Stem cell composition for ovarian repair, preparation and preparation method of stem cell composition

Abstract

The invention provides a stem cell composition for ovarian repair, a preparation and a preparation method thereof, and belongs to the technical field of ovarian repair technology, the stem cell composition for ovarian repair comprises the following components: 4 * 10 < 4 >-8 * 10 < 4 > umbilical cord mesenchymal stem cells, 100-150ng of insulin-like growth factor-1 and 40-60ng of epidermal growth factors. The technical scheme provided by the invention has great significance in remarkably improving the ovarian function recovery effect.

Description

Technical Field

[0001] The present application relates to the technical field of ovarian repair, and more specifically, to a stem cell composition, a preparation and a preparation method thereof for ovarian repair. Background Art

[0002] Premature ovarian failure (POF) is a relatively common disease of the female reproductive system, with premature ovarian function failure (such as oligomenorrhea, amenorrhea, infertility, etc.) as the main clinical manifestation, and reduced estrogen level and elevated gonadotropin level in serum as its main characteristics. The inducements of POF include iatrogenic factors such as anti-tumor treatment, idiopathic factors, genetic and immune factors, etc., and bad living and working styles are also its inducements. For these etiologies and mechanisms, the current main treatment measures for POF include hormone replacement therapy, gonadotropin-releasing hormone receptor agonist therapy, cryopreserved tissue (oocyte, ovarian tissue, embryo) transplantation, immunomodulation, gene therapy, etc. The effects of these treatments mainly focus on improving clinical symptoms, it is difficult to completely restore the normal ovarian function, and there are also obvious adverse reactions, such as hormone replacement therapy increasing the risk of cancer.

[0003] In recent years, stem cell therapy has become a potential solution for the treatment of premature ovarian failure due to its repair and regeneration functions. Mesenchymal stem cells (MSCs) can differentiate into cells that support ovarian function, such as follicular granulosa cells and ovarian stromal cells. However, there are problems such as low cell survival rate, poor migration and colonization effects when using stem cell therapy alone.

[0004] Polypeptides have the effects of promoting cell proliferation, anti-apoptosis and regulating the cell microenvironment, and can improve the effect of stem cell therapy. Therefore, it is of great significance to develop a stem cell preparation that can significantly improve the effect of ovarian function recovery. Summary of the Invention

[0005] In order to improve the effect of ovarian function repair, the present application provides a stem cell composition, a preparation and a preparation method thereof for ovarian repair.

[0006] A stem cell composition for ovarian repair, comprising the following components: 4×10 4 -8×10 4 umbilical cord mesenchymal stem cells, 100-150 ng of insulin-like growth factor-1, 40-60 ng of epidermal growth factor.

[0007] The stem cell composition for ovarian repair, comprising the following components: 4×10 4 -8×10 4 umbilical cord mesenchymal stem cells, 125 ng of insulin-like growth factor-1, 50 ng of epidermal growth factor.

[0008] The stem cell composition for ovarian repair includes the following components: 6×10 4 umbilical cord mesenchymal stem cells, 125 ng of insulin-like growth factor-1, and 50 ng of epidermal growth factor.

[0009] A stem cell preparation for ovarian repair is prepared from raw materials in the following volumes: 4 - 6 volume parts of umbilical cord mesenchymal stem cell suspension, 2 - 3 volume parts of insulin-like growth factor-1 buffer solution, and 2 - 3 volume parts of epidermal growth factor buffer solution.

[0010] Preferably, the stem cell preparation for ovarian repair is prepared from raw materials in the following volumes: 5 volume parts of umbilical cord mesenchymal stem cell suspension, 2.5 volume parts of insulin-like growth factor-1 buffer solution, and 2.5 volume parts of epidermal growth factor buffer solution.

[0011] Among the raw materials of the stem cell preparation for ovarian repair, the concentration of the umbilical cord mesenchymal stem cell suspension is 4×10 4 - 8×10 4 umbilical cord mesenchymal stem cells per 5 mL of umbilical cord mesenchymal stem cell suspension, preferably 6×10 4 umbilical cord mesenchymal stem cells. The umbilical cord mesenchymal stem cell suspension is a physiological saline suspension of umbilical cord mesenchymal stem cells.

[0012] Preferably, the umbilical cord mesenchymal stem cells are P2 generation umbilical cord mesenchymal stem cells.

[0013] Among the raw materials of the stem cell preparation for ovarian repair, the concentration of the insulin-like growth factor-1 buffer solution is 40 - 60 ng / mL, preferably 50 ng / mL. The insulin-like growth factor-1 buffer solution is prepared from insulin-like growth factor-1 and PBS buffer solution.

[0014] Among the raw materials of the stem cell preparation for ovarian repair, the concentration of the epidermal growth factor buffer solution is 40 - 60 ng / mL, preferably 50 ng / mL. The epidermal growth factor buffer solution is prepared from epidermal growth factor and PBS buffer solution.

[0015] The preparation method of the umbilical cord mesenchymal stem cell suspension includes the following steps: 1) Primary treatment of UC-MSCs Wash the umbilical cord, remove the epidermis, artery, and vein to obtain Wharton's jelly; Cut Wharton's jelly into pieces, add serum-free mesenchymal stem cell medium, and perform primary culture in a carbon dioxide incubator; 2) Primary culture of UC-MSCs After culturing the primary UC-MSCs in step 1), when the number of cells crawling out of the clone clusters is relatively large, take them out; 3) Subculture and cryopreservation of UC-MSCs Remove the supernatant and tissue blocks from the UC-MSCs taken out in step 2), add trypsin for digestion, and terminate with trypsin inhibitor when 80% of the cells are digested; filter, centrifuge and separate, discard the supernatant, add normal saline to resuspend; centrifuge and separate, discard the supernatant, add fresh serum-free mesenchymal stem cell medium to resuspend, and culture in a carbon dioxide incubator; When the cell confluence reaches 80-90%, collect the cells, discard the supernatant, add trypsin for digestion, and terminate with trypsin inhibitor when 80% of the cells are digested; filter, centrifuge and separate, discard the supernatant, add normal saline to resuspend; centrifuge and separate, discard the supernatant, and perform the last resuspension to obtain.

[0016] Preferably, the umbilical cord is selected from the umbilical cords of healthy infants born to healthy pregnant women aged 20-30. Remove both ends of the umbilical cord before cleaning. When cleaning, wash it twice with normal saline to remove the blood and dirt on the surface, then wash it once with 75% (v / v) alcohol, and then wash it twice with normal saline. Cut the washed umbilical cord into small sections before removing the umbilical cord epidermis, arteries and veins.

[0017] When judging the number of cells crawling out of the clone clusters in step 2), first take out the cells after 5 days of primary culture to observe whether there are cells crawling out, and perform the first medium change, aspirate the old medium, add fresh medium for culture, and change the medium every 2 days thereafter. When 4-6 dense cell clone clusters crawl out and the clone clusters grow densely, start subculturing.

[0018] In step 3), the trypsin is a 0.4% (m / v) trypsin buffer solution. Centrifuge at 500 g for 5 min during centrifugation. In the last resuspension in step 3), slowly add the cryopreservation solution to resuspend the cells according to the counting result, adjust the cell density to control at 3 million per milliliter, then mix the cells evenly and aliquot them into cryopreservation tubes, and perform programmed cooling of the cryopreserved cells and transfer them to a liquid nitrogen tank.

[0019] In summary, the present application has the following beneficial effects: The present invention provides a preparation for the combined application of stem cells and polypeptides to repair and regenerate ovarian tissue. This preparation acts on the damaged ovary through the regeneration function of stem cells and the biological activity support of polypeptides, promotes ovarian tissue repair, reconstructs the vascular network, restores follicle development and hormone secretion function, and finally realizes the reconstruction of ovarian function. This preparation has broad application prospects in the treatment of premature ovarian failure, not only providing a new solution for infertility patients, but also bringing new breakthroughs in the field of female reproductive health.

[0020] Compared with the prior art, the advantages of the present invention also lie in: 1. Multi-party collaboration: The combined application of UC-MSCs, IGF-1, and EGF can more comprehensively solve problems such as low cell survival rate and poor proliferation ability existing in the repair of ovarian endometrial cells by single stem cells or polypeptides. The combined use of UC-MSCs, IGF-1, and EGF synergistically acts through multiple mechanisms such as cell proliferation, anti-apoptosis, angiogenesis, immunomodulation, ECM remodeling, and exosome transmission, effectively accelerating the repair and regeneration of ovarian endometrial cells. 2. Optimization of the tissue microenvironment: UC-MSCs provide support for ovarian repair, and the loaded IGF-1 and EGF can improve the cell microenvironment and promote the repair and reconstruction of ovarian endometrial cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a microscopic view of ovarian endometrial cells in Example 5 of the present invention; Figure 2 It is a modeling diagram of ovarian endometrial cell injury in Example 5 of the present invention; Figure 3 It is a diagram of reagent A repairing ovarian endometrial cells in Example 5 of the present invention; Figure 4 It is a diagram of reagent B repairing ovarian endometrial cells in Example 5 of the present invention; Figure 5 It is a diagram of reagent C repairing ovarian endometrial cells in Example 5 of the present invention; Figure 6 It is a diagram of reagent D repairing ovarian endometrial cells in Example 5 of the present invention; Figure 7 It is a diagram of reagent E repairing ovarian endometrial cells in Example 5 of the present invention; Figure 8 It is a diagram of reagent F repairing ovarian endometrial cells in Example 5 of the present invention; Figure 9 It is a microscopic view of ovarian endometrial cells in Example 6 of the present invention; Figure 10 It is a modeling diagram of ovarian endometrial cell injury in Example 6 of the present invention; Figure 11 It is a diagram of reagent a repairing ovarian endometrial cells in Example 6 of the present invention; Figure 12 It is a diagram of reagent b repairing ovarian endometrial cells in Example 6 of the present invention; Figure 13 It is a diagram of reagent c repairing ovarian endometrial cells in Example 6 of the present invention; Figure 14 It is a diagram of reagent d repairing ovarian endometrial cells in Example 6 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0022] The following further elaborates on the present application in conjunction with embodiments.

[0023] Umbilical cord mesenchymal stem cells (UC-MSCs) possess strong proliferation, repair, and differentiation capabilities. Compared with other types of stem cells, UC-MSCs exhibit the best performance in promoting the proliferation and repair of ovarian endometrial cells and restoring normal growth. Therefore, UC-MSCs are selected in this patent. UC-MSCs provide a microenvironment conducive to the repair and regeneration of the ovarian endometrium through multiple mechanisms such as paracrine action, immunomodulation, exosome transmission, promotion of angiogenesis, ECM remodeling, and antioxidant effects. These mechanisms work together synergistically to promote the repair and proliferation of endometrial cells, enabling UC-MSCs to show good effects in the treatment of ovarian endometrial injury.

[0024] Insulin-like growth factor-1 (IGF-1) is an important growth factor that can promote cell proliferation, differentiation, and tissue repair. In the ovarian endometrium, IGF-1 can directly stimulate the proliferation of endometrial cells.

[0025] Epidermal growth factor (EGF) is a growth factor composed of small molecular polypeptides, containing 53 amino acid residues. It was initially discovered in the salivary glands of mice and later also found in the human body. EGF can bind to the EGF receptor (EGFR) on the cell membrane, activating a series of intracellular signal pathways, thereby regulating various physiological processes such as cell proliferation, differentiation, and survival.

[0026] The preparation of the present invention is prepared by using UC-MSCs, IGF-1, and EGF in a certain proportion. When umbilical cord mesenchymal stem cells (UC-MSCs) are used in combination with insulin-like growth factor-1 (IGF-1) and epidermal growth factor (EGF), they can act synergistically through multiple mechanisms to effectively promote the repair and proliferation of ovarian endometrial cells. This combined use not only enhances the individual effects of each factor but also constructs a favorable cell microenvironment through mutually enhancing signal pathways, accelerating tissue regeneration and cell differentiation. The specific mechanisms of their combined action are as follows: 1) Promote cell proliferation and differentiation EGF binds to the EGFR receptor, activating signal pathways such as MAPK / ERK, significantly promoting the proliferation and differentiation of endometrial cells and accelerating tissue regeneration.

[0027] IGF-1 activates the PI3K / AKT pathway through the IGF-1R receptor, further promoting cell proliferation and providing anti-apoptotic protection.

[0028] UC-MSCs secrete a variety of growth factors (such as IGF-1, FGF-2, etc.) and cytokines, enhancing the paracrine effect, and acting synergistically with exogenous EGF and IGF-1 to increase the proliferation rate and differentiation potential of ovarian endometrial cells.

[0029] 2) Anti-apoptotic effect The anti-apoptotic effect of IGF-1 mainly activates the PI3K / AKT pathway, inhibits apoptosis-related factors, and ensures a high survival rate of endometrial cells during the repair process.

[0030] EGF reduces apoptotic signals and increases cell viability through the MAPK / ERK pathway.

[0031] Anti-apoptotic factors such as Bcl-2 secreted by UC-MSCs synergize with the anti-apoptotic signals of IGF-1 and EGF to enhance the tolerance and survival ability of ovarian endometrial cells, thus facilitating the continuous proliferation and repair of cells.

[0032] 3) Enhance angiogenesis and improve local nutrient supply UC-MSCs secrete angiogenesis factors such as VEGF, promoting the formation of new blood vessels and increasing local blood flow.

[0033] When EGF acts together with UC-MSCs, it can accelerate the proliferation and differentiation of vascular endothelial cells, providing more nutrients for the repair area.

[0034] The enhanced blood supply provides sufficient nutrients and oxygen for the repair and proliferation of endometrial cells, promoting the normal metabolism and functional recovery of cells.

[0035] 4) Immunomodulatory effect UC-MSCs regulate the immune response by secreting anti-inflammatory factors such as IL-10, reducing local inflammatory responses and creating a microenvironment conducive to repair.

[0036] EGF also has an anti-inflammatory effect to a certain extent, synergizing with the immunomodulatory effect of UC-MSCs to reduce the immune attack on endometrial cells and prevent secondary damage to repair cells by inflammation.

[0037] 5) Extracellular matrix (ECM) remodeling Matrix metalloproteinases secreted by UC-MSCs (such as MMP-2, MMP-9) can remodel the extracellular matrix, providing space for cell migration and proliferation.

[0038] Under the action of EGF and IGF-1, the ECM components secreted by endometrial cells are rebuilt, contributing to the integration and functional recovery of the repaired tissue.

[0039] 6) Synergistic effect of exosomes and miRNAs The exosomes secreted by UC-MSCs contain abundant growth factors, miRNAs, etc., which directly act on endometrial cells to regulate cell proliferation, differentiation, anti-apoptosis, etc.

[0040] EGF and IGF-1 jointly regulate the repair pathway with the miRNAs in exosomes, strengthen the regulation effect of gene expression, and further promote the repair and regeneration of ovarian endometrial cells.

[0041] 7) Comprehensive effect: Multi-pathway synergistic effect When UC-MSCs are combined with EGF and IGF-1, by activating multiple cell signaling pathways such as MAPK / ERK, PI3K / AKT, JAK / STAT, etc., multi-faceted support for cell proliferation, anti-apoptosis, immune regulation, and angiogenesis is achieved.

[0042] The comprehensive activation of these signaling pathways forms a benign cell microenvironment, promoting the rapid repair and proliferation of endometrial cells, and making the restoration of ovarian endometrial tissue more efficient.

[0043] Example 1 Preparation of umbilical cord mesenchymal stem cells (UC-MSCs) 1) Primary treatment of UC-MSCs The umbilical cord is selected from the umbilical cord of a healthy baby born to a healthy pregnant woman aged 20 - 30 years. Remove both ends of the umbilical cord, wash it twice with physiological saline to remove the blood and dirt on the surface; then wash it once with 75% (v / v) alcohol and wash it twice with physiological saline again; Cut the washed umbilical cord into small segments, remove the umbilical cord epidermis, arteries and veins, and only leave Wharton's jelly; cut Wharton's jelly into small pieces, and then aliquot Wharton's jelly into sterile T75 culture flasks, add serum-free mesenchymal stem cell medium, and place it in a carbon dioxide incubator for primary culture.

[0044] 2) Primary culture of UC-MSCs After 5 days of the above primary culture, take it out to observe whether cells have crawled out, and perform the first medium change. Aspirate the old medium and add fresh medium for culture. Subsequently, change the medium every 2 days. When 4 - 6 dense cell clone clusters have crawled out and the clone clusters grow densely, start subculturing.

[0045] 3) Subculture and cryopreservation of UC-MSCs When the primary culture reaches the passage time, remove the supernatant and tissue clumps from the primary culture, add trypsin with a mass-volume fraction of 0.4% for digestion. When 80% of the cells are digested under the microscope, terminate with trypsin inhibitor. Filter the cell suspension into a 50 mL centrifuge tube, centrifuge at 500 g for 5 min. After centrifugation, discard the supernatant, add an appropriate amount of normal saline to resuspend the cells, sample and count. According to the counting result, take the required cells into a new 50 mL centrifuge tube, centrifuge at 500 g for 5 min, discard the supernatant, add fresh serum-free mesenchymal stem cell medium to resuspend, aliquot into sterile T75 culture flasks and supplement the medium, and transfer to a carbon dioxide incubator for culture.

[0046] After culturing for 2 days, start observing under the microscope. When the cell confluence reaches 80 - 90%, start collecting cells. Take out the culture flask, discard the supernatant, add trypsin with a mass-volume fraction of 0.4% for digestion. When 80% of the cells are digested under the microscope, terminate with trypsin inhibitor. Transfer the cell suspension to a 50 mL centrifuge tube, centrifuge at 500 g for 5 min. After centrifugation, discard the supernatant, add a certain amount of normal saline to resuspend the cells, sample and count, supplement normal saline in the centrifuge tube for centrifugation, centrifuge at 500 g for 5 min, discard the supernatant. According to the counting result, slowly add cryopreservation solution to resuspend the cells, adjust the cell density to control at 3 million per milliliter, then mix the cells evenly and aliquot into cryotubes, and cool the cryopreserved cells stepwise and transfer to a liquid nitrogen tank.

[0047] Example 2 Preparation of Insulin-like Growth Factor-1 (IGF-1) Prepare a suspension of insulin-like growth factor-1 (IGF-1) at 50 ng / mL using PBS.

[0048] Example 3 Preparation of Epidermal Growth Factor (EGF) Prepare a suspension of epidermal growth factor (EGF) at 20 ng / mL using PBS.

[0049] Example 4 Preparation of Stem Cell Composition for Repairing Ovarian Endometrial Cells 1) Preparation of Passage 2 Umbilical Cord Mesenchymal Stem Cells Resuscitate Passage 1 umbilical cord mesenchymal stem cells, transfer the cell suspension to a centrifuge tube pre-added with normal saline, centrifuge at 500 g for 5 min, discard the supernatant, add an appropriate amount of serum-free mesenchymal stem cell medium to resuspend the cells, sample and count. According to the counting result, supplement the medium, fully mix the cells and inoculate into the corresponding number of sterile T75 culture flasks and supplement the medium, and transfer to a carbon dioxide incubator for culture.

[0050] After culturing for 2 days, microscopic observation was started. When the cell confluence reached 80 - 90%, the cells were collected. The culture flask was taken out, the supernatant was discarded, and 0.4% (mass / volume) trypsin was added for digestion. When 80% of the cells were digested under the microscope, the trypsin was terminated with a trypsin inhibitor; the cell suspension was transferred to a 50 mL centrifuge tube, centrifuged at 500 g for 5 min, the supernatant was discarded after centrifugation, a certain amount of normal saline was added to resuspend the cells, sampled and counted. According to the counting results, the cell suspension with the required number of cells was drawn into a new centrifuge tube, and normal saline was added to make up the volume and centrifuged at 500 g for 5 min for standby.

[0051] 2) Preparation of the stem cell composition for repairing ovarian endometrial cells For the P2 generation of umbilical cord mesenchymal stem cells prepared in step 1) and kept for standby, the supernatant was discarded, 5 mL of PBS was added to resuspend the cells, and then 5 mL of the prepared IGF-1 was aspirated and added to this cell suspension. After thorough mixing, the stem cell composition for repairing ovarian endometrial cells was obtained.

[0052] Example 5 1) Preparation, culture and injury modeling of human ovarian endometrial cells Resuscitate human ovarian endometrial cells, transfer the cell suspension to a centrifuge tube pre-added with normal saline, centrifuge at 500 g for 5 min, discard the supernatant, add an appropriate amount of serum-free mesenchymal stem cell medium to resuspend the cells, sample and count. According to the counting results, the medium was made up, the cells were thoroughly mixed and inoculated into the corresponding number of sterile T75 culture flasks and the medium was made up, and then transferred to a carbon dioxide incubator for culture; On the 2nd day of culture, observe the ovarian endometrial cells under the microscope and take pictures, as Figure 1 shown.

[0053] Injury modeling of ovarian endometrial cells: Transfer the ovarian endometrial cells cultured for 2 days to a dark box, turn on the ultraviolet lamp and irradiate for 30 min, and then transfer the ovarian endometrium to the microscope to observe whether the modeling is successful and take pictures, as Figure 2 shown.

[0054] Repair test of ovarian endometrial cells Take 6 T75 culture flasks of ovarian endometrial cells with successful modeling and add the following reagents respectively: A. 5 mL PBS B. 5 mL of cell suspension of P2 generation umbilical cord mesenchymal stem cells (resuspended with PBS) 4 P2 generation umbilical cord mesenchymal stem cells C. 5 mL of 50 ng / mL IGF-1 polypeptide solution D. 5 ml of 20 ng / ml EGF polypeptide solution E. 2.5 mL of IGF-1 at 50 ng / mL and combined polypeptide of 2.5 mL of EGF at 20 ng / mL F. 5 mL containing 4×10 4 Composition of P2 cord mesenchymal stem cell suspension added with 2.5 mL of IGF-1 buffer at 50 ng / ml and 2.5 mL of EGF buffer at 20 ng / ml Label these 6 culture flasks respectively as: A. Blank, B. Cord mesenchymal stem cell repair, C. IGF-1 polypeptide repair, D. EGF polypeptide repair, E. Combined polypeptide repair, F. Stem cell composition formula 1 repair; Mix the above culture flasks well and transfer them to the same carbon dioxide incubator for continuous culture.

[0055] After the above ovarian endometrial cells are cultured for another 2 days, take them out and observe and photograph under the microscope, as Figures 3 - 8 shown.

[0056] From Figures 3 - 8 it can be seen that the order of ovarian endometrial repair from the best to the weakest is: stem cell composition formula 1, cord mesenchymal stem cell suspension, IGF-1 polypeptide solution, PBS. Adding stem cell composition formula 1 is the best, adding cord mesenchymal stem cell suspension is the second, adding the combined polypeptide of IGF-1 and EGF has obvious repair, adding IGF-1 or EGF polypeptide solution alone has slight repair, and adding PBS has no repair effect.

[0057] Example 6 1) Preparation, culture and injury modeling of human ovarian endometrial cells Resuscitate human ovarian endometrial cells, transfer the cell suspension to a centrifuge tube pre-added with physiological saline, centrifuge at 500 g for 5 min, discard the supernatant, add an appropriate amount of serum-free mesenchymal stem cell medium to resuspend the cells, take a sample for counting, supplement the medium according to the counting result, mix the cells well and inoculate them into the corresponding number of sterile T75 culture flasks and supplement the medium, then transfer them to the carbon dioxide incubator for culture.

[0058] On the 2nd day of culture, observe and photograph the ovarian endometrial cells under the microscope, as Figure 9 shown.

[0059] Ovarian endometrial cell injury modeling: Transfer the ovarian endometrial cells cultured for 2 days to a dark box, turn on the ultraviolet lamp and irradiate for 30 min, then transfer the ovarian endometrium to the microscope to observe whether the modeling is successful and photograph, as Figure 10 shown.

[0060] 2) Repair test of each formula of stem cell composition for repairing ovarian endometrial cells Take 4 T75 culture flasks of modeled ovarian endometrial cells and add the following reagents respectively: a. 5 ml of PBS b. 5 ml of Stem Cell Composition Formula 1: 5 ml containing 4×10 4 Composition of P2 cord mesenchymal stem cell suspension plus 2.5 ml of 50 ng / ml IGF-1 buffer and 2.5 ml of 20 ng / ml EGF buffer c. 5 ml of Stem Cell Composition Formula 2: 5 ml containing 6×10 4 Composition of P2 cord mesenchymal stem cell suspension plus 2.5 ml of 50 ng / ml IGF-1 buffer and 2.5 ml of 20 ng / ml EGF buffer d. 5 ml of Stem Cell Composition Formula 3: 5 ml containing 8×10 4 Composition of P2 cord mesenchymal stem cell suspension plus 2.5 ml of 50 ng / ml IGF-1 buffer and 2.5 ml of 20 ng / ml EGF buffer And label them with corresponding labels respectively, which are: blank, Stem Cell Composition Formula 1 repair, Stem Cell Composition Formula 2 repair, Stem Cell Composition Formula 3 repair.

[0061] Fully mix the above culture flasks and transfer them to the same carbon dioxide incubator for continuous culture.

[0062] After the above ovarian endometrial cells are cultured for another 2 days, take them out and observe and photograph under a microscope respectively, as Figure 11 shown in Fig. 14.

[0063] It can be seen from Figures 11 - 14 that the order of ovarian endometrial repair from the best to the weakest is: Stem Cell Composition Formula 2, Stem Cell Composition Formula 1, Stem Cell Composition Formula 3, PBS. Adding Stem Cell Composition Formula 2 is the best, adding Stem Cell Composition Formula 1 is the second best, adding Stem Cell Composition Formula 3 is the third best, and adding PBS has no repair effect.

[0064] The specific embodiments of the present invention are only explanations of the present application, and they do not limit the present application. Those skilled in the art can make modifications without creative contributions to the embodiments after reading this specification, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A stem cell composition for ovarian repair, characterized in that, Includes the following components: 4×10 4 -8×10 4 umbilical cord mesenchymal stem cells, 100-150ng of insulin-like growth factor-1, and 40-60ng of epidermal growth factor.

2. The stem cell composition for ovarian repair according to claim 1, wherein: Comprising the following components: 4×10 4 -8×10 4 umbilical cord mesenchymal stem cells, 125 ng of insulin-like growth factor-1, and 50 ng of epidermal growth factor.

3. A stem cell preparation for ovarian repair, characterized in that, Prepared from raw materials including the following volumes: 4 - 6 volume parts of umbilical cord mesenchymal stem cell suspension, 2 - 3 volume parts of insulin-like growth factor-1 buffer solution, and 2 - 3 volume parts of epidermal growth factor buffer solution.

4. The stem cell preparation for ovarian repair according to claim 3, wherein, The concentration of the umbilical cord mesenchymal stem cell suspension is 4×10 4 -8×10 4 umbilical cord mesenchymal stem cells per 5 mL of umbilical cord mesenchymal stem cell suspension.

5. The stem cell preparation for ovarian repair according to claim 3, characterized in that, The umbilical cord mesenchymal stem cells are passage 2 umbilical cord mesenchymal stem cells.

6. The stem cell preparation for ovarian repair according to claim 3, wherein, The concentration of the insulin-like growth factor-1 buffer solution is 40 - 60 ng / mL.

7. The stem cell preparation for ovarian repair according to claim 3, wherein The concentration of the epidermal growth factor buffer solution is 40 - 60 ng / mL.

8. The stem cell preparation for ovarian repair according to claim 3, characterized in that The preparation method of the umbilical cord mesenchymal stem cell suspension includes the following steps: 1) Primary treatment of UC-MSCs Wash the umbilical cord, remove the epidermis, arteries, and veins to obtain Wharton's jelly; Cut the Wharton's jelly into pieces, add a serum-free mesenchymal stem cell medium, and perform primary culture in a carbon dioxide incubator; 2) Primary culture of UC-MSCs After culturing the primary UC-MSCs in step 1), when 4 - 6 dense cell clone clusters crawl out, take them out; 3) Subculture and cryopreservation of UC-MSCs Remove the supernatant and tissue blocks from the UC-MSCs taken out in step 2), add trypsin for digestion, and terminate with a trypsin termination solution when 80% of the cells are digested; filter, centrifuge, discard the supernatant, and resuspend with physiological saline; Centrifuge, discard the supernatant, resuspend with a fresh serum-free mesenchymal stem cell medium, and culture in a carbon dioxide incubator; When the cell confluence reaches 80 - 90%, collect the cells, discard the supernatant, add trypsin for digestion, and terminate with a trypsin termination solution when 80% of the cells are digested; filter, centrifuge, discard the supernatant, and resuspend with physiological saline; Centrifuge, discard the supernatant, resuspend, and obtain.

9. A preparation method of the stem cell preparation for ovarian repair according to claim 3, characterized in that, Including the following steps: preparing an umbilical cord mesenchymal stem cell suspension, preparing an insulin-like growth factor-1 buffer solution, preparing an epidermal growth factor buffer solution, and then mixing the umbilical cord mesenchymal stem cell suspension, insulin-like growth factor-1 buffer solution, and epidermal growth factor buffer solution evenly.

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