Mesenchymal stem cell for resisting ovarian granular cell senescence and preparation method thereof

Abstract

The invention discloses a preparation method of mesenchymal stem cells for resisting ovarian granular cell aging. The preparation method comprises the following steps: providing umbilical cord blood stem cells; carrying out induced culture on the umbilical cord blood stem cells, collecting a supernatant after the culture is completed, and separating the supernatant to obtain an exosome; providing mesenchymal stem cells, and performing induced culture on the mesenchymal stem cells by adding the exosome, so as to obtain the required mesenchymal stem cells for resisting ovarian granular cell senescence. According to the preparation method of the mesenchymal stem cells for resisting ovarian granular cell senescence, the mesenchymal stem cells induced by the umbilical cord blood stem cell exosome can be prepared, and the mesenchymal stem cells can be used for treating premature ovarian failure in a whole body vein use mode. The umbilical cord mesenchymal stem cells induced by the stem cell exosome can exert a better immune regulation function, regulate the balance of an immune system and reduce immune-related inflammation and injury so as to delay the senescence of ovarian granular cells.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a mesenchymal stem cell capable of resisting ovarian granulosa cell aging and a preparation method thereof. Background Art

[0002] Stem cells are cells with self-renewal and multidirectional differentiation potentials, and can differentiate into various types of cells, such as nerve cells, cardiomyocytes, hepatocytes, etc. In the medical field, stem cells are used to treat various diseases, including premature ovarian failure. Premature ovarian failure is a disease that causes a decrease in female fertility, usually due to decreased ovarian function. Ovarian granulosa cells are an important type of ovarian cell. They are located outside the zona pellucida of the oocyte and promote oocyte maturation through paracrine and autocrine mechanisms. They play an important role in regulating oocyte maturation and regulating oocyte growth and development. The aging of ovarian granulosa cells is one of the important reasons for the gradual decline in women's reproductive health. The development of relevant treatment methods targeting its aging mechanism can achieve good treatment effects and solve the problem of premature ovarian failure from the root.

[0003] In the existing technology, there have been many studies showing that various types of stem cells, such as stem cells from bone marrow, fat and umbilical cord, can treat premature ovarian failure. Among them, umbilical cord-derived mesenchymal stem cells are widely used because they are easy to obtain and have a wide range of sources. These stem cells can be used to treat premature ovarian failure through local transplantation or systemic use.

[0004] Although existing technologies have achieved certain results, there are still some problems. First, bone marrow and adipose-derived stem cells need to be obtained through additional surgical operations, which may increase the pain and risk of patients. Secondly, these stem cells do not function well in aging tissues and may not be able to effectively treat premature ovarian failure. In addition, although commonly cultured umbilical cord mesenchymal stem cells (MSC) and other stem cell therapies have certain effects on premature ovarian failure, their stem cells have a short survival time in the body and the effects are not lasting. In addition, due to the limitations of the corresponding mechanism of action, there are large differences in the effects on individuals, and there is no way to solve the problem of ovarian aging from the root. Therefore, it is necessary to find more effective ways to use stem cells to treat premature ovarian failure. Summary of the invention

[0005] Based on this, it is necessary to provide a method for preparing mesenchymal stem cells that resist ovarian granulosa cell aging.

[0006] In addition, it is also necessary to provide mesenchymal stem cells that resist ovarian granulosa cell aging and are prepared by the above-mentioned method for preparing mesenchymal stem cells that resist ovarian granulosa cell aging.

[0007] A method for preparing mesenchymal stem cells that resist ovarian granulosa cell aging comprises the following steps:

[0008] Provide umbilical cord blood stem cells;

[0009] Inducing and culturing the umbilical cord blood stem cells, collecting the supernatant after the culture is completed, and isolating exosomes from the supernatant;

[0010] Mesenchymal stem cells are provided, and the mesenchymal stem cells are induced and cultured by adding the exosomes to obtain the required mesenchymal stem cells that resist ovarian granulosa cell aging.

[0011] In one embodiment, the operation of providing umbilical cord blood stem cells is: after centrifuging the umbilical cord blood, taking out the lower layer of plasma, and separating the umbilical cord blood stem cells from the plasma.

[0012] In one embodiment, the induction culture of the umbilical cord blood stem cells is performed by adding complete culture medium to the umbilical cord blood stem cells to a concentration of 2×10 6 / mL~6×10 6 / mL, and placed in a CO2 incubator at 37°C, saturated humidity, and 5% volume fraction for culture. The culture time is 3 to 7 days, and the medium is changed 1 to 2 times during the culture process, wherein the complete culture medium is DMEM / F12 culture medium + 1v / v% to 2v / v% B-27 serum-free additive + 0.5ng / mL to 1ng / mL of recombinant human bFGF factor + 5ng / mL to 10ng / mL of recombinant human VEGF-165 factor.

[0013] In one embodiment, the operation of separating the umbilical cord blood stem cells from the plasma is: separating the umbilical cord blood stem cells from the plasma using density gradient centrifugation.

[0014] In one embodiment, the operation of isolating exosomes from the supernatant is as follows: taking the supernatant, first centrifuging at 500×g for 30 min at 4°C, then centrifuging at 200×g for 20 min, discarding the precipitate, passing the supernatant through a 0.1 μm filter in a sterile clean bench, and ultracentrifuging at 100,000×g for 1 h. The precipitate is the exosomes.

[0015] In one embodiment, the operation of inducing the mesenchymal stem cells by adding the exosomes is as follows: inoculating the mesenchymal stem cells into a culture bottle, adding a complete medium for mesenchymal stem cells, and when the cells are cultured to 85% confluence, digesting them with 0.05wt% trypsin, collecting P1 generation cells for passaging, and passaging to P3 generation according to the above method to complete the induction culture, wherein the complete medium for mesenchymal stem cells contains 5v / v% to 10v / v% of an exosome solution, and the concentration of the exosomes in the exosome solution is 1×10 10 / mL~10×10 10 Pieces / mL.

[0016] In one embodiment, the mesenchymal stem cells are inoculated into a culture bottle and a complete mesenchymal stem cell culture medium is added. The mesenchymal stem cells are cultured at a concentration of 5×10 3 Pieces / cm 2 ~8×10 3 Pieces / cm 2 The density of the mesenchymal stem cells is 3.5×10 4 / mL~5.6×10 4 pieces / mL pieces / ml.

[0017] In one embodiment, the operation of providing mesenchymal stem cells is: after cleaning the umbilical cord, remove two arteries and one vein, tear off the Wharton's jelly, cut the Wharton's jelly into 1mm pieces, 3 The tissue blocks were inoculated into a container containing complete medium for mesenchymal stem cells and placed in an incubator at 37°C with a volume fraction of 5% CO. 2 After 1 week, the tissue blocks were removed and the culture medium was replaced. The medium was changed every 3 days. When the cells grew to 80% confluence, they were digested with 0.05% trypsin and the P0 cells were collected and cultured at 5×10 3 Pieces / cm 2 The cells were inoculated at a density of 100 μg / cm2 in a culture flask containing 25 mL of the complete medium for mesenchymal stem cells for proliferation and culture. When the cells grew to 85% confluence, they were digested with 0.05% trypsin and the P1 generation cells were collected, which were the mesenchymal stem cells.

[0018] In one embodiment, the umbilical cord blood stem cells are human umbilical cord blood stem cells, and the mesenchymal stem cells are human mesenchymal stem cells.

[0019] A mesenchymal stem cell capable of resisting ovarian granulosa cell aging is prepared by the above-mentioned method for preparing mesenchymal stem cells capable of resisting ovarian granulosa cell aging.

[0020] The preparation method of the mesenchymal stem cells for resisting ovarian granulosa cell aging of the present invention can prepare umbilical cord blood stem cell exosome-induced mesenchymal stem cells, which can be used systemically intravenously to treat premature ovarian failure. Umbilical cord mesenchymal stem cells induced by stem cell exosomes can play a good immunomodulatory function, regulate the balance of the immune system, reduce immune-related inflammation and damage, and delay the aging of ovarian granulosa cells. At the same time, the survival time of the obtained umbilical cord mesenchymal stem cells in the body and the local targeting to the ovary are improved by optimizing the preparation process.

[0021] In addition, the preparation method of the mesenchymal stem cells that resist ovarian granulosa cell aging of the present invention can also obtain induced umbilical cord blood stem cells after induction culture. The induced umbilical cord blood stem cells are rich in various growth factors and cytokines, which can promote the proliferation of ovarian granulosa cells and stimulate their growth and division, thereby increasing the number of healthy ovarian granulosa cells, compensating for the reduction of aging or damaged ovarian granulosa cells, maintaining the stable state of ovarian granulosa cells, and promoting local blood vessel survival, thereby comprehensively improving the microenvironment of ovarian granulosa cells.

[0022] Due to the advanced nature of this patented technology, it can be widely used in the fields of cell therapy, regenerative medicine, and stem cell research. First, in the field of cell therapy, this technical solution proposes a new method for stem cell treatment of premature ovarian failure, which can effectively improve the survival time and therapeutic effect of stem cells by local injection of umbilical cord blood stem cells and umbilical cord blood stem cell exosomes induced by systemic veins. This method provides new ideas and methods for cell therapy and is expected to have broad application prospects in the treatment of other diseases. Secondly, in the field of regenerative medicine, this technical solution improves the microenvironment of the ovary, maintains the number and quality of granulosa cells, promotes oocyte maturation, regulates oocyte growth and development, maintains fertility levels and delays body aging. This series of effects contributes to the recovery and regeneration of ovarian function, which is of great significance to the development of the field of regenerative medicine. Finally, in the field of stem cell research, this technical solution explores a new method for stem cell treatment of premature ovarian failure by improving the cell culture process and the combined application of multiple cells. This study not only enriches the research content of stem cells, but also provides new directions and ideas for the application of stem cells. In general, this technical solution has broad application prospects in the fields of cell therapy, regenerative medicine and stem cell research, and is expected to make important contributions to the development and progress of related fields. At the same time, as society pays more attention to related diseases such as premature ovarian failure, this technical solution also has broad market demand and commercial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] in:

[0025] Figure 1 The present invention is a flow chart of a method for preparing mesenchymal stem cells that resist ovarian granulosa cell aging according to one embodiment.

[0026] Figure 2a and Figure 2b is a microscopic photograph of the umbilical cord blood stem cells in Example 1, wherein: Figure 2a The magnification is 100 times. Figure 2b The magnification is 400 times.

[0027] Figure 3 This is a graph showing the results of flow cytometry detection of CD34+ expression of two cord blood stem cells in Example 1, wherein CD34+ hematopoietic stem cells reached 4.7% and 5.6% respectively.

[0028] Figure 4 To observe cell colonies such as CFU-GM, CFU-M, CFU-GEMM, CFU-E, and CFU-G under a microscope.

[0029] Figure 5 This is a transmission electron microscopy image of the exosomes in Example 2.

[0030] Figure 6a and Figure 6b This is a microscopic photograph of the mesenchymal stem cells that resist ovarian granulosa cell aging in Example 3, wherein: Figure 6a The magnification is 40 times. Figure 6b The magnification is 100 times.

[0031] Figure 7 This is the result of flow cytometry detection of cell surface markers of mesenchymal stem cells that resist ovarian granulosa cell aging.

[0032] Figure 8a This is a staining photograph of the mesenchymal stem cells that resist ovarian granulosa cell aging in Example 3 after being induced and cultured for 21 days using MSC adipose differentiation medium.

[0033] Figure 8b This is a staining photograph of the mesenchymal stem cells that resist ovarian granulosa cell aging in Example 3 after being induced and cultured for 21 days using MSC bone differentiation medium.

[0034] Figure 8c This is a staining photograph of the mesenchymal stem cells that resist ovarian granulosa cell aging in Example 3 after being induced and cultured for 21 days using MSC chondrogenic differentiation medium.

[0035] Figure 9a The following are microscopic photographs of umbilical vein endothelial cells (HUVEC) cultured with MSC-based control medium for 8 hours.

[0036] Figure 9b The following are microscopic photographs of umbilical vein endothelial cells (HUVEC) cultured with MSC-conditioned medium for 8 hours. DETAILED DESCRIPTION

[0037] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] Combination Figure 1 The present invention discloses a method for preparing mesenchymal stem cells that resist ovarian granulosa cell aging, comprising the following steps:

[0039] S10. Provide umbilical cord blood stem cells.

[0040] Umbilical cord blood stem cells can be purchased directly or prepared yourself.

[0041] Preferably, in this embodiment, the operation of providing umbilical cord blood stem cells is: after centrifuging the umbilical cord blood, taking out the lower layer of plasma, and separating the umbilical cord blood stem cells from the plasma.

[0042] Specifically, in this embodiment, the operation of separating and obtaining umbilical cord blood stem cells from plasma is: separating and obtaining umbilical cord blood stem cells from plasma using density gradient centrifugation.

[0043] S20, inducing and culturing the umbilical cord blood stem cells obtained in S10, collecting the supernatant after the culture is completed, and isolating exosomes from the supernatant.

[0044] Preferably, in this embodiment, the operation of inducing the umbilical cord blood stem cells is: adding complete culture medium to the umbilical cord blood stem cells to a concentration of 2×10 6 ~6×10 6 / mL, and cultured in a CO2 incubator at 37°C, saturated humidity, and 5% volume fraction for 3 to 7 days. The medium was changed 1 to 2 times during the culture process. The complete culture medium was DMEM / F12 medium + 1v / v% to 2v / v% B-27 serum-free additive + 0.5ng / mL to 1ng / mL of recombinant human bFGF factor + 5ng / mL to 10ng / mL of recombinant human VEGF-165 factor.

[0045] Preferably, in this embodiment, the operation of isolating exosomes from the supernatant is as follows: take the supernatant, first centrifuge at 500×g for 30 min at 4°C, then centrifuge at 200×g for 20 min, discard the precipitate, pass the supernatant through a 0.1 μm filter in a sterile clean bench, and ultracentrifuge at 100,000×g for 1 h, and the precipitate is the exosomes.

[0046] Specifically, the obtained exosomes can be washed with PBS, and after washing, ultracentrifuged at 100,000×g for 1 h, and finally the precipitate is resuspended in an appropriate amount of sterile PBS to obtain an exosome solution.

[0047] It should be pointed out that in S20, induced umbilical cord blood stem cells can also be obtained after induced culture. The induced umbilical cord blood stem cells are rich in a variety of growth factors and cytokines, which can promote the proliferation of ovarian granulosa cells, stimulate their growth and division, thereby increasing the number of healthy ovarian granulosa cells, making up for the decrease of aging or damaged ovarian granulosa cells, maintaining the stable state of ovarian granulosa cells, and promoting local blood vessel survival, comprehensively improving the microenvironment of ovarian granulosa cells.

[0048] S30, providing mesenchymal stem cells, inducing and culturing the mesenchymal stem cells by adding the exosomes obtained in S20, and obtaining the desired mesenchymal stem cells that resist ovarian granulosa cell aging.

[0049] Mesenchymal stem cells can be purchased directly or prepared by yourself.

[0050] Preferably, in this embodiment, the operation of providing mesenchymal stem cells is as follows: after cleaning the umbilical cord, two arteries and one vein are removed, Wharton's jelly is torn off, and the Wharton's jelly is cut into 1 mm pieces. 3 The tissue blocks were inoculated into a container containing complete medium for mesenchymal stem cells and placed in an incubator at 37°C with a volume fraction of 5% CO. 2 After 1 week, the tissue blocks were removed and the culture medium was replaced. The medium was changed every 3 days. When the cells grew to 80% confluence, they were digested with 0.05% trypsin and the P0 cells were collected and cultured at 5×10 3 Pieces / cm 2The cells were inoculated at a density of 100 μg / mL in a culture flask containing 25 mL of complete mesenchymal stem cell culture medium for expansion and culture. When the cells grew to 85% confluence, they were digested with 0.05% trypsin and the P1 cells were collected, which were mesenchymal stem cells.

[0051] In this embodiment, the complete medium for mesenchymal stem cells can be directly purchased.

[0052] Preferably, in this embodiment, the operation of inducing and culturing mesenchymal stem cells by adding exosomes is as follows: inoculating mesenchymal stem cells into a culture bottle, adding a complete medium for mesenchymal stem cells, and when the cells are cultured to 85% confluence, digesting them with 0.05wt% trypsin, collecting P1 generation cells for passaging, and passaging to P3 generation according to the above method to complete the induction culture, wherein the complete medium for mesenchymal stem cells contains 5v / v% to 10v / v% of an exosome solution, and the concentration of exosomes in the exosome solution is 1×10 10 / mL~10×10 10 Pieces / mL.

[0053] Preferably, in this embodiment, mesenchymal stem cells are seeded into a culture bottle and a complete mesenchymal stem cell culture medium is added. The mesenchymal stem cells are cultured at a concentration of 5×10 3 Pieces / cm 2 ~8×10 3 Pieces / cm 2 After adding the complete medium of mesenchymal stem cells, the density of mesenchymal stem cells was 3.5×10 4 / mL~5.6×10 4 Pieces / mL.

[0054] In this embodiment, the umbilical cord blood stem cells are human umbilical cord blood stem cells, and the mesenchymal stem cells are human mesenchymal stem cells.

[0055] The preparation method of the mesenchymal stem cells for resisting ovarian granulosa cell aging of the present invention can prepare umbilical cord blood stem cell exosome-induced mesenchymal stem cells, which can be used systemically intravenously to treat premature ovarian failure. Umbilical cord mesenchymal stem cells induced by stem cell exosomes can play a good immunomodulatory function, regulate the balance of the immune system, reduce immune-related inflammation and damage, and delay the aging of ovarian granulosa cells. At the same time, the survival time of the obtained umbilical cord mesenchymal stem cells in the body and the local targeting to the ovary are improved by optimizing the preparation process.

[0056] In addition, the method for preparing mesenchymal stem cells that resist ovarian granulosa cell aging of the present invention can prepare umbilical cord blood stem cells that are rich in various growth factors and cytokines, which can promote the proliferation of ovarian granulosa cells and stimulate their growth and division, thereby increasing the number of healthy ovarian granulosa cells, compensating for the reduction of aging or damaged ovarian granulosa cells, maintaining the stable state of ovarian granulosa cells, and promoting local blood vessel survival, thereby comprehensively improving the microenvironment of ovarian granulosa cells.

[0057] Due to the advanced nature of this patented technology, it can be widely used in the fields of cell therapy, regenerative medicine, and stem cell research. First, in the field of cell therapy, this technical solution proposes a new method for stem cell treatment of premature ovarian failure, which can effectively improve the survival time and therapeutic effect of stem cells by local injection of umbilical cord blood stem cells and umbilical cord blood stem cell exosomes induced by systemic veins. This method provides new ideas and methods for cell therapy and is expected to have broad application prospects in the treatment of other diseases. Secondly, in the field of regenerative medicine, this technical solution improves the microenvironment of the ovary, maintains the number and quality of granulosa cells, promotes oocyte maturation, regulates oocyte growth and development, maintains fertility levels and delays body aging. This series of effects contributes to the recovery and regeneration of ovarian function, which is of great significance to the development of the field of regenerative medicine. Finally, in the field of stem cell research, this technical solution explores a new method for stem cell treatment of premature ovarian failure by improving the cell culture process and the combined application of multiple cells. This study not only enriches the research content of stem cells, but also provides new directions and ideas for the application of stem cells. In general, this technical solution has broad application prospects in the fields of cell therapy, regenerative medicine and stem cell research, and is expected to make important contributions to the development and progress of related fields. At the same time, as society pays more attention to related diseases such as premature ovarian failure, this technical solution also has broad market demand and commercial value.

[0058] The present invention also discloses an embodiment of mesenchymal stem cells resistant to ovarian granulosa cell aging prepared by the above-mentioned method for preparing mesenchymal stem cells resistant to ovarian granulosa cell aging.

[0059] The following are specific embodiments.

[0060] Example 1: Preparation of umbilical cord blood stem cells

[0061] 1. Plasma preparation:

[0062] 1) Use a 50ml syringe to transfer the umbilical cord blood of a healthy donor into a 50ml centrifuge tube, pipette up and down to mix, and then take a sample for testing:

[0063] 2) Use a pipette to balance the peripheral blood in the centrifuge tube and centrifuge at 500g for 7 minutes;

[0064] 3) After centrifugation, use a pipette to aspirate the upper yellowish plasma layer and transfer it into two new 50 ml centrifuge tubes and balance them. The remaining liquid is the concentrated blood cells.

[0065] 4) Seal the two centrifuge tubes containing plasma with sealing film and place them in a refrigerator at 2-8°C for future use.

[0066] 2. Isolation and extraction of umbilical cord blood stem cells: density gradient centrifugation method

[0067] 1) Dilution: dilute the concentrated blood cells with normal saline to a final volume of 30 ml per tube;

[0068] 2) Blood layer: Keep the centrifuge tube containing 15ml separation solution at a 45-degree angle to the horizontal plane; use a 10ml electric pipette to draw 10ml of diluted blood cells, and slowly transfer the diluted blood cells to the upper layer of the separation solution at 1cm from the inclined surface of the separation solution. This operation must be gentle and slow, and the flow rate must be controlled well to make the interface between the blood cells and the separation solution obvious;

[0069] 3) Centrifugation: When transferring the last tube of blood cells, slowly straighten the centrifuge tube until the blood cells are transferred and the centrifuge tube is kept at a 90-degree angle to the horizontal plane; use a Pasteur pipette to level each tube of blood cells, cover the lid and seal it, place the centrifuge tube containing blood cells in the centrifuge, centrifuge at 840×g for 20 minutes, and the centrifuge should be raised faster and lowered slower.

[0070] 4) Separation of the buffy coat layer: Open the cap of the centrifuge tube and use a 5ml pipette to aspirate the plasma diluent above the buffy coat layer into the waste liquid tank; then aspirate the buffy coat layer (cord blood stem cells) in the centrifuge tube and transfer it to a new 50ml centrifuge tube (no more than 20ml per centrifuge tube) and add physiological saline to 50ml;

[0071] 5) Centrifugation: Place the centrifuge tube containing umbilical cord blood stem cells in a centrifuge and centrifuge at 1200rpm for 8min. After centrifugation, discard the supernatant, resuspend the cells in each tube with 2.5ml sterile water for injection, pipette rapidly for 15-20 times, and quickly add physiological saline to 50ml per tube, centrifuge at 1000rpm for 10min. After centrifugation, discard the supernatant, resuspend the cells with 8ml physiological saline, add physiological saline to 50ml per tube and mix well. Take 0.5ml from each tube and count to find the average value. Place the resuspended cells in a centrifuge and centrifuge at 1200rpm for 8min.

[0072] 3. Activation and induction of umbilical cord blood stem cells

[0073] 1) Transfer the resuspended cells to a cell culture flask with a pipette and add an appropriate amount of complete culture medium to dilute the cells to 2×10 6 / ml~6×10 6 The complete culture medium is DMEM / F12 culture medium + 1.5v / v% B-27 serum-free additive + 1ng / mL recombinant human bFGF factor + 8ng / mL recombinant human VEGF-165 factor, all of which are American Gibco brands.

[0074] 2) Finally, the culture bottle was placed in a CO2 incubator at 37°C, saturated humidity, and 5.0% volume fraction for culture.

[0075] 3) After the start of cell culture, the cell growth should be observed under an inverted microscope every day, and the medium should be changed 1 to 2 times a week according to the cell growth conditions. After the medium change, the supernatant should be collected, sealed and stored in a -80℃ refrigerator for future use.

[0076] 4. Preparation of cell preparations

[0077] 4) Collect umbilical cord blood stem cells on the 5th to 7th day of induction culture for quality assessment, including observation of cell morphology under a cytometer, flow cytometry, microbial safety inspection, and detection of cell number, activity, cell phenotype, and cell colony formation ability. Figure 2a , Figure 2b , Figure 3 and Figure 4 .

[0078] Combination Figure 2a , Figure 2b , Figure 3 and Figure 4 It can be seen that the induced cord blood stem cells obtained in Example 1 meet the standards of cord blood hematopoietic stem cell preparations.

[0079] Use the plasma in "1. Plasma Preparation" to resuspend the qualified induced umbilical cord blood stem cells to make 10 mL of 1×10 7 Cell preparation suspension of umbilical cord blood stem cells at a concentration of cells / mL.

[0080] Example 2: Method for extracting, preparing and identifying exosomes from umbilical cord blood stem cells

[0081] 1. Extraction of exosomes from umbilical cord blood stem cells

[0082] The supernatant of the umbilical cord blood stem cells obtained in Example 1 was collected and purified by ultrafiltration. The specific operation is as follows: first, centrifuge at 500×g for 30min at 4°C, then centrifuge at 200×g for 20min, discard the precipitate, pass the supernatant through a 0.1μm filter in a sterile clean bench, and ultracentrifuge at 100,000×g for 1h. The precipitate is exosomes. Add 30ml of sterile PBS to the precipitate for washing, and ultracentrifuge again at 100,000×g for 1h. Finally, resuspend the precipitate in an appropriate amount of sterile PBS to make 10mL of 5×10 10 / mL of exosome solution.

[0083] 2. Electron microscopic observation of the morphology of exosomes of umbilical cord blood stem cells

[0084] Exosomes used as electron microscope specimens were placed in 2.5% glutaraldehyde solution, fixed at 4°C overnight, centrifuged the next day, the supernatant was discarded, and the precipitate was embedded in heated and melted agar. After the agar solidified, the part containing the Exosomes precipitate was cut off and transferred to another clean 2.0ep tube. The sample was rinsed with 0.1m, pH7.0 PBS buffer for 3 times, 15min each time, and fixed with 1% osmium acid solution for 1-2h. After absorbing the osmium acid solution, the sample was rinsed with 0.1m, pH7.0 PBS buffer for 3 times, 15min each time. The sample was dehydrated with gradient ethanol (including 50%, 70%, 80%, 90%, 95%), each concentration for 15min, and then dehydrated with 100% ethanol for 20min, and finally treated with pure acetone for 20min. The sample was treated with a mixture of embedding agent and acetone (V / V=1:1) for 1h. The sample was treated with a mixture of embedding agent and acetone (V / V=3:1) for 3 hours. Finally, the sample was treated with pure embedding agent overnight. The sample after infiltration was embedded and heated at 70°C overnight to obtain the embedded sample. The sample was sliced ​​in a Reichert ultrathin slicer to obtain slices with a thickness of 70-90nm. The slices were stained with lead citrate solution and 50% ethanol saturated solution of uranyl acetate for 15 minutes each, and then observed in a transmission electron microscope to obtain Figure 5 .

[0085] Combination Figure 5 It can be seen that the concentration of extracted exosomes is relatively high.

[0086] 3. Specific marker identification of Exosomes:

[0087] Western blot was used to identify exosome-specific markers GRP94, CD9, CD81, CD63 and TSG101. The results are shown in Table 1.

[0088] Table 1

[0089]

[0090] It can be seen from Table 1 that the exosomes extracted in Example 1 have a higher purity.

[0091] Example 3: Preparation and detection of mesenchymal stem cells that resist ovarian granulosa cell aging

[0092] 1. Preparation of Mesenchymal Stem Cell (MSC) Master Cell Bank

[0093] The umbilical cord was removed from the sterile clean bench and rinsed with PBS to remove the residual blood in the umbilical vein and artery. The umbilical cord was torn apart with forceps, two arteries and one vein were removed, Wharton's jelly was torn off, and the Wharton's jelly was cut into pieces of about 1 mm. 3 The tissue blocks were inoculated into T75 plastic culture dishes containing complete mesenchymal stem cell culture medium (Gibco Advanced DMEM / F-12+5% HeliosUltraGRO-Advanced) and cultured in a 37°C, 5% CO2 incubator. After 1 week, the tissue blocks were removed and the culture medium was replaced. The medium was changed every 3 days thereafter. When the cells grew to 80% confluence, they were digested with 0.05% trypsin (the digestion time was controlled under a microscope) and P0 generation MSCs were collected. 5.0×10 3 Pieces / cm 2 The cells were inoculated at a density of 1.50 μg / cm2 in a T175 subculture flask containing 25 ml of complete culture medium for expansion culture. When the cells grew to 85% confluence, they were digested with 0.05% trypsin (the digestion time was controlled under a microscope). The P1 generation MSCs were collected, tested and temporarily stored according to the quality standards of the master cell bank, and transferred to the master cell bank after meeting the quality standards.

[0094] 2. Preparation of mesenchymal stem cells to resist ovarian granulosa cell aging

[0095] Preparation of working cell bank by passaging MSCs recovered from master cell bank, with 5.0×10 3 Pieces / cm 2 The cells were inoculated at a density of 1.50 μg / cm2 in T175 culture flasks, and a mesenchymal stem cell complete medium containing 8 v / v% of the exosome solution in Example 2 was added to each culture flask. When the cells grew to 85% confluence, they were digested with 0.05% trypsin (the digestion time was controlled under a microscope), and the P1 generation MSCs were collected for passaging. The cells were passaged to the P3 generation according to the above passaging method, and the MSCs were collected for testing and temporary storage according to the quality standards of the working cell bank. After meeting the quality standards, they were transferred to the working cell bank.

[0096] 3. Identification of the basic characteristics of mesenchymal stem cells that resist ovarian granulosa cell aging:

[0097] (1) Using an inverted microscope, the morphology of mesenchymal stem cells that resist ovarian granulosa cell aging was observed. Figure 6a and Figure 6b .

[0098] Combination Figure 6a and Figure 6b It can be seen that MSCs grow adherently, appear fibroblast-like, spindle-shaped and orderly arranged, and have good size uniformity.

[0099] (2) Flow cytometry detection of cell surface markers of mesenchymal stem cells that resist ovarian granulosa cell aging. Among them, the positive indicators are: CD73, CD105, CD90 (≥95%); the negative indicators are: HLA-ABC, CD29, CD166, CD34, CD45, CD14, CD19, HLA-DR (≤2%), and the Figure 7 .

[0100] Combination Figure 7 It can be seen that the purity of MSC is very high.

[0101] (3) Three differentiation capacity tests: Commercial MSC adipogenic differentiation medium, MSC osteogenic differentiation medium, and MSC chondrogenic differentiation medium (all purchased from Gibco) were used to culture and observe the ability of anti-ovarian granulosa cell senescence mesenchymal stem cells to differentiate into adipocytes, osteocytes, and chondrocytes. After 21 days of differentiation, they were stained with Alizarin Red S, Oil Red O, and Alcian Blue dyes to obtain Figure 8a , Figure 8b and Figure 8c .

[0102] Combined with 8a, Figure 8b and Figure 8c It can be seen that MSCs have the ability to differentiate into adipogenesis, osteogenesis, and chondrogenesis.

[0103] (4) Cytokine paracrine function detection:

[0104] The evaluation was mainly conducted by detecting the angiogenesis-promoting ability of umbilical cord MSC conditioned medium in promoting umbilical cord vein endothelial cells (HUVEC), namely, preparing MSC conditioned medium concentrate using ultrafiltration centrifuge tubes, stimulating the growth of HUVEC with the concentrate, and studying the angiogenesis-promoting effect of MSC based on the number of nodes or rings formed by HUVEC without conditioned medium (control) and HUVEC with conditioned medium. Figure 9a , Figure 9b .

[0105] Combined with 9a, Figure 9bIt can be seen that MSCs have a good ability to promote angiogenesis, help migrate to the ovary, and improve the local microenvironment of the ovary.

[0106] 4. Preparation of cell preparations

[0107] The plasma obtained in "1. Plasma Preparation" was used to resuspend the mesenchymal stem cells that resist ovarian granulosa cell aging to prepare a concentration of 1×10 7 / ml, 10mL of cell preparation suspension of mesenchymal stem cells that resist ovarian granulosa cell aging.

[0108] Usage of the cell preparation suspension of mesenchymal stem cells that resist ovarian granulosa cell aging: on the day of local ovarian injection, the cells are injected according to the conventional stem cell intravenous injection operating procedures, and the injection frequency is once a week for 2 consecutive injections.

[0109] Test case: Validity study of the rat premature ovarian failure model

[0110] SPF SD rats (purchased from Zhuhai Baishitong Biotechnology Co., Ltd.), female, 7-8 weeks old; SPF SD male rats, 3 months old. The animals were adaptively raised for 7 days, and the estrous cycle of female rats was examined by vaginal exfoliated cell smear (unstained). Rats with normal estrous cycle were enrolled and divided into 4 groups (model group + blank group + ordinary mesenchymal stem cell treatment group and targeted stem cell treatment group), with 6 rats in each group. The model was established by intraperitoneal injection of cyclophosphamide 50 mg / kg (day 1) + intraperitoneal injection of cyclophosphamide 8 mg / kg (day 2-day 15). Vaginal exfoliated cell detection (unstained) was performed every morning from Day 1 at 10:00. When irregular estrous cycle reaction ovarian hypofunction occurred, it was regarded as the standard of successful modeling. On Day 16 and Day 23, both treatment groups were injected with 3×10 6 The mesenchymal stem cells with resistance to ovarian granulosa cell aging obtained in Example 3 and other common mesenchymal stem cells were measured at 100 μg / ml*1 ml. On Day 44, blood was collected from 6 rats in each group and serum was separated and stored at -80°C. On Day 44, 3 rats in each group were euthanized, and bilateral ovarian tissues were taken, weighed, and the organ index was calculated. The tissues were fixed with 10% formalin and stored at room temperature. Serum samples frozen on Day 44 were tested (24 samples in total): ELISA was used to detect FSH, LH, E2 and AMH.

[0111] Fertility assessment Day 44, 4 weeks after stem cell treatment, 3 rats remained in each group (a total of 12 rats), and they were caged in a female-to-male ratio of 3:1, and caged together using the long-term cohabitation method until all matings were completed. The appearance of vaginal plugs in female rats was recorded as the first day of conception (GD0), and the female rats were separated. On GD14, the rats were euthanized and laparotomy was performed, and the pregnancy status or the number of fetuses in the bilateral uterine horns was observed and recorded.

[0112] The results of animal experiments showed that the rats lost weight and had estrous cycle disorders after modeling.

[0113] 1. Body weight and organ index: The organ index results are shown in Table 1 below.

[0114] From Table 1, it can be seen that the stem cell treatment group, model group and blank group showed weight loss and ovarian atrophy, but the body weight and ovarian tissue weight of the two treatment groups were significantly higher than those of the model group.

[0115] Table 1

[0116]

[0117] 2. ELISA detection of FSH, LH, E2 and AMH: The results are shown in Table 2 below.

[0118] Combined with Table 2, it can be seen that compared with the model group, the treatment group showed a regular cycle, and E2, AMH and FSH recovered to varying degrees.

[0119] Table 2

[0120]

[0121] 3. Fertility assessment: See Table 3 below for conception status.

[0122] Table 3

[0123]

[0124] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing mesenchymal stem cells that resist ovarian granulosa cell aging, characterized in that: The steps include: Provide umbilical cord blood stem cells; Inducing and culturing the umbilical cord blood stem cells, collecting the supernatant after the culture is completed, and isolating exosomes from the supernatant; Mesenchymal stem cells are provided, and the mesenchymal stem cells are induced and cultured by adding the exosomes to obtain the required mesenchymal stem cells that resist ovarian granulosa cell aging.

2. The method for preparing mesenchymal stem cells that resist ovarian granulosa cell aging according to claim 1, characterized in that: The operation of providing umbilical cord blood stem cells is: after centrifuging the umbilical cord blood, taking the lower layer of plasma, and separating the umbilical cord blood stem cells from the plasma.

3. The method for preparing mesenchymal stem cells that resist ovarian granulosa cell aging according to claim 2, characterized in that: The operation of inducing the umbilical cord blood stem cells is as follows: adding complete culture medium to the umbilical cord blood stem cells to a concentration of 2×10 6 / mL~6×10 6 / mL, and placed in a CO2 incubator at 37°C, saturated humidity, and 5% volume fraction for culture. The culture time is 3 to 7 days, and the medium is changed 1 to 2 times during the culture process, wherein the complete culture medium is DMEM / F12 culture medium + 1v / v% to 2v / v% B-27 serum-free additive + 0.5ng / mL to 1ng / mL of recombinant human bFGF factor + 5ng / mL to 10ng / mL of recombinant human VEGF-165 factor.

4. The method for preparing mesenchymal stem cells that resist ovarian granulosa cell aging according to claim 3, characterized in that: The operation of separating the umbilical cord blood stem cells from the plasma is: separating the umbilical cord blood stem cells from the plasma using density gradient centrifugation.

5. The method for preparing mesenchymal stem cells that resist ovarian granulosa cell aging according to claim 4, characterized in that: The operation of isolating exosomes from the supernatant is as follows: taking the supernatant, first centrifuging at 500×g for 30 min at 4°C, then centrifuging at 200×g for 20 min, discarding the precipitate, passing the supernatant through a 0.1 μm filter in a sterile clean bench, and ultracentrifuging at 100,000×g for 1 h. The precipitate is the exosome.

6. The method for preparing mesenchymal stem cells that resist ovarian granulosa cell aging according to any one of claims 1 to 5, characterized in that: The operation of inducing the mesenchymal stem cells by adding the exosomes is as follows: inoculating the mesenchymal stem cells into a culture bottle, adding a complete medium for mesenchymal stem cells, and digesting the cells with 0.05wt% trypsin when the cells are cultured to 85% confluence, collecting P1 cells for passaging, and passaging to P3 according to the above method to complete the induction culture, wherein the complete medium for mesenchymal stem cells contains 5v / v% to 10v / v% of an exosome solution, and the concentration of the exosomes in the exosome solution is 1×10 10 / mL~10×10 10 Pieces / mL.

7. The method for preparing mesenchymal stem cells that resist ovarian granulosa cell aging according to claim 6, characterized in that: The mesenchymal stem cells were inoculated into a culture bottle and a complete mesenchymal stem cell culture medium was added. The mesenchymal stem cells were cultured at a rate of 5×10 3 Pieces / cm 2 ~8×10 3 Pieces / cm 2 The density of the mesenchymal stem cells is 3.5×10 4 / mL~5.6×10 4 Pieces / mL.

8. The method for preparing mesenchymal stem cells that resist ovarian granulosa cell aging according to claim 7, characterized in that: The operation of providing mesenchymal stem cells is as follows: after cleaning the umbilical cord, two arteries and one vein are removed, Wharton's jelly is torn off, and the Wharton's jelly is cut into 1 mm 3 The tissue blocks were inoculated into a container containing complete medium for mesenchymal stem cells and placed in an incubator at 37°C and 5% CO 2 After 1 week, the tissue blocks were removed and the culture medium was replaced. The medium was changed every 3 days. When the cells grew to 80% confluence, they were digested with 0.05% trypsin and the P0 cells were collected and cultured at 5×10 3 Pieces / cm 2 The cells were inoculated at a density of 100 μg / cm2 in a culture flask containing 25 mL of the complete medium for mesenchymal stem cells for proliferation and culture. When the cells grew to 85% confluence, they were digested with 0.05% trypsin and the P1 generation cells were collected, which were the mesenchymal stem cells.

9. The method for preparing mesenchymal stem cells that resist ovarian granulosa cell aging according to claim 6, characterized in that: The umbilical cord blood stem cells are human umbilical cord blood stem cells, and the mesenchymal stem cells are human mesenchymal stem cells.

10. A mesenchymal stem cell that resists ovarian granulosa cell aging, characterized in that: The mesenchymal stem cells that resist ovarian granulosa cell aging are prepared by the method for preparing mesenchymal stem cells that resist ovarian granulosa cell aging as claimed in any one of claims 1 to 9.

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