Stem cell preparation for repairing ovarian-bone vascular microenvironment and application thereof

Abstract

The present application relates to a kind of stem cell preparation of repairing ovary-bone vascular microenvironment and its application, belong to biological medicine technical field, solve one of the problems such as lack of functional enhanced stem cell preparation that can simultaneously repair ovary bone vascular microenvironment in prior art, lack POI related osteoporosis standardization animal model simulating clinical real course, clinical treatment only single intervention ovary or bone metabolism and limited curative effect, ordinary mesenchymal stem cells are insufficient in promoting angiogenesis and promoting osteogenesis ability etc.The present application provides a kind of stem cell preparation of repairing ovary-bone vascular microenvironment and its application, including stable high expression CD146 human umbilical cord mesenchymal stem cells;The angiogenesis ability and the osteogenesis ability of human umbilical cord mesenchymal stem cells are significantly higher than ordinary mesenchymal stem cellsThe present application can simultaneously repair ovary microenvironment and bone vascular microenvironment.The present application can improve early-onset ovarian dysfunction related osteoporosis, realize the synergistic therapeutic effect of ovary function recovery and bone quality improvement.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a stem cell preparation for repairing the ovarian-bone vascular microenvironment and its application. Background Technology

[0002] Early-onset ovarian insufficiency refers to the decline or failure of ovarian function in women before the age of 40. Clinically, it is mainly characterized by amenorrhea or oligomenorrhea, decreased estrogen levels, and elevated follicle-stimulating hormone and luteinizing hormone levels. Long-term estrogen deficiency can lead to an imbalance in bone turnover, a significant decrease in bone mineral density, and a significantly increased risk of osteoporosis and fragility fractures, making it a typical type of secondary osteoporosis in clinical practice.

[0003] Current clinical treatments for osteoporosis associated with early-onset ovarian insufficiency have significant limitations. Hormone replacement therapy only provides exogenous estrogen supplementation and cannot repair damaged ovarian tissue. Ovarian function itself cannot be restored, and long-term use may increase the risk of breast and endometrial lesions and thrombotic events. Its use is limited in patients with a history of cancer or high-risk factors. Conventional anti-osteoporosis drugs only act on the end-stage of bone metabolism and cannot fundamentally correct ovarian endocrine imbalances. In cases of severely impaired ovarian function and disruption of the bone vascular microenvironment, it is difficult to achieve stable and lasting bone improvement. Some drugs also pose long-term safety risks such as osteonecrosis of the jaw and atypical femoral fractures. Therefore, current clinical treatments mostly intervene at the level of endocrine or bone metabolism alone, lacking an integrated treatment plan that can simultaneously address ovarian function repair and bone microenvironment improvement.

[0004] In the field of basic research, existing animal models are insufficient to meet the needs of evaluating the mechanisms and efficacy of drugs for osteoporosis associated with early-onset ovarian insufficiency. Bilateral oophorectomy models completely destroy the ovaries. pituitary The dynamic regulatory axis between bones differs significantly from the clinical course of early-onset ovarian failure with partial ovarian tissue preservation, making it impossible to simulate the progressive ovarian dysfunction and bone loss induced by damaging factors such as chemotherapy. Chemotherapy-induced premature ovarian failure models mostly focus on ovarian structure, hormone levels, and reproductive-related indicators, with less systematic assessment of bone mineral density, three-dimensional microstructure of trabecular bone, and bone metabolism markers. A standardized animal model that can stably present both endocrine abnormalities and osteoporosis phenotypes while preserving ovarian tissue has not yet been established.

[0005] Current technologies also have significant limitations in stem cell therapy research. Ordinary mesenchymal stem cells have limited pro-angiogenic and osteogenic repair capabilities, making it difficult to exert a sufficient repair effect in severely damaged ovaries and the bone microenvironment. Most existing mesenchymal stem cell research focuses on a single aspect—ovarian function or bone metabolism—lacking a broader focus on the ovary. bone Systematic design for the regulation of the overall vascular axis. The bone vascular microenvironment plays a key role in maintaining bone remodeling coupling and bone quality. However, in the secondary osteoporosis scenario of early-onset ovarian insufficiency-related osteoporosis, the changes and reversibility of the bone vascular microenvironment have not been systematically studied, and existing interventions have not been specifically designed for the bone vascular microenvironment.

[0006] Currently, there is still a lack of complete technical solutions and supporting evaluation systems for using functionally enhanced mesenchymal stem cells to simultaneously restore ovarian endocrine function and reconstruct the bone vascular microenvironment in a model of osteoporosis associated with early-onset ovarian insufficiency, thereby synergistically improving bone mass and bone microstructure. There is an urgent technical need for this in both clinical and basic research. Summary of the Invention

[0007] Based on the above analysis, the embodiments of the present invention aim to provide a stem cell preparation for repairing the ovarian-bone vascular microenvironment and its application, in order to solve the problem of the lack of existing technologies capable of simultaneously repairing the ovary. One of the problems is the lack of functional stem cell preparations that enhance the bone vascular microenvironment, the lack of standardized animal models of POI-related osteoporosis that simulate the real clinical course, the limited efficacy of clinical treatment that only intervenes in ovarian or bone metabolism, and the insufficient ability of ordinary mesenchymal stem cells to promote angiogenesis and osteoporosis.

[0008] The first aspect of this invention provides a stem cell preparation for repairing the ovarian-bone vascular microenvironment, the stem cell preparation comprising human umbilical cord mesenchymal stem cells that stably and highly express CD146; the angiogenesis-promoting and osteogenic abilities of the human umbilical cord mesenchymal stem cells are significantly higher than those of ordinary mesenchymal stem cells.

[0009] Furthermore, in the stem cell preparation, the human umbilical cord mesenchymal stem cells are stable cells integrated with the CD146 gene via lentivirus-mediated transformation, and their angiogenesis and osteogenic abilities are significantly higher than those of ordinary mesenchymal stem cells.

[0010] Furthermore, in the aforementioned stem cell preparation, the cell concentration is 0.5 × 10⁻⁶. 6 ~5×10 6 per mL.

[0011] Furthermore, in the aforementioned stem cell preparation, the cell survival rate is not less than 90%.

[0012] Furthermore, in the aforementioned stem cell preparation, the human umbilical cord mesenchymal stem cells can simultaneously repair the ovarian microenvironment and the bone vascular microenvironment.

[0013] A second aspect of the present invention also provides the use of the above-mentioned stem cell preparation in the preparation of a drug for treating osteoporosis associated with early-onset ovarian insufficiency.

[0014] Furthermore, in the aforementioned applications, the stem cell preparation is administered via intravenous injection, and the stem cell preparation can simultaneously repair ovarian damage, rebuild the bone vascular microenvironment, and improve bone quality.

[0015] A third aspect of the present invention also provides a method for constructing an animal model of early-onset ovarian insufficiency combined with osteoporosis, comprising the following steps: Step 1: Preserve intact ovarian tissue in female mammals; Step 2: Continuous administration of chemotherapy drugs was used to induce ovarian dysfunction and bone vascular microenvironment damage phenotypes to obtain experimental animal models.

[0016] Furthermore, in the described construction method, the chemotherapy drug is cyclophosphamide.

[0017] Furthermore, in the aforementioned construction method, the damage to the bone vascular microenvironment manifests as decreased intraosseous vascular density, destruction of trabecular bone structure, and / or bone metabolic imbalance.

[0018] Compared with the prior art, the present invention can achieve at least one of the following beneficial effects: 1. The stem cell preparation for repairing the ovarian-bone vascular microenvironment described in this invention uses human umbilical cord mesenchymal stem cells with stable high expression of CD146 as the active ingredient. Compared with ordinary mesenchymal stem cells, it has significantly enhanced angiogenesis and osteogenic capabilities, improving the repair efficiency of damaged tissues at the cellular level. In vitro, it can significantly promote the formation of tubular structures in vascular endothelial cells, and in vivo, it can effectively improve bone remodeling imbalance, thus providing a repair mechanism for the ovarian-bone vascular microenvironment. Synchronous repair of the bone vascular microenvironment provides a more functional and stable cellular basis.

[0019] 2. The stem cell preparation for repairing the ovarian-bone vascular microenvironment described in this invention can act on both the ovary and bone tissue as target organs simultaneously, achieving synergistic regulation of ovarian microenvironment repair and bone vascular microenvironment reconstruction. It can effectively improve endocrine disorders caused by premature ovarian insufficiency, increase follicle reserve and restore estrous cycles, while also increasing bone density, improving the three-dimensional microstructure of bone trabeculae and correcting bone metabolic imbalance, thus overcoming the shortcomings of existing technologies that only intervene in ovarian or bone metabolism and cannot provide holistic treatment.

[0020] 3. The stem cell preparation of the present invention has a controllable cell concentration and a cell survival rate of not less than 90%, which meets the quality control requirements of stem cell drugs. It can be administered systemically by intravenous injection, is easy to operate and has good biocompatibility. It will not cause significant damage to major organs after administration, and has both good efficacy and safety. It has high clinical translational value and can provide a safe and reliable new treatment option for osteoporosis related to early-onset ovarian insufficiency.

[0021] 4. The method for constructing an animal model of early-onset ovarian insufficiency combined with osteoporosis described in this invention, induced by chemotherapy drugs while preserving ovarian tissue, can simultaneously and stably present the phenotypes of ovarian insufficiency and bone vascular microenvironment damage, more closely resembling the true pathological process of clinical chemotherapy-related POIs, and can accurately and objectively evaluate the effects of stem cell preparations on the ovaries. The repair effect on the bone vascular microenvironment provides a standardized and reproducible experimental tool for the efficacy screening and mechanism research of related drugs.

[0022] In this invention, the above-described technical solutions can be combined with each other to achieve more preferred combinations. Other features and advantages of this invention will be set forth in the following description, and some advantages may become apparent from the description or be learned by practicing the invention. The objects and other advantages of this invention can be realized and obtained from what is particularly pointed out in the description and drawings. Attached Figure Description

[0023] The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Throughout the drawings, the same reference numerals denote the same parts. Figure 1 The human umbilical cord mesenchymal stem cells (MSCs) infected with CD146-overexpressing lentivirus in Example 1 showed that the CD146 protein was correctly expressed and located on the cell membrane surface. Figure 2 For blank MSCs and CD146 in Example 1 + Western blot analysis of CD146 protein in MSCs; Figure 3 This is a flow cytometry scatter plot of GFP expression in blank human umbilical cord mesenchymal stem cells in Example 1; Figure 4 MSCs (CD146) infected with CD146-overexpressing lentivirus in Example 1 + Flow cytometry scatter plot of GFP expression in MSCs; Figure 5 For blank MSCs and CD146 in Example 1 + Bar chart showing the percentage of GFP-positive MSCs; Figure 6 For blank MSCs and CD146 in Example 1 + Bar chart of relative expression levels of MCAM (CD146) mRNA in MSCs using RT-qPCR; Figure 7 shows CD146 in Example 1. + Comparison of stable GFP fluorescence expression in MSCs of generations 2 and 5; Figure 8 This is a diagram of endothelial cell tube formation in vitro in Example 1; Figure 9 This is a quantitative bar chart of the in vitro tube formation experiment in Example 1; Figure 10 This is a bar chart showing the serum levels of four reproductive hormones (AMH, LH, FSH, and E2) in the normal control group and the POI model group in Example 2. Figure 11 This is a bar chart showing the number of follicles at each stage in the ovaries of mice in the normal control group (Control) and the POI model group in Example 2; Figure 12 These are two-dimensional and three-dimensional reconstruction images of the femur from the POI model group and the normal control group using micro-CT in Example 2. Figure 13 The groups included in Example 4 were the POI group, PBS control group, MSCs treatment group, and CD146 group. + Line graph showing the changes in the estrous cycle of mice in the MSCs treatment group over 15 days; Figure 14 The four groups of mice in Example 4 (POI, PBS, MSCs, CD146) + A bar chart showing the number of follicles at each stage of MSCs; Figure 15 The images show TUNEL apoptosis fluorescence staining and 3D fluorescence intensity quantification of ovarian tissues from each group in Example 4. Figure 16 This is a bar chart showing the relative quantitative expression of CD31-positive area in each group of ovaries in Example 4. Figure 17 This is a bar chart showing the relative quantitative expression of Ki67-positive area in each group of ovaries in Example 4. Figure 18 For example, the POI group, PBS group, MSCs group, and CD146 group in Example 4 + Bar chart showing the levels of four reproductive hormones (AMH, LH, FSH, and E2) in the serum of mice in the MSCs group; Figure 19 For example, POI, PBS, MSCs and CD146 in Example 5 + Micro-CT two-dimensional sections of distal femur and three-dimensional reconstruction of cancellous bone in four groups of MSCs mice; Figure 20 The graph shows the micro-CT bone analysis parameters of the distal femur of four groups of mice in Example 5. Figure 21 The POI group, PBS group, MSCs group, and CD146 group in Example 5 + Observation of trabecular bone morphology in paraffin sections of distal femur of MSCs group mice stained with HE; Figure 22 For example, the POI group, PBS group, MSCs group, and CD146 group in Example 5 + Observation of the morphology of collagen fibers and trabeculae in the distal femur of mice in the MSCs group using Masson trichrome staining; Figure 23 For example, the POI group, PBS group, MSCs group, and CD146 group in Example 5 + Representative microscopic observation of TRAP-stained osteoclasts in femoral tissue of MSCs group mice; Figure 24 This is a violin plot showing the number of osteoclasts per unit bone surface and the percentage of osteoclast surface area (N.Oc / BS, Oc.S / BS) in Example 5. Figure 25 This is an immunofluorescence staining image of CTSK osteoclasts in the femur from Example 5; Figure 26 This is a CD31 / EMCN double-labeled immunofluorescence image of bone blood vessels in bone tissue from Example 5; Figure 27 This is a bar chart of serum bone resorption marker CTX-1 and osteogenic marker P1NP ELISA quantitative data in Example 5. Figure 28 This is a heatmap of mRNA expression of genes related to ovarian tissue function, aging, and oxidative stress in Example 6. Figure 29 This is a Western blot image of ovarian angiogenesis and Notch pathway proteins from Example 6. Figure 30 This is a heatmap of mRNA expression of genes related to osteogenic, osteoclastic, aging, and oxidative stress in bone tissue in Example 6. Figure 31 Example 6 shows the Western blot bands of osteogenic and estrogen pathway proteins in bone tissue. Detailed Implementation

[0024] Preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, which form part of this application and are used together with the embodiments of the present invention to illustrate the principles of the present invention, but are not intended to limit the scope of the present invention.

[0025] The first aspect of this invention provides a stem cell preparation for repairing the ovarian-bone vascular microenvironment, the stem cell preparation comprising human umbilical cord mesenchymal stem cells that stably and highly express CD146; the angiogenesis-promoting and osteogenic abilities of the human umbilical cord mesenchymal stem cells are significantly higher than those of ordinary mesenchymal stem cells.

[0026] This invention uses human umbilical cord mesenchymal stem cells with stable lentiviral transformation and high expression of CD146 as the core active ingredient. Compared with ordinary umbilical cord mesenchymal stem cells, the in vitro tube formation and in vivo osteogenic properties are significantly improved, breaking through the industry bottleneck of insufficient angiogenesis and osteogenic repair capabilities of ordinary stem cells, and greatly improving the repair efficiency of ovarian and damaged bone vascular microenvironment.

[0027] Furthermore, in the stem cell preparation, the human umbilical cord mesenchymal stem cells are stable cells integrated with the CD146 gene via lentivirus-mediated transformation, and their angiogenesis and osteogenic abilities are significantly higher than those of ordinary mesenchymal stem cells.

[0028] Lentiviral-mediated integration of the CD146 gene into stable transfected cells enables long-term stable expression of the CD146 gene, with no functional decay after continuous passage. This avoids the defects of expression loss and unstable efficacy in transiently transfected cells, resulting in better batch-to-batch consistency of the formulation and meeting the quality control requirements of drug development.

[0029] Furthermore, in the aforementioned stem cell preparation, the cell concentration is 0.5 × 10⁻⁶. 6 ~6×10 6 Cells / mL. For example, the cell concentration in the stem cell preparation is 0.5 × 10⁻⁶ cells / mL. 6 cells / mL, 1×10 6 cells / mL, 1.5×10 6 cells / mL, 2×10 6 cells / mL, 2.5×10 6 cells / mL, 3×10 6 cells / mL, 4.5×10 6 cells / mL, 5×10 6 cells / mL, 5.5×10 6 cells / mL and 6×10 6 per mL.

[0030] Furthermore, in the aforementioned stem cell preparation, the cell survival rate is not less than 90%.

[0031] Furthermore, in the aforementioned stem cell preparation, the human umbilical cord mesenchymal stem cells can simultaneously repair the ovarian microenvironment and the bone vascular microenvironment.

[0032] Stem cells can simultaneously repair the ovarian microenvironment and the bone vascular microenvironment, unlike existing agents that only repair the ovary or simply improve bone metabolism, achieving synergistic repair of both ovarian endocrine and bone vascular microenvironment.

[0033] A second aspect of the present invention also provides the use of the above-mentioned stem cell preparation in the preparation of a drug for treating osteoporosis associated with early-onset ovarian insufficiency.

[0034] This CD146-overexpressing stem cell preparation is used to develop a treatment for osteoporosis associated with early-onset ovarian insufficiency. Unlike traditional hormone replacement therapy and simple anti-bone resorption drugs, it can repair damaged ovaries, restore endogenous hormone secretion, and rebuild bone vascular microstructure. It can simultaneously improve ovarian decline and secondary osteoporosis from the etiological level, rather than just relieving a single symptom.

[0035] Furthermore, in the aforementioned applications, the stem cell preparation is administered via intravenous injection, and the stem cell preparation can simultaneously repair ovarian damage, rebuild the bone vascular microenvironment, and improve bone quality.

[0036] The drug is administered via intravenous injection, which is simple to administer and allows it to accumulate in the ovaries and bone tissues throughout the body. After administration, it simultaneously achieves a triple effect of ovarian damage repair, bone angiogenesis, and increased bone density. This avoids the risks of breast and uterine lesions and thrombosis associated with long-term use of hormones, and also overcomes the shortcoming of conventional anti-osteoporosis drugs that cannot reverse ovarian damage, thus broadening the applicable population for POI patients with osteoporosis.

[0037] A third aspect of the present invention also provides a method for constructing an animal model of early-onset ovarian insufficiency combined with osteoporosis, comprising the following steps: Step 1: Preserve intact ovarian tissue in female mammals; Step 2: Continuous administration of chemotherapy drugs was used to induce ovarian dysfunction and bone vascular microenvironment damage phenotypes to obtain experimental animal models.

[0038] The complete preservation of the ovary and the chemotherapy-induced modeling process overcome the shortcomings of the bilateral ovariectomy model, which completely destroys the ovarian-bone regulatory axis and does not match the clinical chemotherapy-induced POI course. The model animals of this method simultaneously and stably exhibit the dual phenotypes of ovarian failure and bone and blood vessel damage, simulating the real pathological state of early-onset ovarian insufficiency secondary to osteoporosis in clinical practice with partial preservation of ovarian tissue.

[0039] Furthermore, in the described construction method, the chemotherapy drug is cyclophosphamide.

[0040] Furthermore, in the aforementioned construction method, the damage to the bone vascular microenvironment manifests as decreased intraosseous vascular density, destruction of trabecular bone structure, and imbalance of bone metabolism.

[0041] Example 1: CD146-overexpressing mesenchymal stem cells (CD146) + Construction and identification of MSCs 1.1 Human umbilical cord mesenchymal stem cells (hUC) Culture of MSCs The human umbilical cord mesenchymal stem cells (hUC) used in this embodiment MSCs were prepared and provided by the Clinical Stem Cell Research Center of Peking University Third Hospital. All cells were identified through immunophenotyping, differentiation potential, and sterility testing, meeting the typical characteristics and quality standards of mesenchymal stem cells. The hUCs were then... MSCs were inoculated with a solution containing 1% penicillin The streptomycin antibiotic was cultured in serum-free medium (NC103 / NC103.s, Yocon) in a cell culture incubator at 37°C, 5% CO2, and saturated humidity; the medium was replaced with fresh complete medium every 48 hours.

[0042] When the cell confluence reached 80%–90%, the cells were digested and passaged using TrypLE (Thermo Fisher Scientific, 12604021) digestion solution at a passage ratio of 1:2–1:3; hUC cells in good growth condition from passages 3 to 6 were selected. MSCs were used in subsequent experiments.

[0043] 1.2 Construction of CD146 overexpression lentiviral vector and viral packaging A lentiviral expression vector carrying the full-length coding sequence (CDS) of human CD146 was constructed, and the vector also carried a green fluorescent protein (GFP) tag. The recombinant lentiviral vector was mixed with the lentiviral packaging plasmids psPAX2 and pMD2.G at a ratio of 5:1 and co-transfected into HEK293T cells using Lipofectamine 3000 liposome transfection reagent. Cell culture supernatant was collected 48–60 h post-transfection, centrifuged to remove cell debris, filtered through a 0.45 μm filter, and the viral titer was determined for later use.

[0044] 1.3 Lentiviral infection of hUC MSCs Take hUC in the logarithmic growth phase MSCs were seeded in culture dishes, and when the cell confluence reached 60% to 80%, the lentivirus solution prepared above was added, along with polybrene at a final concentration of 5 to 8 μg / mL to improve infection efficiency.

[0045] 1.4 Screening and purification of CD146 overexpression stable transgenes 12-24 hours after infection, the virus-containing culture medium was discarded, and the medium was replaced with complete culture medium containing 2 μg / mL puromycin. After 2 days of selection, surviving CD146 overexpressing hUCs were obtained. MSCs.

[0046] The results were verified by fluorescence microscopy and Western blot analysis, as follows: Figure 1 , Figure 2As shown, CD146 is correctly located on the cell membrane surface and at cell junctions, and possesses biological activity.

[0047] 1.5 CD146 + MSCs expression validation Using flow cytometry and RT qPCR was used to detect CD146 expression levels, and the results are as follows: Figure 3 , Figure 4 , Figure 5 and Figure 6 As shown, functionally enhanced mesenchymal stem cells with stable high expression of CD146 were successfully constructed. During flow cytometry sorting, the target MSC cell population was first delineated using forward scatter (FSC-A) and side scatter (SSC-A) light, and debris was excluded. Subsequently, fluorescence gating was performed on a scatter plot of FSC-A corresponding to GFPFITC-A. Untransfected hUC-MSCs (250121-MSCs) were used as a negative control to set the fluorescence baseline. Positive cells were divided into two subpopulations: those with GFP FITC-A fluorescence intensity at 10... 3 Up to 10 4 The population with a fluorescence intensity greater than 10 is defined as the GFP low population; the population with a GFP FITC-A fluorescence intensity greater than 10 is defined as the GFP low population. 4 (approximately 10) 4 -10 5 The population of CD146 was defined as the GFP high population. Cells within the corresponding gating groups were collected according to subsequent experimental requirements. Flow cytometry results showed that CD146... + The CD146 positivity rate in the MSCs group was significantly higher than that in the conventional hUC-MSCs group (P<0.05). Simultaneously, RT-qPCR was used to detect the mRNA expression level of the CD146 encoding gene MCAM, and the results showed that CD146... + The relative expression level of MCAM in the MSCs group was significantly higher than that in the ordinary hUC-MSCs group (P<0.05). The sorted CD146 high population was expanded and cultured to obtain CD146... + Stable cell lines of MSCs. Figure 7 As shown, after five generations of continuous passage, GFP fluorescence still showed a strong signal, indicating that transgene expression was stable during the amplification process.

[0048] 1.6 Identification of in vitro angiogenesis-promoting function Evaluation of MSCs and CD146 using in vitro tube formation assays +The angiogenesis-promoting capacity of MSCs was assessed. 50 μl of pre-cooled Matrigel (Yeasen, 40186ES08) was added to 24-well plates for coating, ensuring even coverage of the well bottoms. The plates were incubated at 37°C for 30 minutes until gel formation. MSCs and CD146 were then used for further processing. + MSCs were mixed with human umbilical vein endothelial cells (HUVECs) at a 1:1 ratio and seeded in Matrigel-coated 24-well plates. Each well contained either MSCs or CD146. + MSCs and HUVECs were each inoculated at 5 × 10 4 There are [number] cells, with a total cell count of 1 × 10[number] cells. 5 Each well was incubated with serum-free DMEM medium at 37°C, 5% CO2, and saturated humidity for 6 hours. After incubation, 3–5 fields of view were randomly selected from each well and photographed under an inverted phase-contrast microscope (Nikon). The images were quantitatively analyzed using ImageJ image analysis software (National Institutes of Health, USA) to count the number of segments, branches, and nodes of the tubular structure. Three replicates were then used for intergroup comparisons. The results are as follows: Figure 8 , Figure 9 As shown, CD146 + The number of tube segments and branch points formed in the MSCs group was significantly higher than that in the ordinary MSCs group, indicating that CD146 + MSCs have a stronger ability to promote angiogenesis.

[0049] 1.7 Preparation of stem cell preparations Take the logarithmic growth phase CD146 + MSCs were tested and found to be free of bacterial, fungal, and mycoplasma contamination, with a cell viability ≥90%; the cells were resuspended in sterile PBS and the cell concentration was adjusted to 0.5 × 10⁻⁶. 6 ~5×10 6 The stem cell preparation is obtained by obtaining cells / mL.

[0050] Example 2: Construction of a mouse model of POI-related osteoporosis 2.1 Laboratory Animals and Grouping Eight-week-old female C57BL / 6 mice, weighing 18–22 g, were selected and provided by a qualified laboratory animal center. After one week of acclimatization, all animals were randomly divided into 5 groups of 6 mice each. (1) Normal control group (Control); (2) The uninterventional POI model group (POI); (3) POI+PBS control group (POI+PBS); (4) POI + conventional MSCs treatment group (POI + MSCs); (5) POI+CD146 + MSCs treatment group (POI+CD146) + MSCs).

[0051] 2.2 Model Construction Method Except for the normal control group, mice in the other groups received daily intraperitoneal injections of cyclophosphamide (CTX). The CTX (Sigma, PHR1404) was dissolved in sterile physiological saline (0.9% NaCl) to a working concentration of 1 mg / mL; it was administered intraperitoneally at a dose of 40 mg / kg, with a dosing volume of 40 mL / kg, for 14 consecutive days. The normal control group received an equal volume of 0.9% sodium chloride solution concurrently. This method stably establishes a POI-related osteoporosis mouse model that simultaneously exhibits ovarian insufficiency and bone vascular microenvironment damage while preserving ovarian tissue.

[0052] Successful modeling criteria: Mice simultaneously exhibiting ovarian insufficiency phenotypes (decreased E2 and AMH, increased FSH and LH, and reduced follicles). Figure 10 , Figure 11 As shown, the phenotype of bone microenvironment damage is as follows: Figure 12 As shown, and by preserving ovarian tissue, it can be used to evaluate the repair effect of stem cell preparations.

[0053] Example 3 CD146 + Dosing regimen for MSCs in the treatment of POI-related osteoporosis 3.1 Preparation of cell preparations MSCs and CD146 in the logarithmic growth phase + MSCs were tested and found to be free of bacterial, fungal, and mycoplasma contamination, with a cell viability ≥90%. Cells were washed twice with sterile PBS and resuspended to adjust the cell concentration to 5 × 10⁻⁶. 6 Cells / mL were prepared as a suspension for injection.

[0054] 3.2 Administration method The injection was administered via tail vein, with each mouse receiving 1×10⁻⁶ injections per dose. 6Cells were resuspended in 200 μL PBS once a week for 4 consecutive weeks. Cell viability was assessed using the 0.4% trypan blue exclusion method before injection; cells with viability above 90% were used for in vivo infusion. Mice were moderately restrained in a fixation device, their tails were pre-warmed, and the tails were wiped with 70% ethanol to expose the lateral tail vein. Insulin was slowly and evenly injected along the tail vein using a 29G insulin syringe (BD, 328421) within 30 seconds. After injection, pressure was applied to the injection site for 30–60 seconds to stop bleeding, and the mice were observed for 5–10 minutes. Once no reflux, bleeding, or acute adverse reactions were confirmed, the mice were returned to their cages. POI+MSCs group: injected with an equal volume of ordinary MSCs suspension; POI+CD146 + MSCs group: injected with CD146 + MSCs suspension; POI+PBS group: 200 μL of sterile PBS was injected; POI model group: No tail vein injection treatment was performed.

[0055] 3.3 Sample Collection Mice were euthanized using an overdose of anesthesia, and blood was collected via the orbital sinus under deep anesthesia. Whole blood was placed in anticoagulant-free centrifuge tubes and allowed to stand at room temperature for 15–30 minutes until natural coagulation. After coagulation, the blood was centrifuged at 3000 rpm for 10 minutes at 4°C to separate the serum, which was then aliquoted and stored at -80°C for ELISA testing. Subsequently, both ovaries and femurs were isolated. Samples for histological and imaging studies were fixed in 4% paraformaldehyde for 24 hours and then processed according to standard methods. Tissue samples for molecular biological assays were immediately flash-frozen in liquid nitrogen after collection and stored at -80°C for later use.

[0056] Example 4 CD146 + Evaluation of the effect of MSCs on ovarian function repair in POI mice 4.1 Monitoring of the estrous cycle For 15 consecutive days following the intervention, vaginal secretions were collected from mice daily or every other day around 9:00 AM. During sampling, the mice were gently immobilized with their tails slightly elevated. A vaginal irrigation method was used, with 20 μl of room temperature sterile PBS drawn into a narrow-mouthed pipette. The pipette tip was gently placed approximately 1–2 mm from the vaginal opening, and the fluid was slowly rinsed and aspirated 2–3 times. The irrigation fluid was collected, spread evenly on a clean glass slide, and allowed to air dry. After air drying, Wright staining was performed. Commercially available Wright staining solution (Solarbio, G1040) was added to the specimen and stained for 3 minutes to fix the specimen with methanol. Then, an equal volume of pH 6.4 phosphate buffer (or an equal volume of ultrapure water) was added, the slide was gently shaken to mix the stain, and the slide was allowed to stand for 5 minutes. Excess stain was quickly washed away with distilled water, excess water was aspirated from the edges of the slide, and the slide was allowed to air dry before microscopic examination to observe cell morphology.

[0057] The estrous cycle was determined and its recovery status assessed based on the ratio of nucleated epithelial cells, keratinized epithelial cells, and leukocytes. The estrous cycle was determined by the sequential alternation of the four phases: proestrus, estrus, metestrus, and dysestrus. Proestrus was dominated by nucleated epithelial cells; estrus by anucleated keratinized squamous epithelial cells; metestrus by a mixture of leukocytes and epithelial cells, with an increased leukocyte count; and dysestrus was dominated by leukocytes with a small number of nucleated epithelial cells. Results were as follows: Figure 13 As shown, CD146 + After intervention with MSCs, the disordered estrous cycles in mice could be restored to regular cycles.

[0058] 4.2 Ovarian histology and follicle count Ovarian tissue was collected, fixed in 4% paraformaldehyde for 24 hours, then routinely dehydrated, embedded in paraffin, and serially sectioned (5 μm thick). HE staining was performed according to the standard paraffin sectioning procedure: sections were baked, dewaxed in xylene, rehydrated with graded ethanol to distilled water, stained with hematoxylin, rinsed with running water, differentiated with acidic ethanol, blued with ammonia, and counterstained with eosin. The sections were then dehydrated with graded ethanol, cleared with xylene, and mounted. Follicle counting was performed using a whole-ovarian slicing method: after continuous slicing along the full depth of the ovary, one slice was taken every five slices for counting; the counting was based on the whole-slice scan results, counting only follicles with clearly visible oocyte nuclei to reduce duplicate counting; the follicle grading criteria are as follows: Primordial follicle: surrounded by a single layer of flattened, squamous granulosa cells; Primary follicle: surrounded by a single layer of cuboidal granulosa cells; Secondary follicles: two or more layers of cuboidal granulosa cells, without follicular cavity; Antral follicles: There are obvious sinus cavities.

[0059] like Figure 14 As shown in the figure, the normal control group, POI group, POI+PBS group, POI+MSCs group, and POI+CD146 group are respectively displayed.+ The number of primordial follicles, primary follicles, secondary follicles, and antral follicles in the ovaries of mice in the MSCs group was significantly lower than that in the POI+PBS group (POI+CD146). + The number of follicles at each stage was significantly increased in the MSCs group.

[0060] 4.3 Ovarian cell apoptosis detection (TUNEL) After dewaxing and rehydration, ovarian paraffin sections were subjected to antigen retrieval using proteinase K (40 μg / mL, 37℃, 30 min). Following incubation with commercially available TUNEL reaction solution (Solarbio, T2196) at 37℃ for 30 min, the cell nuclei were counterstained with DAPI. Confocal fluorescence microscopy was used for observation. The excitation wavelength for TUNEL positive labeling was 488 nm, and the emission wavelength was 520 nm (FITC labeling); the excitation wavelength for DAPI was 358 nm, and the emission wavelength was 461 nm. The degree of apoptosis in ovarian tissues was assessed by comparing the intensity of green fluorescence. Figure 15 As shown, POI+CD146 + The MSCs group significantly reduced ovarian cell apoptosis.

[0061] 4.4 Ovarian immunohistochemical detection (CD31, Ki67) After dewaxing, rehydration, and antigen retrieval, ovarian paraffin sections were incubated overnight at 4°C with CD31-specific primary antibody (Proteintech, 28083-1-AP, dilution 1:4000) and Ki67-specific primary antibody (Abcam, Ab15580, dilution 1:200). The next day, HRP-labeled secondary antibodies (dilution 1:500) were added and incubated at room temperature for 30 min. DAB staining was then performed (2-5 min, controlled under a microscope until a clear positive signal and low background were observed), followed by hematoxylin counterstaining, dehydration, and mounting. Images were acquired using a digital slide scanning system. Three non-overlapping high-power fields were randomly selected from each slide, and the percentage of CD31 and Ki67 positive areas was analyzed using ImageJ image analysis software. Results are as follows: Figure 16 and Figure 17 As shown: According to the present invention CD146 + After treatment with MSCs, the density of CD31-positive blood vessels in ovarian tissue increased significantly, suggesting that MSCs can promote local angiogenesis and improve ovarian microenvironment perfusion, which is beneficial to follicle survival and functional recovery. At the same time, the positive rate of Ki67 increased, indicating that the ovarian cell proliferation capacity and tissue repair activity were enhanced, which is helpful for follicle development and ovarian function recovery.

[0062] 4.5 Serum sex hormone detection Serum estradiol (E2), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and anti-Müllerian hormone (AMH) concentrations were detected using ELISA kits. The E2 kit was purchased from Elabscience (E-OSEL-M0008, detection range: 3.13–200 pg / mL); the FSH kit was purchased from Cloud-Clone Corp (UCEA830Mu, detection range: 2.22–180 ng / mL); the LH kit was purchased from Cloud-Clone Corp (CEA441Mu, detection range: 370.4–30,000 pg / mL); and the AMH kit was purchased from Cloud-Clone Corp (UCEA228Mu, detection range: 123.5–10,000 pg / mL). Strictly follow the instructions for each kit. Use a microplate reader (BioTeK, Epoch) at 450 nm wavelength, zeroing the instrument with a blank control well, to measure the OD value of each well. Calculate the sample content based on the standard curve to assess the recovery of ovarian endocrine function. Results are as follows: Figure 18 As shown: According to the present invention CD146 + After treatment with MSCs, E2 and AMH levels increased significantly, while FSH and LH levels decreased.

[0063] Example 5 CD146 + Effects of MSCs on the repair of bone microstructure and bone vascular microenvironment in POI mice 5.1 Femoral micro CT scan The right femur of a mouse was harvested, with any attached soft tissue removed while preserving the bone integrity. After fixation, the sample was transferred to 70% ethanol and stored at 4°C until analysis. During scanning, the sample was kept submerged in 70% ethanol to minimize sample drying and displacement artifacts. A high-resolution microarray was used. CT scans were performed with the following parameters: voxel resolution approximately 9 μm, X-ray voltage 50 kV, and current 400 μA. After image reconstruction, regions of interest (ROIs) were selected in the distal femoral metaphysis (cancellous bone region) for analysis. Using the distal femoral epiphyseal plate (the fusion point of the growth plates) as a reference plane, layers 100 to 220 (a total of 121 layers) were selected proximally away from the growth plate as ROI layers. The starting position, length, and analysis conditions of the ROIs were kept consistent for all samples. Bone mineral density (BMD), bone volume fraction (BV / TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), and trabecular connectivity density (Conn.D) were calculated. These parameters were used to quantitatively evaluate CTX-induced bone loss and CD146 levels. + Recovery of bone mass and trabecular structure after MSCs treatment.

[0064] like Figure 19 , Figure 20 As shown, CD146 + MSCs can significantly increase bone mineral density and improve bone microstructure.

[0065] 5.2 Bone histological examination After fixation, the femur was placed in a 10% EDTA decalcification solution for decalcification at room temperature for 21–28 days. The decalcification solution was changed every 2–3 days during decalcification until the bone tissue softened, which was the endpoint of decalcification. After decalcification, the femur was thoroughly rinsed with distilled water or PBS, followed by routine dehydration, clearing, paraffin embedding, and sectioning. The section thickness was 5 μm.

[0066] HE staining was performed according to the standard paraffin sectioning procedure: after dewaxing the sections to distilled water, they were stained with Mayer hematoxylin for about 1 min, rinsed with running water, and then stained with PBS or blue solution for about 1 min, followed by counterstaining with eosin for about 1 min. The sections were then dehydrated with a series of ethanol solutions, cleared with xylene, and mounted. Masson staining was performed according to the standard trichromatic method: after dewaxing the sections to distilled water, they were mordanted with preheated Bouin solution at 56–58°C for about 15 min (or overnight at room temperature), rinsed with water, stained with Weigert iron hematoxylin for about 5 min, differentiated with acidic ethanol for 3–5 s if necessary, then stained with Biebrich scarlet-acid fuchsin for about 5 min, treated with phosphomolybdic acid for about 2–5 min, stained with aniline blue for about 5 min, and treated with 1% acetic acid for about 30 s to 2 min. Finally, the sections were dehydrated, cleared, and mounted. For TRAP staining, after dewaxing and rehydration, TRAP working solution was added to cover the sections, which were then incubated in a humidified chamber at 37°C for 45-60 min. After rinsing with distilled water and draining, the sections were mounted with aqueous mounting medium and observed under a microscope. Immunofluorescence staining was used to detect the osteoclast-specific marker cathepsin K (CTSK): After dewaxing, rehydration, and antigen retrieval, the sections were blocked with blocking solution at room temperature for 30 min; CTSK-specific primary antibody (GB111276, Servicebio) was added at a dilution of 1:1000 and incubated overnight at 4°C; the next day, fluorescently labeled secondary antibody (GB21303, Servicebio) was added at a dilution of 1:300 and incubated at room temperature in the dark for 1 h; DAPI counterstaining of cell nuclei was followed by mounting. Images were observed and acquired using a confocal fluorescence microscope. The excitation wavelength for CTSK positive signals was 555 nm, and the emission wavelength was 570 nm; the excitation wavelength for DAPI was 358 nm, and the emission wavelength was 461 nm. The morphology of osteoclasts and their distribution on the surface of bone trabeculae were observed.

[0067] HE staining was used to observe the morphology and continuity of bone trabeculae; Masson staining was used to observe collagen deposition; TRAP staining was used to label osteoclasts and count the number of positive multinucleated cells per unit bone surface area. CTSK immunofluorescence staining showed a significant decrease in the number of positive osteoclasts. Figure 21 , Figure 22 , Figure 23 , Figure 24 , Figure 25 As shown, CD146 + The MSCs group showed denser, more intact, and more continuous bone trabeculae, and a significantly reduced number of osteoclasts.

[0068] 5.3 Assessment of the bone vascular microenvironment Immunofluorescence was used to detect the expression of CD31 and EMCN in bone tissue, observe the distribution of microvessels in the medullary cavity and around bone trabeculae, and evaluate the repair effect of the bone vascular microenvironment. Paraffin sections were dewaxed, rehydrated, and antigen-retrieved, then blocked at room temperature for 30 min with blocking solution. Subsequently, CD31-specific primary antibody (GB150217, Servicebio) at a dilution of 1:500 and EMCN-specific primary antibody (GB112648, Servicebio) at a dilution of 1:600 ​​were added, and the sections were incubated overnight at 4°C. The next day, Cy3-labeled goat anti-rabbit IgG and FITC-labeled goat anti-rabbit IgG at a dilution of 1:300 were added, and the sections were incubated at room temperature in the dark for 1 h. Cell nuclei were counterstained with DAPI, and the sections were mounted. Images were observed and acquired using confocal fluorescence microscopy. The excitation wavelength for CD31 positive signals was 555 nm, and the emission wavelength was 570 nm; the excitation wavelength for EMCN positive signals was 488 nm, and the emission wavelength was 520 nm; the excitation wavelength for DAPI was 358 nm, and the emission wavelength was 461 nm. Analyze the CD31 and EMCN double-positive vascular structures and compare the differences in microvascular density and morphology in bone tissue among different groups. Figure 26 As shown, CD146 + MSCs can significantly increase the number of CD31 and EMCN double-positive vessels.

[0069] 5.4 Detection of Bone Metabolic Biochemical Indicators The serum type I procollagen N-terminal propeptide (P1NP) and type I collagen cross-linked C-terminal peptide (CTX-1) were detected using an ELISA kit to reflect osteogenic activity and bone resorption levels, respectively, and to clarify CD146. +The regulatory role of MSCs in bone remodeling balance. The P1NP kit was purchased from UpingBio (SYP-M2090, detection range: 2.5 ng / mL–80 ng / mL); the CTX-1 kit was purchased from UpingBio (SYP-M0370, detection range: 62.5 pg / mL–2000 pg / mL). Procedures were strictly followed according to the instructions for each kit. An ELISA reader (BioTeK, Epoch) was used at 450 nm. After zeroing the instrument with blank control wells, the OD values ​​of each well were measured. Sample concentrations were calculated based on the standard curve. Figure 27 As shown, CD146 + MSCs can significantly reduce CTX-1 and increase P1NP.

[0070] Example 6: In vitro "ovary" Research on the mechanism of the "bone axis" Western blot (antibody list in Table 1) and RT-qPCR (primer sequences in Table 2) were used to detect changes in estrogen signaling, angiogenesis, osteogenic / osteoclastogenesis, inflammation-related molecules and pathways in ovarian tissue, bone tissue and related cells, in order to elucidate the role of CD146. + The molecular mechanisms by which MSCs regulate the ovarian-bone axis. For example... Figure 28 , Figure 29 As shown, CD146 + MSCs significantly improved ovarian function in mice with premature ovarian failure, demonstrating good ovarian protection and repair effects. Molecular detection results showed that MSCs in the ovarian tissue after treatment... Amh, Fshr, Cyp19a1, Notch1, Bcl2 and Gpx4 The mRNA expression level of is elevated, while P53, P21, P16, Hmox1 and Bax The mRNA expression level of CD146 decreased, while the protein expression levels of p-VEGFR2, NOTCH1, and CD31 increased. These results suggest that CD146... + MSCs can promote local angiogenesis and microcirculation remodeling in the ovary by activating angiogenesis-related signaling pathways; alleviate granulosa cell senescence and apoptosis by upregulating anti-apoptotic and ovarian function-related factors and downregulating aging and pro-apoptotic factors; and enhance the ovarian tissue's antioxidant capacity by regulating the expression of oxidative stress / lipid peroxidation-related molecules, thereby delaying ovarian function decline, improving the follicular microenvironment, and promoting ovarian function recovery. This invention further demonstrates that CD146... + MSCs not only improve premature ovarian failure, but also significantly alleviate osteoporosis associated with premature ovarian failure. For example... Figure 30 , Figure 31 As shown, in the bone tissue after treatment Runx2, Alp, Opg, Esr1, Nfe2l2 and Hmox1 The mRNA expression level was significantly increased. Sost, Rank, P53 and Nox4 The mRNA expression level of CD146 was significantly decreased, while the expression levels of ALP, RUNX2, ERα, and OPG proteins were significantly increased. These results indicate that CD146... + MSCs can improve ovarian function, promote the recovery of ovarian secretory function, and increase estrogen levels in the body, thereby enhancing the positive regulatory effect of estrogen on bone metabolism, promoting osteoogenesis and inhibiting bone resorption, thus improving bone loss associated with premature ovarian failure; on the other hand, CD146 + MSCs can also directly reduce aging damage and oxidative stress levels in bone tissue, inhibit osteoclast activity, and promote bone formation by downregulating cellular senescence and oxidative stress-related factors. This indicates that CD146... + MSCs can effectively alleviate osteoporosis associated with premature ovarian failure through a dual mechanism of restoring ovarian endocrine function and directly improving the bone metabolic microenvironment, and have good application prospects in the prevention and treatment of premature ovarian failure and its secondary osteoporosis.

[0071] Table 1. Antibody List

[0072] Table 2. Primer Sequences

[0073] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A stem cell preparation for repairing the ovarian-bone vascular microenvironment, characterized in that, The stem cell preparation includes human umbilical cord mesenchymal stem cells that stably and highly express CD146; the angiogenesis and osteogenic abilities of the human umbilical cord mesenchymal stem cells are significantly higher than those of ordinary mesenchymal stem cells.

2. The stem cell preparation according to claim 1, characterized in that, The human umbilical cord mesenchymal stem cells mentioned are stable cells integrated with the CD146 gene via lentivirus-mediated transformation, and their angiogenesis and osteogenic abilities are significantly higher than those of ordinary mesenchymal stem cells.

3. The stem cell preparation according to claim 1, characterized in that, The cell concentration in the stem cell preparation is 0.5 × 10⁻⁶. 6 ~5×10 6 per mL.

4. The stem cell preparation according to claim 1, characterized in that, The cell survival rate in the stem cell preparation is not less than 90%.

5. The stem cell preparation according to claim 1, characterized in that, The human umbilical cord mesenchymal stem cells can simultaneously repair the ovarian microenvironment and the bone vascular microenvironment.

6. The use of a stem cell preparation according to any one of claims 1 to 5 in the preparation of a drug for treating osteoporosis associated with early-onset ovarian insufficiency.

7. The application according to claim 6, characterized in that, The stem cell preparation is administered via intravenous injection and can simultaneously repair ovarian damage, rebuild the bone vascular microenvironment, and improve bone quality.

8. A method for constructing an animal model of early-onset ovarian insufficiency combined with osteoporosis, characterized in that, Includes the following steps: Step 1: Preserve intact ovarian tissue in female mammals; Step 2: Continuous administration of chemotherapy drugs was used to induce ovarian dysfunction and bone vascular microenvironment damage phenotypes to obtain experimental animal models.

9. The construction method according to claim 8, characterized in that, The chemotherapy drug is cyclophosphamide.

10. The construction method according to claim 8, characterized in that, The damage to the bone vascular microenvironment manifests as decreased intraosseous vascular density, destruction of trabecular bone structure, and / or bone metabolic imbalance.

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