Clusterin-overexpressing engineered mesenchymal stem cells, exosomes, and preparation methods and applications thereof

Abstract

The present invention discloses an engineered mesenchymal stem cell overexpressing Clusterin, exosomes, and a preparation method and application thereof. The Clusterin gene is introduced into UC-MSCs via a lentiviral vector, so that the Clusterin protein is efficiently expressed in the cells. By combining ultrafiltration and differential centrifugation techniques, high-purity exosomes of these cells are obtained. UC-MSCs modified with the Clusterin gene not only enhance the anti-apoptosis and pro-proliferation effects of UC-MSCs in the treatment of premature ovarian failure (POF) and improve ovarian function, but also the exosomes derived therefrom have independent therapeutic potential.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority and benefits of Chinese invention patent application No. 202411287356.8 filed on September 13, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the technical field of genetic engineering and biomedical engineering, and in particular to an engineered mesenchymal stem cell overexpressing Clusterin, an exosome, and a preparation method and application thereof. Background Art

[0004] Premature Ovarian Failure (POF), also known as Primary Ovarian Insufficiency (POI), is a common endocrine disease. According to statistics, 1 in 250 women under the age of 35 suffer from POF, and 1 in 100 women under the age of 40 suffer from POF. The clinical characteristics of POF include menstrual disorders, decreased estrogen (E2) levels, increased follicle-stimulating hormone (FSH) levels, and a significant decrease in the number of follicles. These changes not only seriously affect women's fertility, but also cause health problems such as osteoporosis, cardiovascular disease, and menopausal syndrome. The causes of premature ovarian failure are complex and diverse, including genetic factors, autoimmune diseases, iatrogenic factors, and environmental pollutants. At present, a large number of studies have shown that cancer and its treatment methods seriously affect women's reproductive function. Anti-tumor drugs represented by alkylating agents have strong gonadal toxicity, which can directly damage ovarian cells and induce DNA damage, leading to rapid failure of ovarian function. POF has become one of the main sequelae of radiotherapy / chemotherapy in female cancer patients. Statistics show that the incidence of premature ovarian failure in female patients treated with alkylating agents is as high as 42% to 80%. These drugs not only affect the granulosa cells in the growing follicles, causing their abnormal apoptosis, leading to follicular atresia and primordial follicle depletion, but may also damage the interstitial and vascular systems of the ovaries, affect the blood supply and nutritional support of the ovaries, increase the level of oxidative stress in the ovaries and induce inflammatory responses. In addition, the pathological characteristics of premature ovarian failure caused by chemotherapy drugs also include significant atrophy of ovarian tissue, decreased number of follicles and increased fibrosis, and granulosa cell apoptosis is its main pathological feature. Studies have found that ovarian damage induced by chemotherapy drugs tends to worsen over time and leads to irreversible damage to ovarian function. Therefore, it is of great clinical significance to find a treatment method that can effectively repair ovarian damage caused by chemotherapy drugs and restore ovarian function.

[0005] At present, the treatment of POF mainly relies on hormone replacement therapy (HRT), which helps patients maintain secondary sexual characteristics and prevent osteoporosis and cardiovascular disease by supplementing exogenous estrogen and progesterone, but this method has significant limitations and risks. HRT cannot restore the normal function of the ovaries, can only partially relieve symptoms, and cannot improve the patient's fertility. In addition, patients need to take hormone drugs for a long time, which is easy to become dependent and may cause fluctuations in hormone levels. More seriously, HRT increases the risk of breast cancer, endometrial cancer and cardiovascular disease, especially when used for a long time, it also increases the risk of thrombosis, cancer, stroke and other complications.

[0006] In recent years, with the rapid development of regenerative medicine, stem cell therapy has attracted widespread attention as a new treatment for POF. Mesenchymal stem cells (MSCs), especially umbilical cord-derived mesenchymal stem cells (UC-MSCs), are considered to have great application prospects in ovarian tissue repair due to their wide sources, convenient collection, low immunogenicity and strong multidirectional differentiation potential. Studies have shown that UC-MSCs can promote the repair and regeneration of ovarian tissue by secreting a variety of bioactive factors, such as growth factors and cytokines. However, although UC-MSCs have shown certain repair effects in in vitro and in vivo experiments, they still have some significant limitations in clinical applications: (1) Low cell survival rate. In the complex microenvironment in the body, UC-MSCs are easily affected by oxidative stress, inflammatory response and immune rejection, resulting in low survival rate and limiting their effect in repairing ovarian tissue. (2) Limited anti-apoptosis ability: In chemotherapy-induced ovarian damage, apoptosis of ovarian granulosa cells is one of the main pathological characteristics of POF. UC-MSCs have shown a certain anti-apoptosis effect in the premature ovarian failure model, mainly by inhibiting the apoptosis of ovarian granulosa cells. However, studies have shown that this anti-apoptosis ability may not be sufficient to fully restore ovarian function in some cases, especially in severe ovarian damage. (3) Unstable function: The biological activity of UC-MSCs is easily affected by culture conditions, expansion generations and donor differences. There is uncertainty in its function, making it difficult to ensure the consistency and repeatability of clinical application effects.

[0007] Clusterin (CLU) is a highly conserved glycoprotein composed of two chains, α-clusterin (α-Clu) and β-clusterin (β-Clu), which are linked by five disulfide bonds. It is widely expressed in many different tissues and cell types. CLU protein has diverse functions and is involved in multiple biological processes including apoptosis, cell survival, cell adhesion, inflammatory response and tissue remodeling. However, it is still unclear whether Clusterin combined with UC-MSCs can be used as a new treatment for POF. Summary of the invention

[0008] The object of the present invention is to provide an engineered mesenchymal stem cell overexpressing Clusterin, exosomes, and a preparation method and application thereof, so as to solve the problems raised in the above-mentioned background technology.

[0009] To achieve the above object, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a method for preparing engineered mesenchymal stem cells overexpressing Clusterin, comprising the following steps:

[0011] S1: Isolating and subculturing human umbilical cord mesenchymal stem cells in vitro to obtain human umbilical cord mesenchymal stem cells;

[0012] S2: Construction of Clusterin overexpression lentiviral vector;

[0013] S3: The Clusterin overexpression lentiviral vector solution and the Polybrene solution were mixed and added to the mesenchymal stem cell complete medium containing human umbilical cord mesenchymal stem cells obtained in step S1 for viral infection. The culture medium was incubated at 37°C and 5% CO. 2 The cells were cultured in an incubator for 12 hours, and then the culture medium was replaced with new culture medium. After a further 48 hours of culture, the cells and culture supernatant were harvested, and proteins were extracted from the infected UC-MSCs. The overexpression of the CLU gene was detected by Western Blot, and the verified CLU gene-overexpressing UC-MSCs were expanded and cultured in large quantities in serum-free culture medium.

[0014] In one or more embodiments, in step S1, the in vitro separation and subculture of human umbilical cord mesenchymal stem cells comprises the following steps:

[0015] S11: Soak the fresh human umbilical cord in 75% alcohol solution for no less than 1 minute on a sterile operating table for preliminary disinfection to ensure that the solution completely covers the surface of the umbilical cord and achieves the disinfection effect. After the soaking is completed, rinse the umbilical cord with sterile saline to remove the alcohol residue, and use sterile scissors to cut the umbilical cord into small sections of about 2 cm in length. Then, peel off the arteries, veins and epidermis in the umbilical cord, and keep the Wharton's jelly in the middle;

[0016] S12: The peeled Wharton's jelly tissue was cut into small pieces with a side length of 3-5 mm. The cut tissue pieces were inoculated in a 100 mm culture dish. The spacing between the small pieces should be uniform, and the spacing between the small pieces should be 4-7 mm. An appropriate amount of mesenchymal stem cell complete culture medium was added to each dish and incubated at 37°C, 5% CO 2 The culture medium was changed every three days (carefully aspirate and discard the culture supernatant, and slowly add new mesenchymal stem cell complete culture medium along the wall). After 7-10 days of culture, the cells crawled out of the tissue mass to form primary cells P0;

[0017] S13: After the primary cells P0 reach a confluence of more than 85%, they are digested and harvested, specifically:

[0018] Discard the old culture medium in the culture dish and rinse gently with PBS twice to remove the residual culture medium and floating cells. Add an appropriate amount of stem cell mild digestion enzyme (Youkang, catalog number: NC1004.1) to each culture dish and place it at room temperature for digestion for about 3 minutes. Observe under a microscope. When most of the cells are discrete spheres, add 4 mL of complete culture medium to terminate the digestion. Gently blow and mix the cell suspension, and transfer it to a 50 mL centrifuge tube. Rinse the bottom of the culture dish again with PBS, collect all the cell suspension, transfer it to a centrifuge tube, centrifuge at 1300 rpm for 5 minutes, discard the supernatant, and then resuspend the precipitated cells in an appropriate amount of culture medium and count them. The cells harvested in this step are primary cells P0;

[0019] S14: Subculture of UC-MSCs, specifically:

[0020] The primary cells P0 were harvested and plated per cm 2 Inoculate 5000-6000 cells into a new T175 culture flask, add an appropriate amount of culture medium to 20 mL, shake gently to mix, and place in a constant temperature and humidity incubator. Continue to incubate at 5% CO 2, 37°C, and observe the growth status of cells every day until the cell confluence reaches 85%-90%, then digest the cells and pass them to the first generation of cells P1. The passaging operation uses the same steps and digestive enzymes as the primary generation. When the P1 cells reach 90% confluence, pass them to generate the second generation of cells P2. After the harvested P2 cells pass the quality inspection, they are packaged and frozen in liquid nitrogen to establish the main cell bank. The standard for judging the quality of P2 frozen cells is that the cell morphology is long spindle cells with uniform morphology that grow on the wall; and the positive expression rate of CD73, CD90 and CD105 is greater than or equal to 95%, and the positive expression rate of CD11b, CD19, CD34, CD45 or HLA-Ⅱ molecules should not exceed 2%; under in vitro differentiation induction conditions, it has the ability to differentiate into osteoblasts, chondrocytes and adipocytes; the infectious diseases and pathogenic microorganisms test results are all negative, and the endotoxin results are less than 0.25EU / mL. The P2 cells revived from the master cell bank continued to expand and culture, and were subcultured generation by generation to the fifth generation of cells P5. After each subculture, strict quality testing was performed to ensure that the cells had good proliferation ability and multidirectional differentiation potential. When the P5 cells reached the quality standards, they were packaged and frozen in liquid nitrogen to establish a working cell bank. The criteria for determining the quality of P5 cryopreserved cells are that the cell morphology is that the cell is an adherent, uniformly shaped long spindle cell; the positive expression rate of CD73, CD90 and CD105 is greater than or equal to 95%, and the positive expression rate of CD11b, CD19, CD34, CD45 or HLA-II molecules should not exceed 2%; under in vitro differentiation induction conditions, the cell has the ability to differentiate into osteoblasts, chondrocytes and adipocytes; the infectious disease and pathogenic microorganism test results are negative, and the endotoxin result is less than 0.25EU / mL; the inhibition rate of the cell pro-inflammatory lymphocyte Th1 is greater than 50%, the inhibition rate of Th17 is greater than 40%, and the promotion rate of the anti-inflammatory lymphocyte Treg is greater than 40%; the PBMC inhibition rate is greater than 50%.

[0021] In one or more embodiments, in step S2, the Clusterin overexpression lentiviral vector is constructed as follows:

[0022] The full-length sequence of the CLU gene (Gene ID: 1191) was obtained from the NCBI database, and the gene fragment was cloned into the lentiviral expression vector pLV3. The CLU gene was inserted using the multiple cloning site, and the 3Flag tag was ensured to construct the pLV3-CLU-3Flag plasmid. Subsequently, the constructed pLV3-CLU-3Flag plasmid was mixed with the auxiliary plasmid pMD2G (encoding VSV-G envelope protein) and psPAX2 (encoding viral packaging protein) in a ratio of 4:2:1 using EL transfection reagent and co-transfected into 293T cells for virus packaging. After 48 hours of transfection, the supernatant was collected and centrifuged at 300×g for 5 minutes to remove cell debris, followed by ultracentrifugation at 100,000×g for 2 hours to concentrate the virus particles. After the virus was concentrated, it was resuspended in PBS and stored at 4°C for infection of UC-MSCs.

[0023] In one or more embodiments, in step S3, the final concentration of the Polybrene is 5-8 μg / mL.

[0024] In a second aspect, the present invention provides an engineered mesenchymal stem cell overexpressing Clusterin obtained according to the preparation method of the first aspect.

[0025] In a third aspect, the present invention provides a method for preparing Clusterin-overexpressing engineered mesenchymal stem cell exosomes, comprising the following steps:

[0026] S1: amplifying and culturing the engineered mesenchymal stem cells overexpressing Clusterin according to the second aspect of the present invention, and collecting the cell culture supernatant;

[0027] S2: The cell culture supernatant collected in step S1 is purified by combining ultrafiltration and differential centrifugation technology to obtain engineered mesenchymal stem cell exosomes overexpressing Clusterin.

[0028] In one or more embodiments, in step S2, the purification step of combining ultrafiltration and differential centrifugation technology is as follows:

[0029] S21: Removing cell debris and suspended particles from the supernatant collected in S1: Place the supernatant collected in S1 in a 50 mL centrifuge tube, centrifuge at 300 x g for 10 min at 4 °C to remove cell debris and suspended particles. After centrifugation, transfer the supernatant to a new centrifuge tube;

[0030] S22: Ultrafiltration concentration: The supernatant was transferred into a 100 kDa MWCO ultrafiltration tube and centrifuged at 5,000 x g for 30 min at 4 °C. The concentrate after centrifugation, i.e. the liquid above the filter membrane, was the exosome-rich fraction.

[0031] S23: Isolation of exosomes by differential centrifugation: Divide the concentrated supernatant into equal amounts into ultracentrifuge tubes with a difference of no more than 0.02 g to ensure weight balance, adjust with PBS buffer, and then ultracentrifuge at 100,000 x g for 70 min at 4 °C to collect the exosome precipitate at the bottom of the centrifuge tube, gently pour out the supernatant to avoid disturbing the exosome precipitate; gently resuspend the exosome precipitate with 2 mL of sterile PBS buffer; to ensure the purity of the exosomes, centrifuge again at 100,000 x g for 70 min, remove the supernatant, and retain the exosome precipitate at the bottom.

[0032] In a fourth aspect, the present invention provides an engineered mesenchymal stem cell exosome overexpressing Clusterin obtained according to the preparation method of the second aspect.

[0033] In a fifth aspect, the present invention provides use of Clusterin, the Clusterin-overexpressing engineered mesenchymal stem cells described in the second aspect, or the Clusterin-overexpressing engineered mesenchymal stem cell exosomes described in the fourth aspect in the preparation of a drug for treating premature ovarian failure.

[0034] In one or more embodiments, the premature ovarian failure is premature ovarian failure caused by chemotherapy drugs.

[0035] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0036] 1. CLU gene-modified stem cells: The present invention introduces the CLU gene into UC-MSCs through a lentiviral vector, so that the CLU protein is efficiently expressed in the cells. The gene-modified UC-MSCs provided by the present invention have stronger therapeutic effects in tissue repair and functional recovery than traditional unmodified cells. Through histological analysis, UC-MSCs overexpressing the CLU gene significantly increased the number of follicles, reduced atretic follicles, and significantly increased the weight of the ovaries. The structure and function of the ovaries have been fully repaired; the effect of inhibiting the apoptosis of ovarian granulosa cells is also more significant. Through ELISA experiments to detect estradiol (E2) and follicle-stimulating hormone (FSH) in serum, it was found that UC-MSCs overexpressing the CLU gene can significantly increase the level of E2, more significantly reduce the level of FSH, restore the hormone secretion function of the ovaries, and improve the hormone imbalance caused by premature ovarian failure.

[0037] 2. Cell-derived exosomes: In addition to modified UC-MSCs, another key innovation of the present invention is to use exosomes secreted by these cells as an independent treatment method. Exosomes act as nano-scale delivery carriers between cells, carrying CLU proteins and other important bioactive molecules, and are delivered directly to the damaged parts of the ovary, enhancing the regeneration and repair ability of cells. Exosomes have low immunogenicity and high tissue targeting, and can effectively promote the functional recovery of damaged ovarian tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 .Display the microscopic observation results of UC-MSCs P0 cells;

[0039] Figure 2 .Display the microscopic observation results of UC-MSCs P5 cells;

[0040] Figure 3 .The identification results of UC-MSCs surface markers;

[0041] Figure 4 .Indicator diagram for establishing the ovarian granulosa cell injury model;

[0042] Figure 5 . Detection of the effect of CLU recombinant protein on apoptosis and proliferation of KGN cells;

[0043] Figure 6 .The results of MSC-CM inhibiting apoptosis and promoting proliferation of KGN cells;

[0044] Figure 7 .The results of MSC-CM exerting anti-apoptosis and promoting proliferation effects on ovarian granulosa cells through EVs;

[0045] Figure 8 . The result of knocking down CLU protein expression reversed the anti-apoptotic effect of MSC-CM;

[0046] Fig. 9 . The result of knocking down CLU protein expression reversed the proliferation-promoting effect of MSC-CM;

[0047] Fig.10 .Verification diagram of CLU gene overexpression;

[0048] Fig.11 .The results of the enhanced ability of gene-modified MSC-CM to inhibit apoptosis of ovarian granulosa cells;

[0049] Fig.12 .The results of the changes in the ovarian tissue structure of mice induced by CTX / BUS over time;

[0050] Fig.13.Results of UC-MSCs overexpressing CLU protein in treating POF mice;

[0051] Fig.14 .Results of NTA detection of the size and distribution of extracted EVs;

[0052] Fig.15 .The results of transmission electron microscopy observation of EVs morphology and size;

[0053] Fig.16 .Identification of CLU protein overexpression and surface markers in EVs;

[0054] Fig.17 .The results of exosomes extracted from UC-MSCs overexpressing CLU protein in treating POF mice; DETAILED DESCRIPTION

[0055] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0056] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0057] Example 1: Isolation and identification of human umbilical cord mesenchymal stem cells UC-MSCs

[0058] This example describes the isolation, culture, passage and identification methods of human umbilical cord mesenchymal stem cells (UC-MSCs), aiming to obtain high-purity and high-activity UC-MSCs for subsequent gene modification and clinical application.

[0059] 1.1 Acquisition and processing of human umbilical cord tissue

[0060] Fresh human umbilical cords were collected from a tertiary hospital to ensure that the results of microbial tests (including bacteria, fungi, mycoplasma and viruses, etc.) were negative before the samples entered the laboratory. The umbilical cord was soaked in 75% alcohol solution for no less than 1 minute for preliminary disinfection to ensure that the solution completely covered the surface of the umbilical cord and achieved the disinfection effect. After soaking, the umbilical cord was rinsed with sterile saline to remove alcohol residues. Use sterile scissors to cut the umbilical cord into small pieces of about 2 cm long. Next, carefully peel off the arteries, veins and epidermis in the umbilical cord, and retain the Wharton's jelly in the middle. This operation must be performed on a sterile operating table to avoid contamination. The peeled Wharton's jelly tissue was cut into small pieces with a side length of 3-5 mm to facilitate subsequent cell separation and culture operations. The cut tissue blocks were inoculated in 100 mm culture dishes. The spacing between small blocks should be uniform, and the spacing between small blocks is 4-7 mm. 10 mL of mesenchymal stem cell complete culture medium was added to each dish and incubated at 37 ° C, 5% CO 2 The cells were cultured under the conditions of 5% paraformaldehyde until they crawled out. The medium was changed every three days. After 7-10 days of culture, the cells crawled out of the tissue mass and formed primary cells (P0).

[0061] 1.2 Primary culture and subculture of UC-MSCs

[0062] Slowly add an appropriate amount of mesenchymal stem cell complete medium to each culture dish to make the total volume of complete medium reach about 4 mL. This operation needs to be done slowly to avoid strong impact that may cause the tissue blocks to float or shift. Place the culture dish in 5% CO 2 , and culture in a 37°C incubator for 24 h. Figure 1 :Show the microscopic observation results of UC-MSCs P0 cells.

[0063] Before subculturing, it was observed that P0 cells formed multiple large clones in the culture dish. The distribution of these clones and the morphology of the cells could be clearly seen under a microscope. The cells showed good adherent growth ability and high activity.

[0064] When the cell density of the primary culture (P0) reaches more than 85%, cell digestion and harvesting can be performed. Discard the old culture medium in the culture dish and gently rinse twice with PBS to remove residual culture medium and floating cells. Add an appropriate amount of stem cell mild digestion enzyme (Youkang, NC1004.1) to each culture dish and place it at room temperature for about 3 minutes. Observe under a microscope and when most of the cells are discrete spheres, add 4mL of mesenchymal stem cell complete culture medium to terminate digestion. Gently blow the cell suspension to mix and transfer it to a 50mL centrifuge tube. Rinse the bottom of the culture dish with PBS again, collect all the cell suspensions, and transfer them to a centrifuge tube. Centrifuge at 1300rpm for 5 minutes and discard the supernatant. Then, resuspend the precipitated cells in an appropriate amount of mesenchymal stem cell complete culture medium and count them. The cells harvested in this step are primary cells (P0 generation), and the total number of cells can reach millions.

[0065] The harvested primary cells (P0) were plated per cm 2 Inoculate 5000-6000 cells into a new T175 culture flask, add an appropriate amount of culture medium to 20 mL, shake gently to mix, and place in a constant temperature and humidity incubator. Continue to incubate at 5% CO 2, 37°C. Observe the growth status of cells every day until the cell confluence reaches 85%-90%, then digest the cells and pass them to the first generation of cells (P1). The passaging operation uses the same steps and digestive enzymes as the primary generation to ensure that the cells maintain good activity and proliferation ability. When the P1 cells reach 90% confluence, pass the passaging operation to generate the second generation of cells (P2). After the harvested P2 cells pass the quality inspection, they are packaged and frozen in liquid nitrogen to establish a master cell bank. The standard for judging the quality of P2 frozen cells is that the cell morphology is a long spindle-shaped cell with uniform morphology that grows adherently; and the positive expression rate of CD73, CD90 and CD105 is greater than or equal to 95%, and the positive expression rate of CD11b, CD19, CD34, CD45 or HLA-Ⅱ molecules should not exceed 2%; under in vitro differentiation induction conditions, it has the ability to differentiate into osteoblasts, chondrocytes and adipocytes; the infectious diseases and pathogenic microorganisms test results are all negative, and the endotoxin results are less than 0.25EU / mL. Generally, one umbilical cord tissue can culture about (0.5-1.5)×10^9 master cell bank cells. The P2 cells revived from the master cell bank continue to expand and culture, and are subcultured to the fifth generation of cells (P5). After each subculture, strict quality testing is carried out to ensure that the cells have good proliferation ability and multidirectional differentiation potential. When the P5 cells meet the quality standards, they are packaged and frozen in liquid nitrogen to establish a working cell bank. The quality criteria for determining the quality of P5 cryopreserved cells are that the cell morphology is long spindle cells that grow adherently and have uniform morphology; the positive expression rate of CD73, CD90 and CD105 is greater than or equal to 95%, and the positive expression rate of CD11b, CD19, CD34, CD45 or HLA-Ⅱ molecules should not exceed 2%; under in vitro differentiation induction conditions, it has the ability to differentiate into osteoblasts, chondrocytes and adipocytes; the infectious diseases and pathogenic microorganisms test results are all negative, and the endotoxin results are less than 0.25EU / mL; the inhibition rate of the cell pro-inflammatory lymphocyte Th1 is greater than 50%, the inhibition rate of Th17 is greater than 40%, and the promotion rate of anti-inflammatory lymphocyte Treg is greater than 40%; the PBMC inhibition rate is greater than 50%. Generally, one umbilical cord tissue can culture about (0.5-1.5)×10^12 working cell bank cells. Figure 2 :Shows the microscopic observation results of UC-MSCs P5 cells.

[0066] 1.3 Identification of UC-MSCs

[0067] In order to confirm the characteristics of the isolated UC-MSCs, flow cytometry was used to identify their surface markers. The cells were cultured to the fourth generation (P4). When the density reached 80%, 0.125% trypsin was used for digestion. After terminating the digestion, the cell suspension was collected. The cell concentration was adjusted to 5×10 5The cells were stained with antibodies (such as CD105, CD73, CD90, etc.) and then detected by flow cytometry.

[0068] like Figure 3 The results of the identification of UC-MSCs surface markers showed that UC-MSCs showed a high positive rate (>99%) on CD90, CD105 and CD73 markers, while the expression of negative markers such as CD45, CD19, CD34 and HLA-DR was almost zero (<1%). These results confirmed that the isolated UC-MSCs have typical mesenchymal stem cell characteristics.

[0069] Example 2: CLU protein inhibits apoptosis of ovarian granulosa cells and promotes their proliferation

[0070] Clusterin (CLU) is a widely expressed glycoprotein that can be synthesized in a variety of cell types, including hepatocytes, kidney cells, nerve cells, immune cells, etc. Its expression level will increase significantly under stress conditions. CLU can be released outside the cell through the cell's secretory pathway and exists in body fluids such as blood, urine, and cerebrospinal fluid. It is involved in a variety of physiological processes, such as anti-apoptosis, immune regulation, and cell protection, and is therefore considered to play an important role in neurodegenerative diseases, cancer, and cardiovascular diseases. In order to study the protective effect of CLU protein in ovarian granulosa cell injury induced by chemotherapy drugs, the Clusterin recombinant protein used in this study was purchased from MedChemExpress, USA, and an in vitro cell injury model induced by chemotherapy drugs was also established. Since CTX (cyclophosphamide) is inactive under in vitro conditions and its toxic effects on cells cannot be directly simulated, nitrogen mustard (NM), which has a similar chemical structure to CTX and is directly active in vitro, was selected to treat ovarian granulosa cells (KGN cells), thereby effectively simulating the cytotoxic effect of CTX. (Deng T, He J, Yao Q, et al. Human umbilical cord mesenchymal stem cells improve ovarian function inchemotherapy-induced premature ovarian failure mice through inhibiting apoptosis and inflammation via a paracrine mechanism [J]. ReproductiveSciences, 2021, 28: 1718-1732.).

[0071] In the experiment, KGN cells were cultured in DMEM medium (gibco, catalog number: 8123324) (DMEM complete medium) containing 5% fetal bovine serum (FBS) to the logarithmic growth phase to ensure cell viability and proliferation ability. Subsequently, the cells were exposed to NM solution with a final concentration of 2.5 μg / mL and incubated for 48 hours. Nitrogen mustard, as an alkylating agent, can directly react with DNA, causing DNA damage and cross-linking, and then triggering cell cycle arrest and apoptosis. It successfully induced damage and apoptosis of ovarian granulosa cells, and the apoptosis rate of KGN cells in each group was detected by flow cytometry (Annexin V / PI staining method). Figure 4 The cell injury model was successfully established.

[0072] To evaluate the anti-apoptotic and pro-proliferative functions of CLU protein, different concentrations of exogenous CLU recombinant protein (50 ng / mL and 100 ng / mL) were added to KGN cells treated with NM. The experiment was divided into the following groups: blank control NC group, NM control group (only NM treatment), NM+CLU-50 ng / mL group and NM+CLU-100 ng / mL group. The specific operation was as follows: KGN cells were cultured in DMEM complete medium until the logarithmic growth phase, the culture supernatant (KGN-CM) was collected, and the culture medium was replaced with serum-free DMEM medium for starvation treatment, and the cells were incubated at 37°C, 5% CO 2 Incubate for 6 hours. Replace each well with the corresponding group culture medium (2 mL). Except for the NC group, NM (final concentration 2.5 μg / mL) was added to the other groups at the same time as the culture medium was replaced. Incubate at 37°C, 5% CO 2 The cells were cultured in the incubator for 48 h and then harvested.

[0073] The following groups and corresponding media were used:

[0074] (1) NC group: DMEM complete medium.

[0075] (2) NM group: 50% DMEM complete medium + 50% KGN-CM.

[0076] (3) NM+CLU-50 ng / mL group: 50% DMEM complete medium+50% KGN-CM+CLU recombinant protein (50 ng / mL).

[0077] (4) NM+CLU-100 ng / mL group: 50% DMEM complete medium+50% KGN-CM+CLU recombinant protein (100 ng / mL).

[0078] After treatment, flow cytometry (Annexin V / PI staining method) was used to detect the apoptosis rate of KGN cells in each group. Figure 5The results showed that the apoptosis rate of KGN cells in the two groups with the addition of CLU recombinant protein was significantly reduced compared with the control group treated with NM only. In particular, the apoptosis rate of cells in the CLU-100ng / mL treatment group decreased significantly from 30.1±2.9% to 19.7±1.2% (p<0.05), indicating that CLU protein has a significant inhibitory effect on apoptosis induced by nitrogen mustard. At the same time, in order to further verify the proliferation function of CLU protein, CCK-8 cell proliferation experiment was performed. The results showed that the proliferation rate of KGN cells in the CLU-50ng / mL and CLU-100ng / mL treatment groups was significantly higher than that in the control group, among which the proliferation rate of the CLU-100ng / mL treatment group increased significantly from 35.4±13.0% to 61.3±9.2% (p<0.05). These data show that CLU protein can not only effectively inhibit the apoptosis of ovarian granulosa cells induced by chemotherapeutic drugs, but also significantly promote cell proliferation.

[0079] In summary, the results of this experiment show that CLU protein has significant anti-apoptosis and pro-proliferation effects in the ovarian granulosa cell injury model induced by the chemotherapy drug nitrogen mustard. This protective effect of CLU protein provides its application value in the treatment of ovarian function damage caused by chemotherapy.

[0080] Example 3: MSC-CM inhibits apoptosis and promotes proliferation of ovarian granulosa cells

[0081] To investigate the inhibitory effect of umbilical cord mesenchymal stem cell culture supernatant (MSC-CM) on chemotherapeutic drug-induced apoptosis of ovarian granulosa cells (KGN cells) and its promoting effect on cell proliferation. The protective effect of MSC-CM in an in vitro injury model was evaluated by adding MSC-CM to KGN cells. After the injury model was established, the damaged KGN cells were treated with MSC-CM and 293T cell culture supernatant (DMEM complete medium culture, 293T-CM) to compare their effects on cell apoptosis and proliferation.

[0082] The specific operation was as follows: KGN cells were cultured in DMEM complete medium until the logarithmic growth phase, and the medium was replaced with serum-free DMEM medium for starvation treatment. 2 Incubate for 6 hours. Replace each well with the corresponding group culture medium (2 mL). Except for the NC group, NM (final concentration 2.5 μg / mL) was added to the other groups at the same time as the culture medium was replaced. Incubate at 37°C, 5% CO 2 The cells were cultured in the incubator for 48 h and then harvested.

[0083] The following groups and corresponding media were used:

[0084] (1) NC group: DMEM complete medium.

[0085] (2) NM group: 50% DMEM complete medium + 50% KGN-CM.

[0086] (3) NM+MSC-CM group: 50% DMEM complete medium + 50% MSC-CM.

[0087] (4) NM+293T-CM group: 50% DMEM complete medium + 50% 293T-CM.

[0088] Cell apoptosis was detected by flow cytometry (Annexin V / PI staining method). Figure 6 The results showed that the apoptosis rate of KGN cells treated with NM was significantly increased without other treatments (40.4±3.2%). In contrast, MSC-CM treatment significantly reduced the apoptosis rate of KGN cells induced by NM (20.6±2.0%, p<0.01), while the apoptosis rate of cells in the 293T-CM treatment group did not change significantly (41.5±3.6%), indicating that MSC-CM has a significant anti-apoptotic effect. In addition, Western Blot analysis further verified these results. In the NM+MSC-CM treatment group, the expression level of the anti-apoptotic protein BCL-XL in KGN cells was significantly upregulated, while the expression of the pro-apoptotic protein cleaved-caspase3 was significantly downregulated. In contrast, the NM+293T-CM group did not show such changes. In order to evaluate the effect of MSC-CM on the proliferation of KGN cells, a CCK-8 cell proliferation experiment was performed. The results showed that compared with the control group, MSC-CM treatment significantly promoted the proliferation of KGN cells (43.6±3.4% vs. 26.8±6.2%, p<0.01), while 293T-CM treatment did not show a significant pro-proliferation effect.

[0089] In summary, these results indicate that MSC-CM can effectively inhibit NM-induced apoptosis and promote proliferation of KGN cells. Compared with 293T-CM, MSC-CM exhibits unique advantages in cell protection and repair.

[0090] Example 4: MSC-CM inhibits ovarian granulosa cell apoptosis and promotes its proliferation through exosomes (EVs)

[0091] To clarify whether MSC-CM (conditioned medium containing UC-MSCs-derived EVs) exerts its biological effects through EVs, we used GW4869 (an exosome inhibitor) to inhibit the secretion of EVs from UC-MSCs and detected the changes in the role of MSC-CM in inhibiting apoptosis and promoting proliferation of KGN cells.

[0092] At the beginning of the experiment, UC-MSCs were pretreated with GW4869 to inhibit the secretion of EVs. Specifically, UC-MSCs were cultured in a medium containing GW4869 (10 μM, 24 h) to inhibit the release of EVs. Afterwards, the culture supernatant of UC-MSCs treated with GW4869 (referred to as GW4869-CM) and the culture supernatant of UC-MSCs not treated with GW4869 (referred to as MSC-CM) were collected for subsequent experiments. The KGN cells treated with NM were divided into three groups for treatment: control group (NM treatment only), MSC-CM group (NM+MSC-CM) and GW4869-CM group (NM+GW4869-CM). The apoptosis of KGN cells in each group was detected by flow cytometry (Annexin V / PI staining method).

[0093] like Figure 7 The results showed that compared with the control group, MSC-CM significantly inhibited NM-induced apoptosis of KGN cells (23.5±3.1% vs. 52.4±5.1%). However, when the EVs secretion of UC-MSCs was inhibited by GW4869, the apoptosis rate of the GW4869-CM-treated group was significantly increased, similar to that of the control group, indicating that the anti-apoptotic effect of MSC-CM on KGN cells depends on the presence of EVs. Further Western Blot analysis results showed that in the MSC-CM group, the expression of the anti-apoptotic protein BCL-XL in KGN cells was significantly upregulated, and the expression of the pro-apoptotic protein cleaved-caspase3 was significantly downregulated. In contrast, the regulatory effect of the GW4869-CM group on BCL-XL and cleaved-caspase3 in KGN cells was significantly weakened, further supporting the key role of EVs in the anti-apoptotic function of MSC-CM. In addition, CCK-8 cell proliferation assay showed that the MSC-CM group significantly promoted the proliferation of KGN cells (47.1±3.5%), while the proliferation rate of the GW4869-CM group was significantly reduced (31.6±5.1%, p<0.05), which was similar to the proliferation level of the control group. This indicates that the proliferation-promoting effect of MSC-CM also mainly depends on the presence of EVs.

[0094] In summary, the experimental results showed that EVs derived from UC-MSCs played a key role in the anti-apoptotic and pro-proliferative effects of MSC-CM. Inhibition of EV secretion by GW4869 significantly weakened the inhibitory effect of MSC-CM on NM-induced KGN cell apoptosis and the promotion of cell proliferation. These findings confirmed that EVs are the main active ingredients of MSC-CM that play a protective role in the chemotherapeutic drug injury model, providing important evidence for the role of exosomes in intercellular signaling and therapeutic applications.

[0095] Example 5: Knockdown of CLU protein expression reverses the anti-apoptotic and pro-proliferative effects of MSC-CM

[0096] In order to verify the critical role of CLU protein in the anti-apoptosis and pro-proliferation effects of KGN cells in MSC-CM, the present invention knocks down the expression of CLU protein in UC-MSCs by siRNA technology. The specific steps are as follows: design and synthesize siRNA: siNC 5′-TTCTCCGAACGTGTCACGT-3′ and siRNA-CLU 5′-GCATCATAGACGAGCTCTT-3′. Then CLU-specific siRNA (or negative control siRNA, siNC) is transfected into UC-MSCs, and the culture medium is collected 48 hours after transfection to obtain the conditioned medium after CLU knockdown (siCLU-CM) and the conditioned medium of the control group (siNC-CM), respectively. Subsequently, the damaged KGN cells in the ovarian granulosa cell injury model induced by chemotherapy drugs were treated with siNC-CM and siCLU-CM, respectively, to evaluate whether CLU protein is a key component of MSC-CM to exert anti-apoptosis and pro-proliferation effects.

[0097] The apoptosis of KGN cells was detected by flow cytometry. Figure 8 The results showed that under control conditioned medium (siNC-CM) treatment, the apoptosis rate of KGN cells was significantly reduced (19.4±0.33%), indicating that MSC-CM has an anti-apoptotic effect. However, after CLU protein knockdown (siCLU-CM treatment group), the apoptosis rate of KGN cells was significantly increased to 27.0±1.6% (p<0.05), indicating that knocking down CLU protein expression reversed the anti-apoptotic effect of MSC-CM. The CCK-8 cell proliferation experiment verified the pro-proliferation effect of CLU protein in MSC-CM. The experimental results are as follows Fig. 9 The results showed that treatment with control conditioned medium (siNC-CM) significantly promoted the proliferation of KGN cells (70.6±7.0%), while in the siCLU-CM treatment group, the cell proliferation rate significantly decreased to 56.5±7.5% (p<0.05), indicating that knocking down CLU protein expression reversed the pro-proliferation effect of MSC-CM.

[0098] Example 6: Gene modification of CLU gene overexpression in umbilical cord mesenchymal stem cells and preparation of injection solution

[0099] This example introduces the gene modification process of CLU gene overexpressing UC-MSCs, including the construction of CLU gene lentiviral vector, virus packaging and purification, CLU gene infection and screening of UC-MSCs, and preparation of CLU gene overexpressing UC-MSCs injection.

[0100] 1. Construction and packaging of lentiviral vector of CLU gene

[0101] a. Vector construction

[0102] First, the full-length sequence of the CLU gene was obtained from the NCBI database (Gene ID: 1191), and the gene fragment was cloned into the lentiviral expression vector pLV3. The CLU gene was inserted using the multiple cloning site, and the 3Flag tag was ensured to facilitate subsequent detection. As a control, the pLV3-GFP plasmid was also constructed. After the pLV3-CLU-3Flag plasmid was constructed, other reagents were prepared: pMD2G plasmid, psPAX2 plasmid, OPTI-MEM medium (Merco-sigma, USA), and EL transfection reagent (Beijing Quanshijin Company). Subsequently, using the EL transfection reagent, the constructed pLV3-CLU-3Flag plasmid or pLV3-GFP plasmid was mixed with the auxiliary plasmid pMD2G (encoding VSV-G envelope protein) and psPAX2 (encoding viral packaging protein) in a ratio of 4:2:1 and co-transfected into 293T cells for viral packaging. Nucleotide sequence of the Clusterin gene (Nucleotide Sequence (1350nt)):

[0103] ATGATGAAGACTCTGCTGCTGTTTGTGGGGCTGCTGCTGACCTGGGAGAGTGGGCAGGTCCTGGGGGACC

[0104] AGACGGTCTCAGACAATGAGCTCCAGGAAATGTCCAATCAGGGAAGTAAGTACGTCAATAAGGAAATTCA

[0105] AAATGCTGTCAACGGGGTGAAACAGATAAAGACTCTCATAGAAAAAACAAACGAAGAGCGCAAGACACTG

[0106] CTCAGCAACCTAGAAGAAGCCAAGAAGAAGAAAGAGGATGCCCTAAATGAGACCAGGGAATCAGAGACAA

[0107] AGCTGAAGGAGCTCCCAGGAGTGTGCAATGAGACCATGATGGCCCTCTGGGAAGAGTGTAAGCCCTGCCT

[0108] GAAACAGACCTGCATGAAGTTCTACGCACGCGTCTGCAGAAGTGGCTCAGGCCTGGTTGGCCGCCAGCTT

[0109] GAGGAGTTCCTGAACCAGAGCTCGCCCTTCTACTTCTGGATGAATGGTGACCGCATCGACTCCCTGCTGG

[0110] AGAACGACCGGCAGCAGACGCACATGCTGGATGTCATGCAGGACCACTTCAGCCGCGCGTCCAGCATCAT

[0111] AGACGAGCTCTTCCAGGACAGGTTCTTCACCCGGGAGCCCCAGGATACCTACCACTACCTGCCCTTCAGC

[0112] CTGCCCCACCGGAGGCCTCACTTCTTCTTTCCCAAGTCCCGCATCGTCCGCAGCTTGATGCCCTTCTCTC

[0113] CGTACGAGCCCCTGAACTTCCACGCCATGTTCCAGCCCTTCCTTGAGATGATACACGAGGCTCAGCAGGC

[0114] CATGGACATCCACTTCCATAGCCCGGCCTTCCAGCACCCGCCAACAGAATTCATACGAGAAGGCGACGAT

[0115] GACCGGACTGTGTGCCGGGAGATCCGCCACAACTCCACGGGCTGCCTGCGGATGAAGGACCAGTGTGACA

[0116] AGTGCCGGGAGATCTTGTCTGTGGACTGTTCCACCAACAACCCCTCCCAGGCTAAGCTGCGGCGGGAGCT

[0117] CGACGAATCCCTCCAGGTCGCTGAGAGGTTGACCAGGAAATACAACGAGCTGCTAAAGTCCTACCAGTGG

[0118] AAGATGCTCAACACCTCCTCCTTGCTGGAGCAGCTGAACGAGCAGTTTAACTGGGTGTCCCGGCTGGCAA

[0119] ACCTCACGCAAGGCGAAGACCAGTACTATCTGCGGGTCACCACGGTGGCTTCCCACACTTCTGACTCGGA

[0120] CGTTCCTTCCGGTGTCACTGAGGTGGTCGTGAAGCTCTTTGACTCTGATCCCATCACTGTGACGGTCCCT

[0121] GTAGAAGTCTCCAGGAAGAACCCTAAATTTATGGAGACCGTGGCGGAGAAAGCGCTGCAGGAATACCGCA

[0122] AAAAGCACCGGGAGGAGTGA

[0123] Amino acid sequence of Clusterin protein:

[0124] MMKTLLLFVGLLLTWESGQVLGDQTVSDNELQEMSNQGSKYVNKEIQNAVNGVKQIKTLIEKTNEERKTLLSNLE

[0125] EAKKKKEDALNETRESETKLKELPGVCNETMMALWEECKPCLKQTCMKFYARVCRSGSGLVGRQLEEFLNQSSPF

[0126] YFWMNGDRIDSLLENDRQQTHMLDVMQDHFSRASSIIDELFQDRFFTREPQDTYHYLPFSLPHRRPHFFFPKSRI

[0127] VRSLMPFSPYEPLNFHAMFQPFLEMIHEAQQAMDIHFHSPAFQHPPTEFIREGDDDRTVCREIRHNSTGCLRMKD

[0128] QCDKCREILSVDCSTNNPSQAKLRRELDESLQVAERLTRKYNELLKSYQWKMLNTSSLLEQLNEQFNWVSRLANL

[0129] TQGEDQYYLRVTTVASHTSDSDVPSGVTEVVVKLFDSDPITVTVPVEVSRKNPKFMETVAEKALQEYRKKHREE

[0130] b. Virus Collection and Purification

[0131] 48 h after transfection, the culture supernatant was collected and centrifuged at 300 x g for 5 min to remove cell debris. The virus particles were then concentrated by ultracentrifugation at 100,000 x g for 2 h. After the virus was concentrated, it was resuspended in PBS and stored at 4 °C for infection of UC-MSCs.

[0132] 2. Infection of CLU gene and screening of gene-modified UC-MSCs

[0133] a. Viral infection

[0134] Reagents: Polybrene (Wuhan Yishen Biotechnology Co., Ltd.), UC-MSCs (umbilical cord mesenchymal stem cells).

[0135] The packaged pLV3-CLU-3Flag and pLV3-GFP lentiviral particles were used to infect UC-MSCs respectively. During the infection process, 5-8 μg / mL of Polybrene was added to improve the infection efficiency. After the virus particles were mixed with Polybrene, they were added to the complete mesenchymal stem cell culture medium of UC-MSCs and incubated at 37°C and 5% CO 2 The cells were cultured for 12 h under the same conditions, and then the culture medium was replaced with fresh medium. After a further 48 h of culture, the cells and culture supernatant were harvested and aliquoted and frozen at -80°C.

[0136] b. Verification of CLU gene overexpression

[0137] Reagents: Flag antibody, Western Blot reagent.

[0138] For infected UC-MSCs, the overexpression of CLU gene was detected by Western Blot. After protein extraction, the Flag tag expression in cells was detected using anti-Flag antibody to verify whether CLU protein was successfully overexpressed. Fig.10 The results showed that these cells significantly expressed Flag-tagged protein, proving that the CLU gene was successfully overexpressed.

[0139] 3. Preparation of CLU gene overexpression UC-MSCs injection

[0140] a. Cell Expansion and Collection

[0141] The verified CLU gene overexpressing UC-MSCs were cultured in a large number in a complete mesenchymal stem cell culture medium. When the number of cells reached the required standard for treatment, the cells and culture supernatant (CLU-CM) were collected. The collected cells were washed with PBS to remove the residual culture medium and other impurities.

[0142] b. Preparation of injection solution

[0143] After washing, count the cells and suspend them in PBS solution to prepare a sterile cell suspension. Adjust the cell concentration to ensure that each ml of injection solution contains 2x10 7 UC-MSCs cells are used to make the final CLU gene overexpression UC-MSCs injection. The preparation process of the CLU gene overexpression UC-MSCs injection undergoes strict aseptic operation and quality control and is stored under low temperature conditions to ensure cell activity and safety. During the preparation of the injection, the trypan blue staining method is used to detect cell survival rate to ensure that the cell survival rate is above 90%. At the same time, the injection must undergo sterility testing to ensure that there is no bacterial, fungal and mycoplasma contamination.

[0144] Example 7: Genetically modified MSC-CM enhances the ability to inhibit apoptosis of ovarian granulosa cells

[0145] In this example, UC-MSCs modified by CLU gene (see Example 6 for details) were used to prepare CLU-CM to treat KGN cells induced by NM damage (see Example 2 for details) to evaluate its protective effect in an in vitro damage model. The experiment set up a control group (NC), an NM damage group (NM), an NM+GFP-CM group (GFP-CM of unmodified UC-MSCs was added while the cells were damaged), and an NM+CLU-CM group (CLU-CM of UC-MSCs modified by CLU gene was added while the cells were damaged) to compare their effects on cell apoptosis. After 48h treatment, flow cytometry (Annexin V / PI staining method) was used to detect the apoptosis of KGN cells. Fig.11 As shown, CLU-modified MSC culture supernatant (CLU-CM) significantly reduced the apoptosis rate of KGN cells, and the effect was better than that of the unmodified GFP-CM group.

[0146] Example 8: Establishment of POF mouse model

[0147] The present invention uses cyclophosphamide (CTX) and busulfan (BUS) to induce a POF mouse model. The specific steps are as follows:

[0148] 1. Experimental Animals and Grouping

[0149] Seventy 7-week-old C57BL / 6 female mice were randomly divided into two groups, NC group (normal control group) and POF group (premature ovarian failure model group), with 35 mice in each group.

[0150] 2. Drug treatment

[0151] The mice in the POF group were intraperitoneally injected with BUS (30 mg / kg) and CTX (120 mg / kg) according to their body weight to induce premature ovarian failure, while the mice in the NC group were injected with an equal volume of PBS buffer as a control.

[0152] 3. Model Validation

[0153] On days 3, 6, 9, 12, 15, 18, and 21 after drug injection, 5 mice in each group were killed, and eye blood and bilateral ovaries were obtained for histological analysis and hormone level detection. Fig.12 As shown in the figure, HE staining results showed that over time, the ovarian tissue of mice in the POF group was significantly atrophied, and the number of follicles was significantly reduced, especially on day 9, the number of primordial follicles, primary follicles, and secondary follicles was significantly reduced, while the number of atretic follicles was significantly increased. These results showed that the POF model was successfully established.

[0154] Example 9: Application of Genetically Modified Umbilical Cord Mesenchymal Stem Cells in the Treatment of POF

[0155] In this example, the effect of umbilical cord mesenchymal stem cells (UCMSCs-CLU) with overexpression of CLU gene in the treatment of premature ovarian failure (POF) was studied, and the therapeutic effects of UCMSCs-CLU and ordinary UCMSCs in the POF mouse model induced by cyclophosphamide (CTX) and busulfan (BUS) were compared. 25 7-week-old C57BL / 6 female mice were randomly divided into 5 groups, with 5 mice in each group: NC group (healthy control group), POF group (receiving PBS injection), POF+MSCs (receiving uninfected UC-MSCs treatment), POF+MSCs (GFP) treatment group (receiving control UC-MSCs infected with GFP) and POF+MSCs (CLU) treatment group (receiving CLU gene overexpression UC-MSCs treatment). On the 6th day after the mice received intraperitoneal injection of CTX / BUS chemotherapy drugs, each mouse was injected with 200 μL of the corresponding MSCs (2×10 7 After the experiment, the number of follicles, ovarian weight, serum hormone levels, ovarian tissue repair, granulosa cell apoptosis and proliferation of each group of mice were measured and analyzed.

[0156] After two weeks of treatment, the estrous cycles of all mice were detected, and the mice were killed when they were in diestrus, and ovarian samples were collected and analyzed. The results of HE staining of the ovarian structure showed that the ovarian structure of POF mice treated with UC-MSCs was repaired to a certain extent ( Fig.13 A), the number of primordial follicles, primary follicles and secondary follicles in the ovaries of mice in the treatment group were significantly higher than those in the POF group, and the number of atretic follicles was significantly reduced. Compared with the MSCs group, the MSCs (CLU) treatment group had a greater advantage in restoring the number of primordial follicles and primary follicles ( Fig.13 B), which indicates that UC-MSCs with overexpression of CLU gene showed better effect in repairing ovarian structure. The results of weighing the bilateral ovaries of mice in each group showed that the ovarian weight of mice in the MSC (CLU) treatment group was significantly higher than that in the POF group and the MSCs treatment group ( Fig.13 C), which further indicates that overexpression of the CLU gene helps to restore ovarian function more effectively. The levels of serum hormones estradiol (E2) and follicle-stimulating hormone (FSH) in each group of mice were further tested. The results of ELISA analysis showed that the E2 level in the MSCs (CLU) treatment group was significantly higher than that in the POF and MSCs treatment groups, and the FSH level was significantly lower than that in the POF and MSCs treatment groups ( Fig.13 D), indicating that UC-MSCs with overexpression of CLU gene are more effective in restoring hormone levels. The apoptosis of granulosa cells was detected by TUNEL staining. The results showed that the apoptosis of ovarian granulosa cells in the MSCs (CLU) treatment group was significantly reduced, and the apoptosis inhibition effect was better than that in the MSCs and MSCs (GFP) treatment groups ( Fig.13 E), indicating that overexpression of the CLU gene enhanced the anti-apoptotic effect of UC-MSCs. In addition, the expression of PCNA, a proliferation marker of granulosa cells, was detected by immunofluorescence staining. It was found that compared with the POF group, the proliferation of granulosa cells in the MSCs (CLU) treatment group was significantly increased, and the proliferation level was better than that in the MSCs and MSCs (GFP) treatment groups ( Fig.13 F), further indicating that UC-MSCs overexpressing CLU gene have significant advantages in promoting the proliferation of ovarian granulosa cells.

[0157] In summary, the therapeutic effect of UC-MSCs with overexpression of CLU gene in POF mouse model is better than that of ordinary UC-MSCs. UC-MSCs with overexpression of CLU gene can more effectively repair ovarian tissue, restore ovarian function, improve hormone levels, inhibit apoptosis of ovarian granulosa cells, and significantly promote their proliferation. This research result provides a strong experimental basis for the application of CLU gene-modified UC-MSCs in the treatment of premature ovarian failure, indicating that overexpression of CLU gene can significantly enhance the therapeutic effect of UC-MSCs.

[0158] Example 10: Isolation and identification of exosomes

[0159] This example describes in detail how to isolate exosomes from CLU gene-modified umbilical cord mesenchymal stem cells (UC-MSCs) and identify the exosomes. This process includes specific operating steps, required reagents, and equipment used to ensure that precise operations can be performed to obtain high-purity exosomes.

[0160] 1. Isolation of Exosomes

[0161] a. Cell culture and supernatant collection

[0162] Reagents: UC-MSCs, complete medium for mesenchymal stem cells.

[0163] The CLU gene-overexpressing UC-MSCs constructed in Example 6 were expanded and cultured, and the cell culture supernatant was collected for subsequent isolation of exosomes.

[0164] b. Extraction of exosomes

[0165] Equipment and reagents: ultracentrifuge, 100 kDa MWCO ultrafiltration tube, PBS buffer.

[0166] Steps: Preliminary centrifugation: Place the collected supernatant in a 50mL centrifuge tube and centrifuge at 300xg at 4°C for 10 minutes to remove cell debris and suspended particles. After centrifugation, transfer the supernatant to a new centrifuge tube. Ultrafiltration concentration: Transfer the supernatant to a 100kDa MWCO ultrafiltration tube and centrifuge at 5,000xg for 30 minutes at 4°C. The concentrate (liquid above the filter membrane) after centrifugation is the exosome-rich fraction. Differential centrifugation to separate exosomes: Divide the concentrated supernatant into equal amounts in ultracentrifuge tubes, ensure weight balance (difference does not exceed 0.02g), and level with PBS buffer. Then ultracentrifuge at 100,000xg at 4°C for 70 minutes to collect the exosome pellet at the bottom of the centrifuge tube. Gently pour off the supernatant to avoid disturbing the exosome pellet. Gently resuspend the exosome pellet with 2mL of sterile PBS buffer. To ensure the purity of the exosomes, the cells were centrifuged again at 100,000 × g for 70 min, the supernatant was removed, and the exosome pellet at the bottom was retained.

[0167] c. Storage of exosomes

[0168] The exosome pellet was resuspended in 100-200 μL sterile PBS. The separated exosomes were packaged in sterile EP tubes and stored at 4°C for short term and in a -80°C refrigerator for long term.

[0169] 2. Identification of Exosomes

[0170] a. Particle size analysis and particle concentration detection

[0171] Equipment and reagents: Nanoparticle Tracking Analyzer (NTA, ZetaView PMX 110, ParticleMetrix, Germany), PBS buffer.

[0172] Procedure: Calibrate the NTA instrument using 110nm polystyrene microspheres to ensure accurate measurements. Dilute the separated exosome sample with PBS to an appropriate concentration to avoid particles that are too dense or over-diluted and affect the detection. Add the sample to the NTA sample pool, run the program, and analyze the particle size distribution and concentration of the exosomes. The typical exosome particle size range is 30-150nm, and the concentration is recorded based on the data displayed by the detection instrument.

[0173] like Fig.14 The size and distribution of EVs extracted by NTA detection are shown. The particle concentration of MSC-EVs (GFP) is 3.1×10 6 / mL, the peak particle size was 116.6nm, and the MSC-EVs (CLU) particle concentration was 3.4×10 6 / mL, and the peak particle size is 116.6nm.

[0174] b. Transmission electron microscopy (TEM) observation of exosome morphology

[0175] Equipment and reagents: transmission electron microscope (HT7700, Hitachi), 200-mesh carbon-coated copper grid, 2% phosphotungstic acid or 1% uranic acid.

[0176] Steps: Take 10μL of exosome sample and drop it onto a 200-mesh carbon-film copper grid. Let it stand for 5 minutes to allow the liquid to spread evenly. Carefully use filter paper to absorb excess liquid to ensure that the sample is spread evenly. Add 10μL of phosphotungstic acid or uranic acid to the surface of the exosomes on the copper grid and let it stand for 2 minutes to negatively stain the exosomes. Gently use filter paper to absorb excess staining liquid. After the sample is completely dry, place the copper grid under a transmission electron microscope for observation.

[0177] like Fig.15 Transmission electron microscopy observations showed that both exosomes, MSC-EVs (GFP) and MSC-EVs (CLU), presented a typical round double-layer membrane vesicle morphology with a diameter ranging from 50-150 nm.

[0178] c. Western Blot detection of surface markers and CLU protein overexpression in EVs

[0179] Equipment and reagents: SDS-PAGE electrophoresis equipment (Bio-Rad), PVDF membrane, CD9 antibody, Flag antibody.

[0180] Steps: Sample preparation: Mix the exosome protein sample with SDS-PAGE Loading Buffer in a ratio of 4:1, and heat at 95°C for 5 minutes to denature the protein. Electrophoresis: Perform SDS-PAGE protein electrophoresis, load the exosome sample and prestained protein Marker into the electrophoresis tank, set the voltage to 60V for 30 minutes, and adjust the voltage to 120V after the sample enters the separation gel, and continue electrophoresis until the separation is complete. Transfer: Transfer the protein after electrophoresis to the PVDF membrane, and use a current of 150mA for 90 minutes. Blocking and antibody incubation: Block the PVDF membrane in 5% skim milk powder or BSA solution for 1 hour. Then the antibody (CD9, Flag) is diluted in proportion, and the membrane is incubated with the antibody at 4°C overnight. Secondary antibody incubation and development: After the primary antibody is recovered the next day, wash the membrane three times with TBST, each time for 10 minutes. Then add HRP-labeled goat anti-rabbit secondary antibody, incubate at room temperature for 1 hour, and wash the membrane three times with TBST. Finally, ECL luminescent reagent was used for development, and protein bands were recorded by chemiluminescent imaging system.

[0181] Western blot test results Fig.16 As shown in the figure, both MSC-EVs (GFP) and MSC-EVs (CLU) expressed the exosome marker transmembrane protein CD9, and compared with MSC-EVs (GFP), MSC-EVs (CLU) significantly expressed Flag tag protein, proving that the constructed gene-modified UC-MSCs can highly express CLU protein and secrete it into exosomes.

[0182] Example 11: Application of EVs derived from genetically modified umbilical cord mesenchymal stem cells in the treatment of POF

[0183] To investigate the effect of exosomes (EVs) derived from umbilical cord mesenchymal stem cells (UC-MSCs) with overexpression of CLU gene in the treatment of premature ovarian failure (POF), 25 7-week-old C57BL / 6 female mice were randomly divided into 5 groups, with 5 mice in each group: NC group (healthy control group), POF group, POF+MSC-EVs treatment group (exosomes extracted from culture supernatant of UC-MSCs without infection operation), POF+MSC-EVs(GFP) treatment group (exosomes extracted from culture supernatant of UC-MSCs infected with GFP control), and POF+MSC-EVs(CLU) treatment group (exosomes extracted from culture supernatant of UC-MSCs with overexpression of CLU gene). On the 6th day after the mice received intraperitoneal injection of CTX / BUS chemotherapy drugs, 50 μg of exosomes were injected into each mouse through the tail vein, and the analysis was performed after two weeks of observation.

[0184] The results of HE staining of ovarian structure showed that compared with the POF group, the ovarian atrophy of mice treated with each therapy was significantly improved. The number of primordial follicles and primary follicles in the MSC-EVs (CLU) treatment group increased significantly, and the number of atretic follicles decreased significantly, which was better than that in the EVs treatment group ( Fig.17 A and 17B). Compared with the POF group, the ovarian weight of mice in the MSC-EVs (CLU) treatment group was significantly increased ( Fig.17 C). In addition, the results of the detection of estradiol (E2) and follicle-stimulating hormone (FSH) levels showed that compared with the POF group, the E2 level of mice in the MSC-EVs (CLU) treatment group was significantly increased, and the FSH level was significantly decreased ( Fig.17 D). Further testing revealed that the MSC-EVs (CLU) treatment group significantly inhibited the apoptosis of ovarian granulosa cells and significantly promoted the proliferation of granulosa cells ( Fig.17 E and 17F).

[0185] Based on the above experimental results, exosomes derived from UC-MSCs with overexpression of CLU gene showed significant therapeutic effect in repairing ovarian function of POF mice, and the therapeutic effect was better than that of ordinary EVs in restoring the number of primordial follicles and primary follicles, reducing the number of atretic follicles and inhibiting granulosa cell apoptosis. This study shows that exosomes derived from UC-MSCs with overexpression of CLU gene have broad clinical application prospects in the treatment of premature ovarian failure.

[0186] The present invention illustrates the process method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, that is, it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for preparing engineered mesenchymal stem cells overexpressing Clusterin, characterized in that: The following steps are involved: S1: Isolating and subculturing human umbilical cord mesenchymal stem cells in vitro to obtain human umbilical cord mesenchymal stem cells; S2: Construction of Clusterin overexpression lentiviral vector; S3: The Clusterin overexpression lentiviral vector solution and the Polybrene solution were mixed and added to the complete culture medium of human umbilical cord mesenchymal stem cells obtained in step S1 for virus infection, and cultured in an incubator at 37°C and 5% CO2 for 12 hours. After that, the culture medium was replaced, and the cells and culture supernatant were harvested after a further 48 hours of culture. Protein was extracted from the infected UC-MSCs, and the overexpression of the CLU gene was detected by Western Blot. The verified CLU gene overexpression UC-MSCs were cultured in large quantities in the complete culture medium of mesenchymal stem cells.

2. The preparation method according to claim 1, characterized in that: In step S1, the in vitro separation and subculture of human umbilical cord mesenchymal stem cells comprises the following steps: S11: Soak the fresh human umbilical cord in 75% alcohol solution for no less than 1 minute on a sterile operating table for preliminary disinfection to ensure that the solution completely covers the surface of the umbilical cord and achieves the disinfection effect. After the soaking is completed, rinse the umbilical cord with sterile saline to remove the alcohol residue, and use sterile scissors to cut the umbilical cord into small sections of about 2 cm in length. Then, peel off the arteries, veins and epidermis in the umbilical cord, and keep the Wharton's jelly in the middle; S12: The peeled Wharton's jelly tissue was cut into small pieces with a side length of 3-5 mm. The cut tissue pieces were inoculated in a 100 mm culture dish. The spacing between the small pieces should be uniform, and the spacing between the small pieces should be 4-7 mm. An appropriate amount of mesenchymal stem cell complete medium (mesenchymal stem cell serum-free medium (Youkang, catalog number: NC0103) containing mesenchymal stem cell culture factors (Youkang, catalog number: NC0103.S)) was added to each dish, and cultured at 37°C and 5% CO2. The culture medium was replaced every 3 days (carefully aspirate and discard the culture supernatant, and slowly add new mesenchymal stem cell complete medium along the wall). After 7-10 days of culture, the cells crawled out of the tissue block to form primary cells P0; S13: Primary cells P0 are digested and harvested after reaching a confluence of more than 85%, specifically: The old culture medium in the culture dish was discarded, and the dish was gently rinsed twice with PBS to remove the residual culture medium and floating cells. An appropriate amount of stem cell mild digestion enzyme was added to each dish, and the dish was placed at room temperature for digestion for about 3 minutes. When most of the cells were discrete spheres, 4 mL of mesenchymal stem cell complete culture medium was added to terminate the digestion. The cell suspension was gently blown and mixed, and transferred to a 50 mL centrifuge tube. The bottom of the dish was rinsed with PBS again, and all the cell suspensions were collected and transferred to a centrifuge tube. Centrifuged at 1300 rpm for 5 minutes, the supernatant was discarded, and then the precipitated cells were resuspended in an appropriate amount of mesenchymal stem cell complete culture medium and counted. The cells harvested in this step are primary cells P0; S14: Subculture of UC-MSCs, specifically: The primary cells P0 were harvested and plated per cm 2 5000-6000 cells were inoculated into a new T175 culture flask, and an appropriate amount of culture medium was added to 20 mL. After gently shaking to mix, the flask was placed in a constant temperature and humidity incubator and cultured under 5% CO2 and 37°C. The growth status of the cells was observed every day until the cell confluence reached 85%-90%. The cells were digested and passaged to the first generation of cells P1. The passage operation used the same steps and digestive enzymes as the primary generation. When the P1 cells reached 90% confluence, the second generation of cells P2 was generated. After the harvested P2 cells passed the quality inspection, they were packaged and frozen in liquid nitrogen to establish the master cell bank. The P2 cells revived from the master cell bank continued to expand and culture, and were passaged generation by generation to the fifth generation of cells P5. After each passage, strict quality inspection was carried out to ensure that the cells had good proliferation ability and multidirectional differentiation potential. When the P5 cells reached the quality standard, they were packaged and frozen in liquid nitrogen to establish the working cell bank.

3. The preparation method according to claim 1, characterized in that: In step S2, the Clusterin overexpression lentiviral vector is constructed, specifically: The full-length sequence of the CLU gene (Gene ID: 1191) was obtained from the NCBI database, and the gene fragment was cloned into the lentiviral expression vector pLV3. The CLU gene was inserted using the multiple cloning site, and the 3Flag tag was ensured to construct the pLV3-CLU-3Flag plasmid. Subsequently, the constructed pLV3-CLU-3Flag plasmid was mixed with the auxiliary plasmid pMD2G (encoding VSV-G envelope protein) and psPAX2 (encoding viral packaging protein) in a ratio of 4:2:1 using EL transfection reagent and co-transfected into 293T cells for virus packaging. After 48 hours of transfection, the supernatant was collected and centrifuged at 300×g for 5 minutes to remove cell debris, followed by ultracentrifugation at 100,000×g for 2 hours to concentrate the virus particles. After the virus was concentrated, it was resuspended in PBS and stored at 4°C for infection of UC-MSCs.

4. The preparation method according to claim 1, characterized in that: In step S3, the final concentration of the Polybrene is 5-8 μg / mL.

5. An engineered mesenchymal stem cell overexpressing Clusterin obtained according to the preparation method according to any one of claims 1 to 4.

6. A method for preparing Clusterin-overexpressing engineered mesenchymal stem cell exosomes, characterized in that: The following steps are involved: S1: amplifying and culturing the engineered mesenchymal stem cells overexpressing Clusterin according to claim 5 of the present invention, and collecting the cell culture supernatant; S2: The cell culture supernatant collected in step S1 is purified by combining ultrafiltration and differential centrifugation technology to obtain engineered mesenchymal stem cell exosomes overexpressing Clusterin.

7. The method for preparing Clusterin-overexpressing engineered mesenchymal stem cell exosomes according to claim 6, characterized in that: In step S2, the steps of combining ultrafiltration and differential centrifugation technology for purification are as follows: S21: Removing cell debris and suspended particles from the supernatant collected in S1: Place the supernatant collected in S1 in a 50 mL centrifuge tube, centrifuge at 300 x g for 10 min at 4 °C to remove cell debris and suspended particles. After centrifugation, transfer the supernatant to a new centrifuge tube; S22: Ultrafiltration concentration: The supernatant was transferred into a 100 kDa MWCO ultrafiltration tube and centrifuged at 5,000 x g for 30 min at 4 °C. The concentrate after centrifugation, i.e. the liquid above the filter membrane, was the exosome-rich fraction. S23: Isolation of exosomes by differential centrifugation: Dispense the concentrated supernatant into ultracentrifuge tubes in equal amounts, with a difference of no more than 0.02 g, to ensure weight balance, and level with PBS buffer, then ultracentrifuge at 100,000 x g at 4 °C for 70 min, collect the exosome precipitate at the bottom of the centrifuge tube, and gently pour out the supernatant to avoid disturbing the exosome precipitate; gently resuspend the exosome precipitate with 2 mL of sterile PBS buffer; To ensure the purity of the exosomes, the cells were centrifuged again at 100,000 × g for 70 min, the supernatant was removed, and the exosome pellet at the bottom was retained.

8. An engineered mesenchymal stem cell exosome with overexpression of Clusterin obtained according to the preparation method of claim 6 or 7.

9. Use of Clusterin, the engineered mesenchymal stem cells overexpressing Clusterin according to any one of claims 1 to 4, or the exosomes of the engineered mesenchymal stem cells overexpressing Clusterin according to claim 6 or 7 in the preparation of a drug for treating premature ovarian failure.

10. The use according to claim 9, characterized in that: The premature ovarian failure is premature ovarian failure caused by chemotherapy drugs.

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