to effectively apply these data to the regulation of BMSCs still needs further exploration.

[0007] 3. Limitations and challenges of gene editing technology

[0008] Construction of gene knockout models: Although the gene knockout technology based on the Cre-LoxP system is relatively mature, there are still many challenges in constructing specific gene knockout mouse models of specific cell types (such as BMSCs). How to ensure the specificity, efficiency and stability of gene knockout is a key issue in current research.

[0009] Potential off-target effects of gene editing: While gene editing technologies (such as CRISPR / Cas9) can edit target genes efficiently and accurately, they can also cause off-target effects that interfere with experimental results. Therefore, when constructing and applying gene knockout models, experimental conditions need to be strictly controlled to ensure the accuracy and reliability of the results.

[0010] 4. Limitations of BMSCs functional studies

[0011] Differences between in vitro culture and in vivo environment: There are significant differences in the behavior of BMSCs under in vitro culture conditions and in the in vivo microenvironment. This difference limits the direct application of in vitro research results in vivo. Therefore, how to simulate the in vivo microenvironment in vitro to more accurately study the function of BMSCs is an important topic in current research.

[0012] Complexity of immunosuppressive mechanisms: BMSCs are immunosuppressive cells, and their immunosuppressive mechanisms involve multiple molecules and cell-cell interactions. Although current research has revealed some key molecules and mechanisms, there are still a large number of unknown factors to be explored.

[0013] In summary, there are still many problems and challenges in the field of BMSCs research with existing technologies. Future research needs to further explore the differentiation regulation mechanism of BMSCs, the specific mechanism of the effect of hypoxia on it, the optimization of gene editing technology, and the in vivo and in vitro consistency of BMSCs functional research, so as to promote the widespread application of BMSCs in regenerative medicine, tissue engineering and other fields. Summary of the invention

[0014] In view of the problems existing in the prior art, the present invention provides a method for constructing a bone marrow mesenchymal stem cell-specific HIF-2α gene knockout mouse model and its application based on Cre-LxoP+ technology.

[0015] The present invention is achieved by a method for constructing a bone marrow mesenchymal stem cell-specific HIF-2α gene knockout mouse model based on Cre-LxoP+ technology, the method comprising:

[0016] S1: BMSC-specific HIF-2α knockout mice (Prx1-Cre; Hif-2α fl / fl Mice) were used for in vivo experiments, and age- and sex-matched wild-type (WT) littermates (Hif-2α fl / fl ) was used as a control;

[0017] S2: Hif-2α fl / fl and Prx-1-Cre;Hif-2α fl / fl Mice were subjected to ovariectomy, dexamethasone (DEX) treatment, or semilethal irradiation, and mice of the same age (6–8 weeks), sex, and genotype were randomly grouped for subsequent experiments (the researchers were not blinded);

[0018] S3: In Prx-1-Cre; Hif-2α fl / fl No sex-related differences were observed in mice.

[0019] Furthermore, the mice with conditional knockout of HIF-2α in the BMSCs (Prx-1-Cre; Hif-2αfl / fl Mouse) is Hif-2α fl / fl Mice were generated by mating with Prx-1-Cre transgenic mice.

[0020] Furthermore, all animals in this method were housed under specific pathogen free conditions and fed a standard diet.

[0021] Another object of the present invention is to provide an application of the method for constructing a bone marrow mesenchymal stem cell-specific HIF-2α gene knockout mouse model based on Cre-LxoP+ technology to evaluate the physiological function of HIF-2α in the early osteoblast lineage.

[0022] Another object of the present invention is to provide a bone marrow mesenchymal stem cell (BMSCs) specific HIF-2α gene knockout mouse model constructed using Cre-LoxP technology. fl / fl The mice were mated with Prx-1-Cre transgenic mice. The Prx-1 promoter specifically expressed Cre recombinase in BMSCs, achieving specific knockout of the HIF-2α gene in BMSCs.

[0023] The mouse model is used to study the biological function of the HIF-2α gene in bone marrow mesenchymal stem cells and its role under different physiological or pathological conditions, including ovariectomy, dexamethasone (DEX) treatment or semi-lethal radiation.

[0024] Another object of the present invention is to provide a method for studying gene function based on the mouse model of claim 1, comprising the following steps:

[0025] a) Select 6-8 week old Prx-1-Cre; Hif-2α fl / fl Mice with age- and sex-matched Hif-2α fl / fl Mice served as controls;

[0026] b) Simulating different physiological or pathological conditions through treatments such as ovariectomy, dexamethasone treatment or semi-lethal irradiation;

[0027] c) Bone marrow samples were collected from mice, bone marrow mesenchymal stem cells were isolated, the expression of HIF-2α gene was detected, and the proliferation, differentiation and apoptosis of cells were evaluated.

[0028] Furthermore, this mouse model was used to evaluate the potential application of HIF-2α gene knockout on bone marrow mesenchymal stem cells in regenerative medicine, bone tissue engineering and anti-aging research, including but not limited to detecting the biological responses of cells under different stimuli and their role in tissue repair.

[0029] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0030] First, the present invention constructs genetically engineered mice with BMSC-specific HIF-2α knockout and uses them to evaluate the physiological function of HIF-2α in the early osteoblast lineage.

[0031] The present invention effectively solves the problem of quite low absolute pO2 in the bone marrow, constructs mice with BMSC-specific HIF-2α knockout, and is used to evaluate the physiological function of HIF-2α in the early osteoblast lineage.

[0032] The present invention provides a method for constructing a bone marrow mesenchymal stem cell (BMSCs) specific HIF-2α gene knockout mouse model using Cre-LoxP technology, which effectively solves the problem of insufficient specificity and controllability in studying the function of the HIF-2α gene in the prior art. Traditional gene knockout methods often lead to systemic gene knockout and cannot specifically study the gene function in a specific cell or tissue. The present invention uses the Cre-LoxP system to achieve specific knockout of the HIF-2α gene in BMSCs, ensuring the accuracy and pertinence of the experimental results.

[0033] In the experiment, mice with specific HIF-2α knockout (Prx1-Cre; Hif-2α fl / fl mice) were compared with age- and sex-matched wild-type littermates (Hif-2α fl / fl ) as a control, and experiments such as ovariectomy, dexamethasone treatment, and semi-lethal irradiation were performed. A significant advancement of this method is that by specifically knocking out the HIF-2α gene, researchers can more accurately study the effects of HIF-2α on BMSCs function under different stress conditions, overcoming the complexity and uncertainty brought about by systemic gene knockout.

[0034] Another technical advancement of the present invention is the rationality and scientificity of the experimental design. In the experiment, all mice were randomly grouped according to the same age (6-8 weeks), gender and genotype, ensuring the repeatability and reliability of the experimental results. At the same time, the researchers were not blinded during the experiment, ensuring the accuracy of data collection and analysis. Through these strict experimental control conditions, the research results of the present invention have a high degree of credibility and scientific value.

[0035] The present invention uses Cre-LoxP technology to construct a BMSCs-specific HIF-2α gene knockout mouse model, which not only solves the specificity and controllability problems in traditional gene knockout methods, but also obtains reliable research data through rigorous experimental design and execution. This technology significantly improves the accuracy and effectiveness of studying the function of the HIF-2α gene, and provides a powerful tool for further exploring the biological function of HIF-2α in BMSCs, with broad application prospects and important scientific significance.

[0036] Second, the expected benefits and commercial value after the technical solution of the present invention is transformed as follows: The present invention is an embodiment to construct a bone marrow mesenchymal stem cell (BMSCs) specific HIF-2α gene knockout mouse model. The working principle of this technology is mainly based on the Cre-LoxP recombination system, which is a site-specific gene recombination technology and is widely used in studies such as gene knockout, insertion, flipping and translocation. Gene knockout technology is divided into complete gene knockout and conditional gene knockout. The former eliminates the activity of individual target genes by homologous recombination. Because the HIF-2α gene is essential for embryonic development, this technology will cause mice to be unable to survive to adulthood, so we use the Cre-loxP recombinase system in the latter to overcome this defect. The Cre recombinase consists of 343 amino acids and specifically recognizes loxP sites to achieve excision or reversal of specific genes. LoxP is a 34bp long palindromic DNA fragment containing 2 inverted repeat sequences and 1 separator. The two loxP sequences are placed in the introns at both ends of the target gene by homologous recombination, without affecting the expression of the target gene. Cre enzyme mediates sequence recombination between two loxP sites. When the two loxP sequences are in reverse, the direction of the target gene fragment is reversed, and when they are in the same direction, the target gene fragment between them is removed by Cre enzyme. Cre / loxP recombinase system is widely used in gene knockout, does not affect the normal expression regulation of genes, and has the advantages of strong specificity, high efficiency and specific action.

[0037] The present invention uses Cre-LoxP technology to successfully construct a bone marrow mesenchymal stem cell (BMSCs) specific HIF-2α gene knockout mouse model. This model mouse provides an animal experimental basis for in-depth exploration of the relationship between hypoxia and osteoporosis and the mechanism of action of HIF-2α in the occurrence and development of BMSCs, and is used to evaluate the physiological function of HIF-2α in the early osteoblast lineage, which is expected to achieve considerable benefits and commercial value.

[0038] Third, the present invention provides a method for constructing a bone marrow mesenchymal stem cell (BMSCs)-specific HIF-2α gene knockout mouse model using Cre-LxoP+ technology. This method achieves specific knockout of the HIF-2α gene in BMSCs through precise gene editing, providing a powerful tool for studying the function of this gene in specific cell types.

[0039] The core of this technology is to use the Cre-LoxP recombination system, which can perform precise gene manipulation at specific sites. fl / fl We successfully constructed Prx1-Cre; Hif-2α fl / fl Mouse model. Among them, the Prx-1 promoter ensures the specific expression of Cre recombinase in BMSCs, and then achieves the knockout of HIF-2α gene by recognizing LoxP sites.

[0040] To comprehensively evaluate the role of HIF-2α in BMSCs, we performed Prx-1-Cre; Hif-2α fl / fl The mice were subjected to a variety of physiological and pathological mimicking treatments, including ovariectomy, dexamethasone treatment, and semi-lethal irradiation. These treatments helped us gain a deeper understanding of the function and impact of HIF-2α in different physiological states. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 It is a flow chart of a method for constructing a bone marrow mesenchymal stem cell-specific HIF-2α gene knockout mouse model based on Cre-LxoP+ technology provided in an embodiment of the present invention;

[0042] Figure 2 It is a flow chart of the identification method of mice with bone marrow mesenchymal stem cell-specific HIF-2α knockout provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0044] like Figure 1 As shown, the embodiment of the present invention provides a method for constructing a bone marrow mesenchymal stem cell-specific HIF-2α gene knockout mouse model based on Cre-LxoP+ technology, the method comprising:

[0045] S1: BMSC-specific HIF-2α knockout mice (Prx1-Cre; Hif-2α fl / flMice) were used for in vivo experiments, and age- and sex-matched wild-type (WT) littermates (Hif-2α fl / fl ) was used as a control;

[0046] S2: Hif-2α fl / fl and Prx-1-Cre;Hif-2α fl / fl Mice were subjected to ovariectomy, dexamethasone (DEX) treatment, or semilethal irradiation, and mice of the same age (6–8 weeks), sex, and genotype were randomly grouped for subsequent experiments (the researchers were not blinded);

[0047] S3: In Prx1-Cre;Hif-2α fl / fl No sex-related differences were observed in mice.

[0048] The BMSCs were subjected to conditional knockout of HIF-2α in mice (Prx-1-Cre; Hif-2α fl / fl Mouse) is Hif-2α fl / fl Mice were generated by mating with Prx-1-Cre transgenic mice.

[0049] The present invention is an embodiment to construct a bone marrow mesenchymal stem cell (BMSCs) specific HIF-2α gene knockout mouse model. The working principle of this technology is mainly based on the Cre-LoxP recombination system, which is a site-specific gene recombination technology and is widely used in research on gene knockout, insertion, flipping and translocation.

[0050] About Prx-1-Cre; Hif-2α fl / fl The construction and working principle of the mouse, the specific steps are as follows:

[0051] 1) Mouse model construction: Hif-2α fl / fl The mice (i.e. mice with two floxed HIF-2α genes) were mated with Prx-1-Cre transgenic mice. Prx-1 is a promoter that is specifically expressed in BMSCs, so the Cre recombinase will be specifically expressed in these cells.

[0052] 2) Cre-LoxP system function: When Cre recombinase is expressed in BMSCs under the control of Prx-1 promoter, it will recognize and bind to the LoxP sites on both sides of the floxed HIF-2α gene. The LoxP site is a specific DNA sequence recognized by Cre recombinase.

[0053] 3) Gene knockout: Once the Cre recombinase binds to the two LoxP sites, it will catalyze the recombination and deletion of the DNA fragment between the two sites, thereby achieving specific knockout of the HIF-2α gene in BMSCs.

[0054] 4) Follow-up experiments: Researchers can study the effects of HIF-2α knockout on BMSCs function by treatments such as ovariectomy, dexamethasone (DEX) treatment, or semi-lethal irradiation. These treatments can simulate different physiological or pathological conditions, thereby more comprehensively evaluating the role of HIF-2α in BMSCs.

[0055] In step S3 it is mentioned that “In Prx-1-Cre; Hif-2α fl / fl No sex-related differences were observed in mice," which means that the knockout effect of the HIF-2α gene in BMSCs is consistent regardless of the sex of the mice, which facilitates subsequent research.

[0056] By utilizing the specificity and efficiency of the Cre-LoxP recombination system, researchers can construct mouse models in which specific genes are knocked out in specific cells or tissues, thereby conducting more in-depth studies on the functions and roles of these genes in organisms.

[0057] All animals in this method were housed under specific pathogen free conditions and fed a standard diet.

[0058] The embodiment of the present invention provides an application of the method for constructing a bone marrow mesenchymal stem cell-specific HIF-2α gene knockout mouse model based on Cre-LxoP+ technology, and its application in evaluating the physiological function of HIF-2α in the early osteoblast lineage.

[0059] like Figure 2 The figure shows the construction of mice with bone marrow mesenchymal stem cell-specific HIF-2α knockout. A: Mouse mating process; B: Prx-1 Cre gene identification; 100bp is the positive band; C: HIF-2α gene identification; 220bp is the target band, 182bp is the WT band; D: From Prx1-Cre; Hif-2α fl / fl and Hif-2α fl / fl Protein expression differences in primary BMSCs isolated from mice; EL: Prx1-Cre; Hif-2α fl / fl and Hif-2α fl / fl HIF-2α (E) and HIF-2α exon 2 (I) transcript levels in mouse BMSCs; Prx1-Cre; Hif-2α fl / fl and Hif-2α fl / fl HIF-2α (F) and HIF-2α exon 2 (J) transcript levels in the spleen of mice; Prx1-Cre; Hif-2α fl / fl and Hif-2α fl / flHIF-2α (G) and HIF-2α exon 2 (K) transcript levels in muscle of mice; Prx1-Cre; Hif-2α fl / fl and Hif-2α fl / fl HIF-2α (H) and HIF-2α exon 2 (L) transcript levels in mouse liver; aP<0.05. BMSC: bone marrow mesenchymal stem cells; HIF-2α: hypoxia-inducible factor 2α.

[0060] The present invention constructs a mouse model with BMSC-specific HIF-2α knockout. In order to evaluate the physiological function of HIF-2α in the early osteoblast lineage, Prx-1-Cre; Hif-2α fl / fl In brief, HIF-2αfloxed mice were mated with Prx1-Cre transgenic mice to generate Prx1-Cre; Hif-2α fl / fl Mice were then treated with Hif-2α fl / fl Mating of mice to produce Hif-2α fl / fl Mouse, Prx1-Cre; Hif-2α fl / fl Mice and Prx1-Cre;Hif-2α fl / fl Mice. Littermate mice were used in all experiments. DNA genotyping results are shown in Figure 2 B and C. Mice of these genotypes were born in normal Mendelian ratios and showed no significant differences in weight or development ( Figure 2 A and B). To confirm that Prx1-Cre;Hif-2α fl / fl Knockout of HIF-2α in BMSCs. Analysis of HIF-2α protein expression in BMSCs revealed that Prx1-Cre; Hif-2α fl / fl HIF-2α was effectively knocked out in most BMSCs of mice ( Figure 2 D) In ​​addition, quantitative RT-PCR analysis of total RNA extracted from BMSCs, spleen, muscle and liver showed that Prx1-Cre; Hif-2α fl / fl HIF-2α mRNA in mouse BMSCs ( Figure 2 E) and HIF-2α exon 2 ( Figure 2 I) expression level was significantly lower than that of Hif-2α fl / fl BMSCs in mice, but not in the spleen ( Figure 2 F and J), muscle ( Figure 2 G and K) and liver ( Figure 2 These levels did not differ between the two genotypes of mice in H and L).

[0061] Example 1: Study on the role of HIF-2α gene in BMSCs after ovariectomy

[0062] Step 1: Experimental animal preparation

[0063] Using Prx1-Cre;Hif-2α fl / fl Mice (BMSCs-specific HIF-2α knockout mice) and Hif-2α fl / fl Experiments were performed with mice (wild-type littermates), all 6-8 weeks old and sex-matched.

[0064] Step 2: Oophorectomy

[0065] The above mice were subjected to ovariectomy, and the mice recovered in a sterile environment after the operation, and their postoperative response and survival were observed.

[0066] Step 3: Sample collection and analysis

[0067] Four weeks after surgery, bone marrow samples were collected from mice and BMSCs were isolated.

[0068] The expression of HIF-2α gene in BMSCs was detected by qPCR and Western blot, and the knockout groups (Prx1-Cre; Hif-2α fl / fl ) and control group (Hif-2α fl / fl ) at the gene expression and protein levels.

[0069] Cell proliferation and differentiation experiments were further performed to evaluate the effect of HIF-2α gene knockout on the function of BMSCs.

[0070] Experimental results: The results showed that after ovariectomy, Prx1-Cre; Hif-2α fl / fl The expression of HIF-2α gene in BMSCs of mice was significantly decreased, while that in wild-type mice remained at normal levels.

[0071] The proliferation and differentiation abilities of BMSCs in the knockout group mice were significantly reduced, indicating that HIF-2α plays an important regulatory role in the biological functions of BMSCs after ovariectomy.

[0072] Example 2: Effect of dexamethasone treatment on the function of BMSCs in HIF-2α knockout mice

[0073] Step 1: Experimental animal preparation

[0074] Select Prx1-Cre;Hif-2α fl / fl Mice and Hif-2α fl / fl Mice were 6-8 weeks old and gender matched.

[0075] Step 2: Dexamethasone treatment

[0076] The mice were divided into two groups. The mice in the experimental group were treated with dexamethasone (DEX), and the mice in the control group were injected with an equal amount of normal saline. The treatment period was 4 weeks.

[0077] Step 3: Sample collection and analysis

[0078] After treatment, bone marrow samples were collected from mice and BMSCs were isolated.

[0079] The expression of HIF-2α in BMSCs was detected by qPCR and Western blot techniques, and the changes in gene expression and protein levels in mice of different genotypes were compared before and after dexamethasone treatment.

[0080] Flow cytometry and apoptosis detection experiments were used to evaluate the effects of dexamethasone treatment on the proliferation and apoptosis of BMSCs in HIF-2α knockout mice.

[0081] The results showed that after dexamethasone treatment, Prx1-Cre; Hif-2α fl / fl The expression of HIF-2α gene in BMSCs of mice was significantly decreased, while there was no significant change in the control group mice.

[0082] BMSCs from HIF-2α knockout mice in the dexamethasone-treated group showed significant proliferation inhibition and increased apoptosis, indicating that HIF-2α plays an important role in the regulation of dexamethasone-induced BMSC function.

[0083] These two examples demonstrate the application of the BMSCs-specific HIF-2α gene knockout mouse model constructed using Cre-LoxP technology in studying the function of the HIF-2α gene. By analyzing gene expression and cell function under different experimental conditions, the important biological role of HIF-2α in BMSCs is revealed.

[0084] Application Example 1: Study on the effect of HIF-2α gene knockout on the proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs)

[0085] 1. Construction of mouse model:

[0086] Use of Hif-2α fl / fl Mice were mated with Prx-1-Cre transgenic mice to obtain Prx-1-Cre; Hif-2α fl / fl Mice (BMSC-specific HIF-2α knockout mice).

[0087] Age- and sex-matched Hif-2α fl / fl Mice were used as wild-type controls.

[0088] 2. Experimental treatment:

[0089] Age of mice: 6-8 weeks.

[0090] Prx-1-Cre;Hif-2α fl / fl Mice and control mice were ovariectomized (OVX) to simulate the osteoporotic state after menopause.

[0091] Experimental groups: 10 mice in each group were randomly assigned to knockout group and control group.

[0092] 3. BMSC isolation and culture:

[0093] Bone marrow cells were extracted from mouse femurs and tibias, and BMSCs were isolated by density gradient centrifugation.

[0094] The separated BMSCs were seeded into culture flasks and cultured using DMEM medium containing 10% fetal bovine serum.

[0095] 4. Proliferation and differentiation assays:

[0096] When investigating the proliferation ability of bone marrow mesenchymal stem cells (BMSCs), although the traditional MTT method is a commonly used cell activity detection method, experiments used to specifically evaluate the proliferation ability of BMSCs usually use the colony forming unit-fibroblast (CFU-F) experiment of BMSCs combined with crystal violet staining.

[0097] CFU-F assay and crystal violet staining of BMSCs:

[0098] 1) Principle of CFU-F experiment:

[0099] The CFU-F assay is based on the ability of BMSCs to form colonies under in vitro culture conditions. Each colony is derived from the proliferation of one or more BMSCs, so the number and size of colonies can reflect the proliferation capacity of BMSCs.

[0100] 2) Experimental steps:

[0101] First, BMSCs were isolated from bone marrow and seeded in appropriate culture dishes.

[0102] Under certain culture conditions (such as suitable temperature, humidity and nutritional environment), BMSCs will begin to proliferate and form colonies.

[0103] After a period of culture, the formation of colonies can be observed under a microscope.

[0104] 3) Crystal violet staining:

[0105] To more intuitively evaluate the colony formation and proliferation ability of BMSCs, crystal violet staining can be used.

[0106] Crystal violet is a commonly used cell stain that dyes DNA within cells purple, making colonies more clearly visible.

[0107] After staining, the number of colonies can be observed and counted under a microscope, and the size of the colonies can be measured.

[0108] 4) Data analysis:

[0109] According to the number and size of colonies, the proliferation capacity of BMSCs can be evaluated.

[0110] The large number and uniform size of colonies indicated that BMSCs had strong proliferation ability.

[0111] On the contrary, a small number or uneven size of colonies indicated that the proliferation ability of BMSCs was weak.

[0112] The CFU-F experiment of BMSCs combined with crystal violet staining is an effective method to evaluate the proliferation ability of BMSCs. Through this method, we can more accurately understand the proliferation characteristics of BMSCs under in vitro culture conditions, providing strong support for subsequent cell therapy and tissue engineering research. At the same time, this method also provides an important experimental means for us to further study the differentiation regulation mechanism of BMSCs and the impact of hypoxic environment on them.

[0113] BMSCs were induced to differentiate into osteoblasts and adipocytes, and the differentiation effects were detected by Alizarin red staining and Oil red O staining, respectively.

[0114] Results and Analysis:

[0115] Proliferation ability: MTT test results showed that Prx-1-Cre; Hif-2α fl / fl The proliferation capacity of BMSCs in mice was significantly lower than that in the control group.

[0116] Differentiation ability:

[0117] Osteogenic differentiation: Alizarin red staining results showed that the osteogenic differentiation ability of the HIF-2α gene knockout group was significantly decreased.

[0118] Adipogenic differentiation: Oil red O staining results showed that the adipogenic differentiation ability of the HIF-2α gene knockout group was significantly enhanced.

[0119] Application Example 2: Study on the effect of HIF-2α gene knockout on the antioxidant capacity of bone marrow mesenchymal stem cells (BMSCs)

[0120] 1. Construction of mouse model:

[0121] Use of Hif-2α fl / fl Mice were mated with Prx-1-Cre transgenic mice to obtain Prx-1-Cre; Hif-2α fl / fl Mice (BMSC-specific HIF-2α knockout mice).

[0122] Age- and sex-matched Hif-2α fl / fl Mice were used as wild-type controls.

[0123] 2. Experimental treatment:

[0124] Age of mice: 6-8 weeks.

[0125] Prx-1-Cre;Hif-2α fl / fl Mice and control mice were treated with dexamethasone (DEX) to induce oxidative stress.

[0126] Experimental groups: 10 mice in each group were randomly assigned to knockout group and control group.

[0127] 3. BMSC isolation and culture:

[0128] Bone marrow cells were extracted from mouse femurs and tibias, and BMSCs were isolated by density gradient centrifugation.

[0129] The separated BMSCs were seeded into culture flasks and cultured using DMEM medium containing 10% fetal bovine serum.

[0130] Through these two specific examples, we can observe the significant effects of HIF-2α gene knockout on the proliferation, differentiation and antioxidant capacity of BMSCs. The importance of HIF-2α gene in maintaining the function of BMSCs has been verified, which provides an experimental basis for further studying the role of HIF-2α in bone marrow mesenchymal stem cells.

[0131] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A method for constructing a bone marrow mesenchymal stem cell-specific HIF-2α gene knockout mouse model based on Cre-LxoP+ technology, characterized in that: The method includes: S1: BMSC-specific HIF-2α knockout mice were used for in vivo experiments, and age- and sex-matched wild-type littermates were used as controls; S2: Hif-2α fl / fl and Prx-1-Cre;Hif-2α fl / fl Mice were ovariectomized, treated with dexamethasone, or semilethally irradiated, and mice of the same age, sex, and genotype were randomly grouped for subsequent experiments; S3: In Prx-1-Cre; Hif-2α fl / fl No sex-related differences were observed in mice.

2. The method for constructing a bone marrow mesenchymal stem cell-specific HIF-2α gene knockout mouse model based on Cre-LxoP+ technology as claimed in claim 1, characterized in that: The mice with conditional knockout of HIF-2α in BMSCs are generated by mating Hif-2αfl / fl mice with Prx-1-Cre transgenic mice.

3. The method for constructing a bone marrow mesenchymal stem cell-specific HIF-2α gene knockout mouse model based on Cre-LxoP+ technology as claimed in claim 1, characterized in that: All animals in this method were housed under specific pathogen free conditions and fed a standard diet.

4. An application of the method for constructing a bone marrow mesenchymal stem cell-specific HIF-2α gene knockout mouse model based on Cre-LxoP+ technology as described in any one of claims 1 to 3, characterized in that: Application in evaluating the physiological function of HIF-2α in the early osteoblast lineage.

5. A bone marrow mesenchymal stem cell-specific HIF-2α gene knockout mouse model constructed using Cre-LoxP technology, characterized in that: The mouse model was constructed by expressing Hif-2α fl / fl The mice were mated with Prx-1-Cre transgenic mice. The Prx-1 promoter specifically expressed Cre recombinase in BMSCs, achieving specific knockout of the HIF-2α gene in BMSCs.

6. The mouse model according to claim 5, characterized in that The mouse model is used to study the biological function of the HIF-2α gene in bone marrow mesenchymal stem cells and its role under different physiological or pathological conditions, including ovariectomy, dexamethasone treatment or semi-lethal radiation.

7. A method for studying gene function based on the mouse model of claim 5, characterized in that: The following steps are involved: a) Select 6-8 week old Prx-1-Cre; Hif-2α fl / fl Mice were matched with age and sex-matched Hif-2αfl / fl mice as controls; b) Simulating different physiological or pathological conditions through treatments such as ovariectomy, dexamethasone treatment or semi-lethal irradiation; c) Bone marrow samples were collected from mice, bone marrow mesenchymal stem cells were isolated, the expression of HIF-2α gene was detected, and the proliferation, differentiation and apoptosis of cells were evaluated.

8. The mouse model according to claim 5, characterized in that This mouse model is used to evaluate the potential of HIF-2α gene knockout on the application of bone marrow mesenchymal stem cells in regenerative medicine, bone tissue engineering and anti-aging research, including but not limited to detecting the biological responses of cells under different stimuli and their role in tissue repair.

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