Pharmaceutical composition containing high-activity umbilical cord MSC

Abstract

The invention discloses a pharmaceutical composition containing high-activity umbilical cord MSC (mesenchymal stem cells). On the basis of HA-MSC, by reasonably compounding components such as cell growth factors, estrogen and nicotinamide, the disadvantages of high price, large side effect and the like of hormone products are effectively overcome, and the purpose of effectively preventing and / or treating ovarian aging is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of human umbilical cord stem cells, and in particular to a pharmaceutical composition containing highly active umbilical cord MSCs for treating ovarian aging. Background Art

[0002] Premature ovarian failure (POF) usually refers to symptoms of female endocrine disorders or abnormalities such as amenorrhea, significantly increased FSH levels, and significantly decreased estrogen levels in women before the age of 40, which lead to ovarian dysfunction, decline, or even complete failure. At present, the common treatment strategy is estrogen and progesterone therapy. This treatment method is actually a replacement therapy that simulates the ovarian secretion and regulation function of the normal body through the introduction of exogenous hormones to make up for the defects of ovarian dysfunction, but it must be taken until the normal menopausal age of women. As artificially synthesized hormone products, they are often expensive, and long-term use may further aggravate the adverse effects of ovarian dysfunction and endocrine disorders.

[0003] Mesenchymal stem cells (MSC) are a group of adult pluripotent stem cells with the ability to self-renew and differentiate. They are widely available, and the techniques and equipment for their separation and cultivation are becoming increasingly mature. Existing clinical trials have shown the great therapeutic potential of MSC in tissue damage and various degenerative diseases. CN2024111959044 prepared HA-MSC, which has high proliferation activity, small size, high nuclear-cytoplasmic ratio, high expression of embryonic stem cell-related marker antigens SOX2, Nanog, OCT4, and has the potential to differentiate into neural, myocardial, and hepatic and intestinal progenitor cells derived from the three germ layers. Summary of the invention

[0004] The object of the present invention is to provide a composition for preventing and / or treating ovarian aging, characterized in that the active ingredient thereof comprises highly active MSC (HA-MSC).

[0005] More preferably, the active ingredients also include: epidermal growth factor (EGF) and transforming growth factor (TGF).

[0006] More preferably, the active ingredient further comprises estradiol valerate.

[0007] More preferably, the active ingredient further comprises niacinamide.

[0008] More preferably, the active ingredient is a pharmaceutical active ingredient; most preferably, the active ingredient is a pharmaceutical active ingredient for preventing and / or treating ovarian aging.

[0009] Further preferably, the active ingredients thereof include highly active MSC (HA-MSC), epithelial growth factor (EGF), transforming growth factor (TGF), estradiol valerate and niacinamide.

[0010] More preferably, the combination may also contain a stabilizer.

[0011] Further preferably, the stabilizer comprises DPBS (Ca 2+ / Mg 2+ ).

[0012] Further preferably, the stabilizer is DPBS (Ca 2+ / Mg 2+ ).

[0013] Further preferably, the composition comprises the following active pharmaceutical ingredients: highly active MSC (HA-MSC), epithelial growth factor (EGF), transforming growth factor (TGF), estradiol valerate, and nicotinamide.

[0014] More preferably, the composition contains 10 high activity MSC (HA-MSC). 3 -10 10 cells / ml; more preferably 10 5 -10 6 cells / ml HA-MSC; most preferably 10 5 or 10 6 cells / ml HA-MSC.

[0015] Further preferably, the composition contains 10-50 ng of epithelial growth factor (EGF) per ml of highly active MSC; more preferably 10, 15, 20, 25, 30, 35, 40, 45, 50 ng of EGF; most preferably 35 ng of EGF.

[0016] Further preferably, the composition contains 800-1000 ng transforming growth factor (TGF) per ml of highly active MSC; more preferably 800, 850, 900, 950, 1000 ng TGF; most preferably 950 ng TGF.

[0017] Further preferably, the composition contains 0.01-2 mg estradiol valerate per ml of highly active MSC; more preferably 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 1.0, 1.5, 2.0 mg estradiol valerate; most preferably 0.3 mg estradiol valerate.

[0018] Further preferably, in the composition, 0.1-0.3 mg nicotinamide is compounded per ml of highly active MSC; more preferably 0.1, 0.15, 0.2, 0.25, 0.3 mg nicotinamide; most preferably 0.25 mg nicotinamide.

[0019] More preferably, in the composition, the high activity MSC (HA-MSC) is 10 5 -10 6 cells / ml, and each ml of highly active MSCs is compounded with 35ng epithelial growth factor (EGF), 950ng transforming growth factor (TGF), 0.3mg estradiol valerate and 0.25mg niacinamide.

[0020] More preferably, the composition contains 1×PBS or 1×DPBS (Ca 2+ / Mg 2+ ).

[0021] More preferably, in the composition, the high activity MSC (HA-MSC) is 10 5 -10 6 cells / ml, and each ml of high-activity MSC is compounded with 35ng of epithelial growth factor (EGF), 950ng of transforming growth factor (TGF), 0.3mg of estradiol valerate and 0.25mg of nicotinamide; in addition to the above active ingredients, the composition may also contain sterile triple distilled water, physiological saline, 1×PBS or 1×DPBS (Ca 2+ / Mg 2+ ), the latter can be used as a buffer and stabilizer. Most preferably, the composition no longer contains other components (except inevitable impurities).

[0022] Further preferably, the preparation method of the above-mentioned human umbilical cord highly active MSCs refers to the embodiment of CN2024111959044; more preferably, the details of the primary and passage operations refer to the preparation method of embodiment 3 thereof.

[0023] Further preferably, the method for preparing the above-mentioned human umbilical cord highly active MSCs comprises the following steps: The following steps are involved: S1. Reagent preparation; S2, primary culture; S3, subculture; Preferably, it also includes S4, high-activity MSC cryopreservation.

[0024] Further preferably, S1 includes: S11. Preparation of high-activity MSC complete medium: Add TeSR-E8Supplement to TeSR-E8 Basal Medium, invert to mix, then add CloneR, adjust pH to 7.2-7.4 with 6.6% NaHCO3, and store at 4°C for later use; S12. Preparation of high-activity MSC culture flask: Slowly thaw the laminin LN521 stock solution at 2-8°C and add 1× DPBS (Ca 2+ / Mg 2+ ) Dilute the laminin LN521 stock solution to make the coating solution, add the coating solution to the culture bottle to evenly cover the entire bottom of the bottle, and incubate at 2-8℃ for later use; Further preferably, S2 includes: S21, washing the collected human umbilical cord to remove residual blood; S22, breaking down the umbilical cord into small tissue pieces and transferring them into a high-activity MSC culture bottle; S23, add high-activity MSC complete medium to the high-activity MSC culture bottle, shake the bottle slowly, and place it in an incubator at 37°C and 5% CO2 for primary culture; S24, on the 3rd day, add high-activity MSC complete medium, and then change the medium every 3-4 days S25. During subculturing, the detached tissue pieces are collected and secondary adherent culture can be performed according to the above method.

[0025] Further preferably, S3 includes: S31. Take out the cells from the incubator, discard the old culture medium, and gently rinse once with saline; S32, primary high-activity MSCs were added with Recombinant Trypsin-EDTA Solution and placed in an incubator at 37°C and 5% CO2 for 3-5 minutes; S33, after the cells were completely detached, an equal amount of highly active MSC complete medium was added to terminate the digestion, the cells were collected and added with normal saline, and centrifuged at 1500 rpm for 4 mins; S34, discard the supernatant after centrifugation, add high-activity MSC complete medium, gently pipette to mix the cells, and count the number of live cells with trypan blue staining; S35, according to 5000 cells / cm 2 The cells were seeded into new high-activity MSC culture flasks at a seeding density of S36, placed in an incubator at 37°C and 5% CO2 for expansion culture; Further preferably, S4 comprises: selecting highly active MSCs in the logarithmic growth phase and digesting them with Recombinant Trypsin-EDTA Solution, and after the cells shrink and become round, adding highly active MSC complete culture medium to terminate the process, centrifuging at 1500 rpm for 4 minutes, discarding the supernatant, gently blowing and beating the cells to make them uniform, and then packing them into sterile cryopreservation tubes, placing the cryopreservation tubes containing the cells in a programmed cooling box in a -80°C refrigerator, and after 4 hours, storing them in a -196°C liquid nitrogen tank for later use.

[0026] Further preferably, in S4, after centrifugation and discarding the supernatant, MSCs are added to the prepared freezing solution (containing 10% DMSO and 5% glycerol) to adjust the cell density to 4×10 6 / ml, gently blow with a pipette to make the cells uniform, and then divide them into sterile cryopreservation tubes, adding 1.8ml of liquid to each cryopreservation tube.

[0027] Preferably, S4 is washed with 1× DPBS (Ca 2+ / Mg 2+ ) and resuspended; more preferably, the freezing solution (containing 10% DMSO and 5% glycerol) is prepared after resuspending.

[0028] Further preferably, in step S11, the ratio of TeSR-E8 Basal Medium, TeSR-E8 Supplement and CloneR is 24:1:1.

[0029] More preferably, in step S12, the laminin LN521 stock solution is diluted to 5 μg / ml.

[0030] Further preferably, in step S21, the human umbilical cord is repeatedly rinsed with physiological saline containing 100u / ml dual antibody.

[0031] More preferably, in the primary culture of S2, stem cells are cultured using a whole tissue adherence method without stripping off blood vessels and capsules.

[0032] Further preferably, in the subculture of S3, subculture is performed when the primary cells grow to a 80% confluence state.

[0033] Further preferably, the primary culture of human umbilical cord HA-MSC includes: culturing two types of stem cells by the whole tissue adhesion method without stripping off the blood vessels and capsule, and in the culture system of TeSR-E8 Medium containing 5% ClonR, umbilical cord HA-MSC is in a culture bottle coated with laminin LN 521, and cells are observed to grow scattered around the tissue block on the 8th day. The umbilical cord HA-MSC is small in shape, short and spindle-shaped, and has a high nuclear-to-cytoplasmic ratio. On the 12th day after the fluid was changed, a large number of cells were observed to swim out from the periphery of the tissue block. On the 10th day, umbilical cord MSC cells were observed to grow sporadically around the tissue block in an elongated spindle shape, and on the 15th day, they grew in a vortex-like fusion. Compared with MSC, HA-MSC is smaller in size, has a higher nuclear-to-cytoplasmic ratio, and has fewer miscellaneous cells in primary cells.

[0034] Further preferably, the subculture of human umbilical cord HA-MSC includes: when the primary cells grow to a fusion state of about 80%, subculture is performed. The morphology of the two stem cells subcultured to P3 remains unchanged and the growth condition is good. Compared with cells from the same umbilical cord, HA-MSCs subcultured to P9 have maintained a short rod-shaped morphology and good activity; the P9 cells of MSC increase in volume, gradually become wider and flatter in morphology, some cells are polygonal, and the number of granules in the cytoplasm increases, showing an aging state.

[0035] Further preferably, the ovarian aging is severe decline, functional degeneration and / or organ damage.

[0036] More preferably, the composition is a pharmaceutical composition. More preferably, the effective unit dose of the pharmaceutical composition is 1 ml.

[0037] Further preferably, the above composition is a pharmaceutical composition for preventing, improving and / or treating ovarian aging.

[0038] More preferably, the human umbilical cord highly active MSCs are in the form of cell culture or lyophilized powder.

[0039] More preferably, the dosage form of the above-mentioned pharmaceutical composition is selected from injection.

[0040] Further preferably, the above-mentioned pharmaceutical composition is used to prepare a medicament for preventing, improving or treating physiological and / or pathological aging / functional weakening of the skin, liver, heart, ovary, uterus, testicles, muscles, nerves, kidneys, immunity, metabolism, or systemic aging / functional weakening.

[0041] Further preferably, the above pharmaceutical composition is used for preparing a preparation for preventing, improving and / or treating ovarian aging.

[0042] Further preferably, the above pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients, diluents or carriers.

[0043] A pharmaceutical composition, the active ingredients of which are prepared by any of the above-mentioned methods for preparing human umbilical cord highly active MSCs; preferably, further comprising one or more pharmaceutically acceptable excipients, diluents or carriers.

[0044] Compared with the prior art, the present invention has the following beneficial effects: First, improving the culture medium and the supporting culture conditions is simple and easy, with high cell activity and simple operation.

[0045] Second, the use of composite cell preparations is closer to industry needs and can simultaneously maintain cell proliferation activity, which is the key basis for cell preparation products to function smoothly in the body.

[0046] Third, significantly reduce the dosage of hormones to avoid excessive interference with the body's endocrine system. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The morphology of umbilical cord HA-MSCs under an inverted phase contrast microscope (×40).

[0048] Figure 2 This is a diagram of the tail vein injection operation in mice. DETAILED DESCRIPTION

[0049] The present invention will be described in detail below in conjunction with examples, but the examples provided herein are only for illustrative purposes and are not intended to limit the present invention.

[0050] the term “HA-MSC” and “highly active MSC” and “highly active mesenchymal stem cells” are used interchangeably throughout the text.

[0051] "Anti-aging" means reversing, preventing, alleviating or inhibiting the progression of aging and aging-related diseases, disorders or conditions, or one or more symptoms of a disease, disorder or condition.

[0052] "Treatment" refers to slowing, alleviating, ameliorating or reducing at least one symptom of a disease, or reversing its onset after it has occurred. "Prevention" refers to the process of acting before the onset of a disease to prevent the disease from developing or to reduce its severity or slow its development.

[0053] "Pharmaceutical composition" refers to a composition at any stage of the manufacturing process, including the final pharmaceutically acceptable product and any process intermediates thereof.

[0054] "Excipients" are additives other than the main drug in pharmaceutical preparations, and can also be called auxiliary materials. For example, preservatives, antioxidants, flavoring agents, aromatics, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc.

[0055] The various terms and phrases used in the present invention have general meanings known to those skilled in the art. Even so, the present invention still hopes to provide a more detailed description and explanation of these terms and phrases herein. If the mentioned terms and phrases are inconsistent with the known meanings, the meanings expressed in the present invention shall prevail.

[0056] Example 1: Establishment of a mouse model of premature ovarian failure 1 Materials and reagents 1.1 Experimental animals 140 C57BL / 6, SPF-grade female mice, weighing about 30 g.

[0057] 1.2 Experimental Reagents Cisplatin solution for injection (45 μg / mL), FSH immunoassay kit, E2 immunoassay kit.

[0058] 2 Experimental methods 140 mice were divided into blank control group (n=20), PBS treatment group (n=20), and positive control group (n=100). The PBS treatment group and the positive control group were intraperitoneally injected with 1 mL of PBS and cisplatin solution for injection (45 μg / mL) every day for 7 consecutive days.

[0059] On the 8th day, the mice (20 in the blank control group, 20 in the PBS treatment group, and 20 in the randomly selected positive control group) were anesthetized, and 1 mL of blood was collected from the retrobulbar vein of each mouse; after disinfection, the bilateral ovaries were dissected and removed.

[0060] After routine treatment of venous blood, the supernatant was centrifuged and the levels of FSH and E2 were determined by ELISA. The ovaries fixed with 4% paraformaldehyde were routinely paraffin-embedded, and 4 μm sections were prepared. After HE staining, various types of follicles were counted under a light microscope, and the total number of follicles, the number of growing follicles, and the ratio of atretic follicles to the total number of follicles were calculated.

[0061] 3 Experimental Results The external state of the blank control group and the PBS-treated group was normal, and the distribution of estrogen and follicles in ovarian tissue was normal. The weight of the positive control group decreased slightly, the estrogen level decreased significantly, the ovarian morphology was more seriously damaged, the total number of follicles and the number of growing follicles decreased significantly, and the proportion of atretic follicles increased significantly. The above results show that the mouse model of premature ovarian failure was successfully established, and the remaining 80 cisplatin-treated model mice were used for subsequent experiments.

[0062] Example 2: Preparation method of human umbilical cord highly active MSC (HA-MSC) Human umbilical cord high-activity MSCs were prepared according to the conventional steps of Example 1 of CN2024111959044 and the primary and passage details disclosed in Example 3. The details are as follows: 1 Materials 1.1 Source of human umbilical cord The umbilical cords in this experiment were obtained from the Department of Obstetrics and Gynecology of the 920th Hospital of the Joint Logistics Support Force of the Chinese People's Liberation Army. The selected pregnant women were required to have no infectious diseases, no tumor diseases, no family history of genetic diseases, and no congenital diseases in the full-term fetus. With the consent of the pregnant women and their families, the "Informed Consent Form for Umbilical Cord Collection" was signed, and the umbilical cord was collected during delivery. The umbilical cord collection was approved by the Ethics Committee of the 920th Hospital of the Joint Logistics Support Force of the Chinese People's Liberation Army, with the approval number: 2019-036 (Section) -01.

[0063] 1.2 Main Reagents

[0064] 2. Experimental Methods The following steps are involved: S1. Reagent preparation S1-1. Screening and preparation of culture medium: S1-1-1. Based on DMEM / F12, gradually enrich the components of the culture medium, such as replacing bovine serum with human platelet lysate, replacing bovine serum albumin with human serum albumin, and gradually replacing components from animal origin with components from human origin, thereby reducing the immune rejection reaction caused by differences in species origin; there is also the practice of completely abandoning ingredients of animal origin based on DMEM / F12, that is, completely free of ingredients of animal origin (including those of human origin), adding trace elements such as selenium, vitamins, transferrin, insulin, basic fibroblast growth factor, as well as buffer pairs to maintain the pH stability of the solution, antioxidants and other substances. We tried to use DMEM / F12 as the basis, adding human platelet lysate to replace FBS, and observed that the cell proliferation ability remained good, but the in vitro osteogenic and adipogenic abilities were reduced. Then we continued to add transferrin, insulin, basic fibroblast growth factor, etc., and observed that the cell morphology was closer to the original stem cells, the cells were small and translucent, with clear outlines, a high nuclear-cytoplasmic ratio, and the in vitro tri-lineage differentiation ability remained good. The above additives played a key role in maintaining the stemness of stem cells; CloneR was also added to prevent and control the cloning efficiency of single stem cells, and it was observed that the cells still maintained a good proliferation ability at a low density of seeding. Finally, the HA-MSC complete medium described in S1-1-2 was determined.

[0065] S1-1-2. Preparation of HA-MSC complete culture medium: Add 20 ml TeSR-E8 Supplement to 480 ml TeSR-E8 Basal Medium, invert to mix, then add 20 ml CloneR, adjust the pH value to 7.2-7.4 with 6.6% NaHCO3, and store in a refrigerator at 4°C until use.

[0066] S1-2. Optimization of the culture environment S1-2-1, Optimization and screening of coating solution: Starting from how to create a primitive microenvironment for stem cell growth, considering the optimization of 2D space, the original use of serum and / or mouse embryonic fibroblast trophoblast was changed to gelatin, Matrigel, and LN521 for pre-treatment of culture flasks. After repeated experiments, it was found that primary cells rarely grew after gelatin coating when they were separated. First, the gelatin coating treatment was excluded, and Matrigel and LN521 extracellular matrices were studied. It was found that both can maintain a relatively primitive cell state for the separated cells. Finally, the coating solution and culture flask treatment method in S1-2-2 were determined.

[0067] S1-2-2. Coating solution for HA-MSC culture flasks: slowly thaw the laminin LN 521 stock solution at 2-8°C and add 1× DPBS (Ca 2+ / Mg 2+ ) Dilute the laminin LN521 stock solution to 5μg / ml to prepare the STSC coating solution. 2 Add 5 ml of coating solution to evenly cover the entire bottom of the bottle, incubate at 2-8°C overnight, and record the bottle as LN521-CoatedT75cm 2 .

[0068] S2. Primary culture of human umbilical cord HA-MSCs The stem cells were cultured using the whole tissue adherence method without stripping off the blood vessels and capsule; S2-1. In a clean bench, repeatedly rinse the collected human umbilical cord with physiological saline containing 100u / ml of dual antibody, remove the residual blood and place it in a 10cm culture dish.

[0069] S2-2, first use tissue scissors to cut the umbilical cord into 1mm pieces 3 Each small tissue block was transferred to a 1.5 ml EP tube. The tissue in the EP tube was separated with iris scissors until it became a paste and then transferred to LN521-Coated T75cm 2 In culture flask.

[0070] S2-3. After adding 3 ml of HA-MSC complete culture medium to each bottle, shake the bottle slowly to evenly disperse the microtissue blocks. Mark the culture time, cell name and generation number on the bottle and place it in an incubator at 37°C and 5% CO2 for primary culture.

[0071] S2-4, on the 3rd day, add 3 ml of complete culture medium, and then change the medium every 3-4 days, and observe the cell growth under an inverted phase contrast microscope. When changing the medium, gently suck up the old culture medium with a pipette and discard it. Move gently and try not to move the tissue blocks attached to the wall.

[0072] S2-5. During subculturing, the detached tissue pieces are collected and secondary adherent culture can be performed according to the above method.

[0073] S3. Subculture of HA-MSCs When the primary cells grow to about 80% fusion, they need to be subcultured. The specific steps are as follows: S3-1. Take out the cells from the incubator, discard the old culture medium in the clean bench, and gently rinse once with saline.

[0074] S3-2. Add 4 ml of Recombinant Trypsin-EDTA Solution to each bottle of primary HA-MSCs and place in an incubator at 37°C and 5% CO2 for 3-5 minutes.

[0075] S3-3. After the cells are completely detached under a microscope, add an equal amount of HA-MSC complete medium to terminate digestion. Collect the cells into a 50 ml centrifuge tube, add physiological saline, and centrifuge at 1500 rpm for 4 minutes.

[0076] S3-4. After centrifugation, discard the supernatant, add 2 ml of complete culture medium, gently pipette to mix the cells, and count the number of living cells by trypan blue staining.

[0077] S3-5, according to 5000 cells / cm 2 The cells were seeded into new LN521-Coated T75 culture flasks at a seeding density of

[0078] S3-6. Mark the culture time, cell name and generation number on the bottle and place it in a 37°C, 5% CO2 incubator for expansion culture. Take photos of each generation of cells.

[0079] S4. Highly active MSC cryopreservation Select HA-MSCs in the logarithmic growth phase and digest them with Recombinant Trypsin-EDTA Solution. After the cells shrink and become round, add the corresponding complete culture medium to terminate the process. Centrifuge at 1500rpm for 4 minutes and discard the supernatant. Add NutriFreezTMD10 Cryopreservation Medium to HA-MSCs, gently blow with a pipette to make the cells uniform, and then divide them into sterile cryopreservation tubes, adding 1.8ml of liquid to each cryopreservation tube. Write the cell name, batch number and freezing time on the cryopreservation tube and keep a record. Place the cryopreservation tube containing cells in a programmed cooling box in a -80℃ refrigerator, and place it in a -196℃ liquid nitrogen tank for 4 hours for storage.

[0080] Example 3: Preliminary study on the treatment of premature ovarian failure 1 Materials and reagents 1.1 Experimental animals Example 1 80 mice were successfully modeled.

[0081] 1.2 Experimental Reagents Highly active MSCs (1×10 10 cells / mL; prepared according to Example 2 of this application, i.e., the conventional steps of Example 1 of CN2024111959044, and the primary and subculture details disclosed in Example 3, the morphology is shown in Figure 1 ), in order to avoid contamination of animal-derived components brought by culture medium components, cells in the logarithmic growth phase were selected, digestion was terminated, and after centrifugation, the cells were gently washed with physiological saline and gently blown to mix the cells for later use; 1×PBS; estradiol valerate.

[0082] 2 Experimental methods The 80 model mice described in Example 1 were randomly divided into 8 groups with 10 mice in each group. According to the dosing schedule (as shown in the following table), each mouse was injected with 0.5 mL of the drug into the tail vein once in the morning and evening every day (the total dose reached the dose set in Table 1 twice in the morning and evening). Figure 2 ), and the injection was continued for 5 days. On the 6th day, the serum hormone level was measured according to the method described in Example 1.

[0083] Table 1: Preliminary experimental dosing regimen

[0084] 3 Experimental Results As shown in Table 2, compared with the blank control and negative control groups, each experimental group was able to significantly reduce FSH levels and increase E2 levels at the same time. However, there was still a certain gap compared with the positive control group.

[0085] Table 2: Preliminary experimental results

[0086] Example 4: Optimization of treatment regimen for premature ovarian failure In order to further improve the treatment effect, the treatment plan was further optimized based on the plan described in Example 3. Based on the development experience accumulated in the early stage, an attempt was made to use epithelial growth factor (EGF) and transforming growth factor (TGF) that can promote MSC proliferation in combination; at the same time, nicotinamide was used in combination to improve the product stability of MSC products. 8On the basis of cells highly active MSC, EGF, TGF, nicotinamide and estradiol valerate were compounded respectively. This application obtained the optimal single agent concentration through preliminary exploration and setting of dose escalation test screening, specifically: EGF 35ng, ​​TGF 950ng, nicotinamide 0.25mg, estradiol valerate 0.3 mg. On this basis, the optimized experimental scheme shown in Table 3 below was designed. In this scheme, in order to maintain and improve the activity of MSC treatment products in vivo, EGF and TGF were added at the same time. Modeling was carried out according to the scheme described in Example 1, and 150 mice were obtained, which were randomly divided into the following 15 groups at 10 / group. Administration was carried out according to the scheme of Example 3, and serum hormone levels and follicular status were determined according to the method of Example 1.

[0087] Table 3: Optimized dosing regimen

[0088] Remark: Experimental results: As shown in Table 4, compared with the blank control and negative control groups, each experimental group was able to significantly reduce FSH levels, increase E2 levels, and significantly improve the follicle state. 8 cells, 10 6 cells, 10 5 Under the conditions of low cell concentration, the follicle improvement effect can be achieved that is equivalent to or even significantly better than that of the positive control group. The use of low cell concentration not only reduces the cost of the product, but also significantly reduces the dosage of estradiol, which is beneficial to reduce the interference with the endocrine conditions of the body.

[0089] Table 4: Experimental results of optimization scheme

[0090] Example 5: Product Development Formulation Optimization Strategy Under long-term storage conditions, the instability of the therapeutic effect was found. The preparation stabilizer was tried to obtain a more easily preserved preparation formula. The cells were prepared according to the method of Example 2. The cells in the logarithmic growth phase were selected, digested and centrifuged, and then washed with physiological saline (Group 1) or 1×DPBS (Ca 2+ / Mg 2+ ) (Group 2) Wash gently and then gently pipette to mix the cells for later use.

[0091] The samples were stored at room temperature (25 °C), refrigerated (4 °C), and frozen (-70 °C, containing 10% DMSO, 5% glycerol, 4 × 10 6 / ml) for 1, 2, 3, 4, 5, 6, 7, 30, 60, and 90 days, and then 3D holographic living cell dynamic observation and transmission electron microscopy observation were performed according to the method disclosed in Example 1 of CN2024111959044.

[0092] The experimental results showed that both Group 1 and Group 2 could not be stored for a long time at room temperature. After 1 day, the cell body edges became obviously irregular, the cell membrane became incomplete, intracellular vacuolation increased, and re-culture became difficult. Under refrigerated conditions, Group 1 and Group 2 could maintain their morphological integrity for about 4 days and 6 days, respectively. Under frozen conditions, there was no significant difference in the morphological integrity of Group 1 and Group 2 after 1, 2, 3, 4, 5, 6, 7, 30, and 60 days of resuscitation and re-culture; there was still no significant difference in the morphology of the two groups of cells after 90 days of resuscitation, but after one subculture, a small amount of cell nucleus irregularity appeared in the cells of Group 1; the two groups of cells in the logarithmic growth phase were taken, and the two groups of cells were divided into two equal parts and then centrifuged, and then washed with 1×PBS or 1×DPBS (Ca) 2+ / Mg 2+ ) were gently washed and then gently blown to mix the cells, thus obtaining groups 1A, 1B, 2A, and 2B.

[0093] The pharmaceutical composition was prepared according to the experimental group 9 of Example 4, and the effect on FSH and E2 levels was determined according to the method of Example 3. The results are shown in Table 5. The results showed that the addition of 1×DPBS (Ca 2+ / Mg 2+ ) can maintain the cell cryopreservation morphology stability and effect stability for a longer period of time, and is also helpful for improving the in vivo effect of HA-MSC.

[0094] Table 5: Preliminary experimental results

[0095] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A composition for preventing and / or treating ovarian aging, characterized in that: Its active ingredients include highly active MSC (HA-MSC), epithelial growth factor (EGF), transforming growth factor (TGF), estradiol valerate, and niacinamide; The method for preparing the highly active MSC comprises the following steps: S1. Reagent preparation S11. Preparation of high-activity MSC complete medium: Add TeSR-E8 Supplement to TeSR-E8 Basal Medium, invert and mix, then add CloneR, adjust the pH value to 7.2-7.4 with 6.6% NaHCO3, and store at 4°C for future use; the ratio of TeSR-E8 Basal Medium, TeSR-E8 Supplement, and CloneR is 24:1:1; S12. Preparation of high-activity MSC culture flask: Slowly thaw the laminin LN521 stock solution at 2-8°C and add 1× DPBS (Ca 2+ / Mg 2+ ) Dilute the laminin LN521 stock solution to make the coating solution, add the coating solution to the culture bottle to evenly cover the entire bottom of the bottle, and incubate at 2-8℃ for later use; S2. Primary Culture S21, washing the collected human umbilical cord to remove residual blood; S22, breaking down the umbilical cord into small tissue pieces and transferring them into a high-activity MSC culture bottle; S23, add high-activity MSC complete medium to the high-activity MSC culture bottle, shake the bottle slowly, and place it in an incubator at 37°C and 5% CO2 for primary culture; S24, on day 3, add high-activity MSC complete medium, and then change the medium every 3-4 days; S25. During the passage, the detached tissue pieces were collected and cultured for the second time according to the above method; S3. Subculture S31. Take out the cells from the incubator, discard the old culture medium, and gently rinse once with saline; S32, primary high-activity MSCs were added with Recombinant Trypsin-EDTA Solution and placed in an incubator at 37°C and 5% CO2 for 3-5 minutes; S33, after the cells were completely detached, an equal amount of highly active MSC complete medium was added to terminate the digestion, the cells were collected and added with normal saline, and centrifuged at 1500 rpm for 4 mins; S34, discard the supernatant after centrifugation, add high-activity MSC complete medium, gently pipette to mix the cells, and count the number of live cells with trypan blue staining; S35, according to 5000 cells / cm 2 The cells were seeded into new high-activity MSC culture flasks at a seeding density of S36. Place in an incubator at 37°C and 5% CO2 for expansion culture.

2. The composition according to claim 1, characterized in that The high activity MSC (HA-MSC) is 10 3 -10 10 cells / ml, and each ml of highly active MSCs is compounded with 10-50 ng epithelial growth factor (EGF), 800-1000 ng transforming growth factor (TGF), 0.01-2 mg estradiol valerate and 0.1-0.3 mg nicotinamide.

3. The composition according to claim 2, characterized in that The high activity MSC (HA-MSC) is 10 5 -10 6 cells / ml, and each ml of highly active MSCs is compounded with 35ng epithelial growth factor (EGF), 950ng transforming growth factor (TGF), 0.3mg estradiol valerate and 0.25mg niacinamide.

4. The composition according to claim 3, characterized in that The method for preparing highly active MSCs further comprises S4, cryopreservation of highly active MSCs, Select highly active MSCs in the logarithmic growth phase and digest them with Recombinant Trypsin-EDTA Solution. After the cells shrink and become round, add highly active MSC complete culture medium to terminate the process. Centrifuge at 1500rpm for 4 minutes, discard the supernatant, gently blow to make the cells uniform, and then divide them into sterile cryopreservation tubes. Put the cryopreservation tubes containing cells into a programmed cooling box in a -80℃ refrigerator. After 4 hours, store them in a -196℃ liquid nitrogen tank for later use.

5. The composition according to claim 4, characterized in that In step S12, the laminin LN521 stock solution is diluted to 5 μg / ml; in step S21, the human umbilical cord is repeatedly rinsed with physiological saline containing 100u / ml double antibody.

6. The composition according to any one of claims 1 to 5, characterized in that In the primary culture of S2, stem cells are cultured by the whole tissue adherence method without stripping off blood vessels and capsules; in the subculture of S3, subculture is performed when the primary cells grow to a 80% confluence state.

7. Use of the composition according to any one of claims 1 to 6 in the preparation of a medicine for preventing and / or treating ovarian aging, wherein the medicine further comprises one or more pharmaceutically acceptable excipients, diluents or carriers.

8. The use according to claim 7, wherein the ovarian aging is severe decline, functional degeneration and / or organ damage.

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