Empowered mesenchymal stem cell, empowered mesenchymal stem cell composition, and use thereof in field of regenerative medicine

Abstract

The present invention relates to the technical field of biomedicine, and specifically provides an empowered mesenchymal stem cell, an empowered mesenchymal stem cell composition, and use thereof in the preparation of a drug for treating organ dysfunction. The empowered mesenchymal stem cell is obtained by treating human mesenchymal stem cells with ingenol HEP14. The empowered mesenchymal stem cell composition comprises the empowered mesenchymal stem cell and an HEP14 / PLGA microsphere having a high HEP14 loading capacity and a long-acting HEP14 sustained release prepared by using poly(lactic-co-glycolic acid) (PLGA) as a carrier. The empowered mesenchymal stem cell has the effects of enhancing the viability and stemness of mesenchymal stem cells, resisting tissue fibrosis, promoting tissue angiogenesis, and promoting tissue regeneration and functional recovery. The combined use of the empowered mesenchymal stem cell and the HEP14 / PLGA microsphere enhances the effects of retention and transdifferentiation of the empowered mesenchymal stem cell in tissues, thereby further enhancing the function of the empowered mesenchymal stem cell and the use thereof in the field of regenerative medicine.

Description

Empowered mesenchymal stem cells, empowered mesenchymal stem cell compositions and their use in the field of regenerative medicine TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, in particular to an empowered mesenchymal stem cell, an empowered mesenchymal stem cell composition and its use in the preparation of a drug for treating ovarian insufficiency or other tissue and organ insufficiency. BACKGROUND

[0002] Ovarian insufficiency is caused by the development disorder and dysfunction of ovarian follicles due to the decrease or depletion of the reserve of follicle pool in the ovary, including premature ovarian insufficiency (POI) and age-related ovarian aging (Age-related Ovarian Aging), i.e. ovarian aging. Premature ovarian insufficiency refers to the functional failure of the ovary caused by various reasons, mainly chemotherapy or / and radiotherapy leading to ovarian damage in women of childbearing age, i.e. amenorrhea occurs before the age of 40, which is the main cause of female infertility. Ovarian aging is a natural law, i.e. normal ovarian function begins to decline gradually with age until ovarian function is depleted. The menopausal syndrome and related degenerative diseases caused by ovarian insufficiency seriously damage the health of women. At present, there is no effective treatment for ovarian insufficiency at home and abroad. The most commonly used hormone replacement therapy for treating ovarian insufficiency can only alleviate the symptoms of low estrogen levels, and has a large side effect, and has no substantial therapeutic effect on improving ovarian reproductive function. Other available treatment methods, such as cryopreservation and transplantation of ovarian tissue, oocytes or embryos, have low efficacy and lack of ovarian tissue resources. Therefore, it is urgent to find / explore a more effective method for treating ovarian insufficiency in the field, which has been one of the most concerned research and development hotspots internationally.

[0003] Stem cells, especially mesenchymal stem cells (MSCs) of various types, are currently a hot research topic in the field of regenerative medicine, especially in the field of reproductive medicine. Therapeutic strategies based on (mesenchymal) stem cells and their exosomes are currently recognized as the most promising means of treating ovarian insufficiency. By promoting the regeneration of ovarian tissue and the development of ovarian follicles, the reproductive and fertility functions of the ovary can be promoted, i.e. ovarian insufficiency can be cured or improved. Compared with other sources of (mesenchymal) stem cells, adipose-derived mesenchymal stem cells (ADSCs) have many significant advantages and characteristics, including that human adipose-derived stem cells (hADSCs) are easy to obtain, have little damage to patients, have enough adipose tissue, can be rapidly expanded in vitro, have high biological safety, and make autologous stem cell transplantation possible, etc. hADSCs have become one of the most promising sources of stem cells and have attracted more and more attention in recent years. Animal transplantation experiments and a recent preliminary clinical trial have shown that ADSCs are effective in treating premature ovarian failure, but the therapeutic effect is not sufficient to meet the purpose of clinical translation for treatment. The main limitations of using ADSCs or other sources of mesenchymal stem cells to treat ovarian or other tissue and organ insufficiency are (ovarian) tissue fibrosis, ischemia in the injection area, and differentiation or aging of the 'transplanted' stem cells. In order to overcome these obstacles, improved stem cell-based treatment regimens have been under development, including pretreatment of stem cells with cytokines, use of collagen scaffolds combined with stem cells to enhance treatment efficacy, etc. Unfortunately, so far, the development regimens reported have not made breakthrough progress in promoting the recovery of ovarian and / or other tissue and organ function or / and elucidating the treatment mechanism. To solve this clinical challenge, basic research and clinical trials around stem cells in regenerative medicine applications are still ongoing.

[0004] HEP14 is a 5β-O-angeloyl-20-deoxymegastigmanol compound extracted from the whole plant of Euphorbia peplus Linn. and is a new activator of the protein kinase C (PKC) pathway. At present, in addition to the effect of regulating lysosome generation that has been reported, other action pathways of HEP14 have not been reported. In the process of exploring the therapeutic effect of HEP14 and its potential mechanism, in order to overcome the poor water solubility of HEP14 and obtain sustained and stable release of HEP14 in the circulation in vivo, we use poly (lactic-co-glycolic acid) (PLGA) as a drug carrier to prepare HEP14-loaded PLGA microspheres, which are called HEP14 / PLGA microspheres. PLGA is a biodegradable copolymer composed of polylactic acid and polyglycolic acid, and is widely used as a carrier to deliver therapeutic drugs due to its safety, operable biodegradation rate and excellent drug loading performance. By adjusting the monomer ratio of lactic acid and glycolic acid, the degradation time of PLGA can be changed, which is particularly important in the field of biomedicine.

[0005] PLGA is a drug carrier approved by the U.S. Food and Drug Administration (FDA).

[0006] SUMMARY

[0007] In view of the fact that no breakthrough has been made in the promotion of ovarian function or / and the recovery of functions of other tissues and organs and the elucidation of the treatment mechanism by the existing mesenchymal stem cell treatment alone or combined treatment, the enabled mesenchymal stem cells of the present application have significantly enhanced survival and stemness, have the effects of resisting tissue fibrosis of ovarian tissue, liver tissue, lung tissue, brain tissue, heart tissue, muscle tissue, bone tissue, kidney tissue, skin tissue, etc., promoting tissue angiogenesis, promoting tissue regeneration and functional recovery, and have the effects of treating organ dysfunction of ovary, liver, lung, brain, heart, muscle, bone, kidney, skin, etc.; the combination of the enabled mesenchymal stem cells and the HEP14 / PLGA microsphere composition further strengthens the retention and transdifferentiation of the enabled mesenchymal stem cells in the organ tissue, so that the combination of the composition further strengthens the effects of resisting tissue fibrosis, promoting tissue angiogenesis, promoting tissue regeneration and functional recovery, and treating organ dysfunction of the enabled mesenchymal stem cells.

[0008] In particular in the ovary, the enabled mesenchymal stem cells have significantly enhanced viability and stemness, and have the effects of resisting ovarian tissue fibrosis, promoting ovarian tissue angiogenesis, promoting ovarian regeneration, follicle development, and improving ovarian endocrine function and / or fertility function (including improving the endocrine function and fertility function of the ovary of the childbearing age, and improving the endocrine function of the ovary of the old age); the combination of the enabled mesenchymal stem cells and the HEP14 / PLGA microsphere composition further strengthens the retention of the enabled mesenchymal stem cells in the ovarian tissue and the effect of transdifferentiating into follicular granulosa cells and theca cells, so that the combination of the composition further strengthens the effects of the enabled mesenchymal stem cells on resisting ovarian tissue fibrosis, promoting ovarian tissue angiogenesis and ovarian regeneration, follicle development, and improving ovarian endocrine function and / or fertility function.

[0009] The present application solves the technical problems by adopting the following technical solutions:

[0010] In one aspect, the present application provides an enabled mesenchymal stem cell, which is obtained by treating human mesenchymal stem cells with HEP14, and the HEP14 is a 5β-O-angeloyl-20-deoxylarge gordonol compound with the following structural formula:

[0011] Preferably, the step of treating human mesenchymal stem cells with HEP14 comprises the following steps:

[0012] (1) Culturing and expanding human mesenchymal stem cells;

[0013] (2) Treating human mesenchymal stem cells with HEP14 at a concentration of 1.25-20 μM in the culture medium for 12-48 hours;

[0014] (3) Digesting human mesenchymal stem cells to obtain a single cell suspension;

[0015] (4) Eluting the human mesenchymal stem cells with physiological saline to obtain the enabled mesenchymal stem cells suspended in physiological saline.

[0016] More preferably, the step (2) of treating human mesenchymal stem cells with HEP14 is treating human mesenchymal stem cells with HEP14 at a concentration of 2.5 μM in the culture medium for 32 hours.

[0017] Preferably, the human mesenchymal stem cells are derived from bone marrow, fat, placenta, umbilical cord, skin, amniotic membrane, and dental pulp.

[0018] More preferably, the human mesenchymal stem cells are derived from adipose tissue.

[0019] Another aspect of the present application provides an enabled mesenchymal stem cell composition, which comprises:

[0020] An enabled mesenchymal stem cell, which is obtained by treating human mesenchymal stem cells with HEP14.

[0021] HEP14 / PLGA microspheres, which are HEP14 / PLGA microspheres with high HEP14 loading and long-term HEP14 sustained release prepared using polylactic-co-glycolic acid (PLGA) as a carrier.

[0022] Preferably, the step of treating human mesenchymal stem cells with HEP14 comprises the following steps:

[0023] (1) culturing and expanding human mesenchymal stem cells;

[0024] (2) treating human mesenchymal stem cells with HEP14 at a concentration of 1.25-20 μM in a culture medium for 12-48 hours;

[0025] (3) digesting human mesenchymal stem cells into a single cell suspension;

[0026] (4) eluting the human mesenchymal single cells with normal saline to obtain the enabled mesenchymal stem cells suspended in normal saline.

[0027] More preferably, the step (2) of treating human mesenchymal stem cells with HEP14 is treating human mesenchymal stem cells with HEP14 at a concentration of 2.5 μM in a culture medium for 32 hours.

[0028] Preferably, the HEP14 / PLGA microspheres with high HEP14 loading and long-term HEP14 sustained release are prepared by the following method:

[0029] In a dark environment, polylactic-co-glycolic acid (PLGA) and HEP14 are dissolved in dichloromethane at a mass ratio of 5-10:1, and a magnetic stirrer is used to stir at a speed of 400-500 rpm, then the obtained solution is added to an aqueous solution containing 1-2% (w / v) polyvinyl alcohol, and dichloromethane is volatilized by stirring at a speed of 300-500 rpm with a magnetic stirrer at 25°C for at least 12 hours, then the microspheres are collected by centrifugation, and repeatedly washed with distilled water to completely remove residual polyvinyl alcohol, finally the microspheres are placed in a freeze dryer for freeze-drying treatment to obtain HEP14 / PLGA microspheres.

[0030] More preferably, the polylactic-co-glycolic acid and HEP14 are dissolved in dichloromethane at a mass ratio of 5:1.

[0031] Preferably, the human mesenchymal stem cell sources comprise mesenchymal stem cells from different human tissue sources of bone marrow, fat, placenta, umbilical cord, skin, amniotic membrane, dental pulp.

[0032] More preferably, the human mesenchymal stem cells are mesenchymal stem cells from adipose tissue.

[0033] Another aspect of the present application provides the use of the enabled mesenchymal stem cells in the preparation of a medicament for treating tissue fibrosis.

[0034] Another aspect of the present application provides the use of the enabled mesenchymal stem cells in the preparation of a medicament for promoting tissue angiogenesis.

[0035] Another aspect of the present application provides the use of the enabled mesenchymal stem cells in the preparation of a medicament for promoting tissue regeneration and functional recovery.

[0036] Preferably, the tissue is one of ovarian tissue, liver tissue, lung tissue, brain tissue, heart tissue, muscle tissue, bone tissue, kidney tissue, skin tissue.

[0037] Another aspect of the present application provides the use of the enabled mesenchymal stem cells in the preparation of a medicament for treating organ dysfunction.

[0038] Preferably, the organ dysfunction is caused by impaired regeneration function or aging of an organ, and the organ is one of ovary, liver, lung, brain, heart, muscle, bone, kidney, skin.

[0039] More preferably, the organ is ovary.

[0040] Preferably, the physiological saline solution containing the enabled mesenchymal stem cells is locally injected into the ovary of a subject in need, including any one of the following uses:

[0041] (1) the use in the preparation of a medicament for treating premature ovarian failure in women of childbearing age;

[0042] (2) the use in the preparation of a medicament for treating ovarian function decline in elderly women and its caused complications;

[0043] (3) the use in the preparation of a medicament for restoring endocrine and / or reproductive function in the ovary.

[0044] Another aspect of the present application provides the use of the enabled mesenchymal stem cell composition in the preparation of a medicament for treating tissue fibrosis.

[0045] Another aspect of the present application provides the use of the enabled mesenchymal stem cell composition in the preparation of a medicament for promoting tissue angiogenesis.

[0046] In another aspect, the present application provides the use of the enabled mesenchymal stem cell composition in the preparation of a medicament for promoting tissue regeneration and functional recovery.

[0047] Preferably, the tissue is one of ovarian tissue, liver tissue, lung tissue, brain tissue, heart tissue, muscle tissue, bone tissue, kidney tissue, and skin tissue.

[0048] In another aspect, the present application provides the use of the enabled mesenchymal stem cell composition in the preparation of a medicament for treating organ dysfunction.

[0049] Preferably, the organ dysfunction is caused by impaired regenerative function of an organ, which is one of ovary, liver, lung, brain, heart, muscle, bone, kidney, and skin.

[0050] More preferably, the organ is ovary.

[0051] Preferably, the subject in need is locally injected with a saline solution containing the enabled mesenchymal stem cells in the ovary, and is also injected with a saline solution containing the HEP14 / PLGA microspheres, including any one of the following uses:

[0052] (1) the use in the preparation of a medicament for treating premature ovarian failure in a woman of childbearing age;

[0053] (2) the use in the preparation of a medicament for treating ovarian function decline and its resulting complications in an elderly woman;

[0054] (3) the use in the preparation of a medicament for restoring endocrine and / or reproductive function of the ovary.

[0055] Fibrosis and ischemia of ovarian tissue or other organ tissue are important causes of loss of transplanted stem cells and failure of cell therapy. The number of transplanted stem cells and the cytokines secreted by them significantly affect the therapeutic effect. Local injection of a saline solution containing the enabled mesenchymal stem cells in the ovary, combined with injection of a saline solution containing the HEP14 / PLGA microspheres into the subject in need, has the following advantages: first, using different administration routes helps to provide more space for local injection of enabled mesenchymal stem cells. Second, this administration route is more conducive to direct, timely and effective interaction between enabled mesenchymal stem cells, follicular cells, cytokines and extracellular matrix. Therefore, it promotes the survival and survival ability of transplanted enabled mesenchymal stem cells in the ovary and / or the corresponding treated organ tissue. Third, this administration method can achieve multiple administrations, and combined with the long-acting and sustained-release characteristics of HEP14 / PLGA microspheres, it can maintain stable and sustained HEP14 blood drug concentration, ultimately improve the treatment of enabled mesenchymal stem cell composition for ovarian dysfunction and / or dysfunction of other organ tissues, especially for severe ovarian dysfunction.

[0056] More preferably, the injection route of the physiological saline solution containing the HEP14 / PLGA microspheres includes any one of intraperitoneal injection, subcutaneous injection, intramuscular injection.

[0057] The treatment of ovarian dysfunction includes promoting the regeneration and functional recovery of damaged ovarian tissue, specifically at least one of the following: (1) ovarian follicle regeneration and development, and an increase in the number of ovarian follicles at each stage; (2) an increase in blood estrogen E2 and AMH levels and a decrease in FSH levels, and an improvement in ovarian endocrine function; (3) a significant increase in pregnancy rate and fertility rate.

[0058] The ovarian follicle regeneration and development includes the development and / or maturation of ovarian mesenchymal tissue and follicles at various levels.

[0059] The present application research found that: HEP14 stimulates the secretion of a variety of factors, including STC1, MMP1 and PDGFD, by activating the PKC-ERK1 / 2 signaling pathway, which synergistically act to improve the microenvironment of organ tissue by strengthening the anti-tissue fibrosis and pro-angiogenic effects of transplanted enabled mesenchymal stem cells through MMP1 and PDGFD; in the ovary, STC1 stimulates the secretion of CYP19A1, which synergistically stimulates the growth of ovarian follicle granules and foam cells, thus promoting follicle development and improving ovarian hormone secretion and endocrine function. In summary, these results show that the enabled mesenchymal stem cells and the enabled mesenchymal stem cell composition of the present application have excellent effects in promoting the regeneration and functional recovery of damaged organ tissue.

[0060] Compared with the prior art, the present application has the following beneficial effects:

[0061] The enabled mesenchymal stem cells of the present application have significantly enhanced viability and stemness, and have the effects of anti-tissue fibrosis, promoting tissue angiogenesis, promoting tissue regeneration and functional recovery, and treating organ dysfunction, such as ovarian, liver, lung, brain, heart, muscle, bone, kidney, skin, etc. The combination of enabled mesenchymal stem cells and HEP14 / PLGA microspheres further enhances the retention and transdifferentiation of enabled mesenchymal stem cells in organ tissue, thus further enhancing the anti-tissue fibrosis, pro-angiogenic effects, and the effects of promoting tissue regeneration and functional recovery and treating organ dysfunction of the combination of enabled mesenchymal stem cells.

[0062] In particular in the ovary, the enabed mesenchymal stem cells have significantly enhanced viability and stemness, and have the effects of anti-ovarian tissue fibrosis, promoting ovarian tissue angiogenesis, promoting ovarian regeneration, follicle development, and improving ovarian endocrine function and / or reproductive function (including improving the endocrine function and reproductive function of the ovary in the reproductive age, and improving the endocrine function of the ovary in the elderly); the combination of enabed mesenchymal stem cells and HEP14 / PLGA microsphere composition further strengthens the retention of enabed mesenchymal stem cells in the ovarian tissue and the effect of transdifferentiating into follicular granulosa cells and theca cells, so that the combination of the composition further strengthens the effects of enabed mesenchymal stem cells on anti-ovarian tissue fibrosis, promoting ovarian tissue angiogenesis and ovarian regeneration, follicle development, and improving ovarian endocrine function and / or reproductive function. BRIEF DESCRIPTION OF DRAWINGS

[0063] Figure 1 is that HEP14 treatment enhances the stemness and proliferation and survival ability of hADSCs.

[0064] Figure 2 is the gene expression profile of HEP14-induced hADSCs and its analysis, and the differentially expressed genes are enriched in signal pathways such as regulation of extracellular matrix (ECM) receptor interaction, cell fate determination, vascularization, ovarian steroid hormone production, and MAPK activity activation, and these biological functions are crucial for the remodeling, regeneration and functional recovery of organ tissues, especially the ovary.

[0065] Figure 3 is the morphology and property analysis of HEP14 / PLGA microspheres.

[0066] Figure 4 is the establishment of doxorubicin-induced ovarian dysfunction model mice, and the results show that a single intraperitoneal injection of doxorubicin at a dose of 12 mg / kg can cause significant damage to the structure and function of the mouse ovary.

[0067] Figure 5 is that enabed adipose stem cells, enabed adipose stem cell composition treatment restores the premature ovarian function in the reproductive age, and enabed adipose stem cells alone or in combination with HEP14 / PLGA microsphere composition treatment both have the significant effects of reducing ovarian tissue fibrosis, promoting ovarian tissue angiogenesis, promoting ovarian follicle development in POI mice, and restoring reproductive and endocrine functions, and the combined treatment of the composition is superior to the treatment effect of enabed hADSCs alone.

[0068] Figure 6 is that enabed adipose stem cells, enabed adipose stem cell composition treatment the decline of ovarian function in the elderly and its complications, and enabed adipose stem cells alone or in combination with adipose stem cell / PLGA microsphere composition both have the significant effects of reducing ovarian tissue fibrosis, promoting ovarian tissue angiogenesis, promoting ovarian follicle development and regeneration in the elderly mice, and the effect of the combined application of the composition is obviously superior to that of enabed hADSCs alone.

[0069] Figure 7 is a mechanism study of the treatment of early ovarian function decline in the reproductive age by the enabled adipose stem cells and the enabled adipose stem cell composition. HEP14 can stimulate the secretion of various factors, particularly including STC1, CYP19A1, MMP1 and PDGFD, which can resist ovarian tissue fibrosis, promote ovarian angiogenesis, improve ovarian tissue microenvironment, stimulate ovarian follicle granules and foam cell growth, and thus promote follicle development and improve ovarian hormone secretion through synergistic exocrine and endocrine mechanisms. DETAILED DESCRIPTION

[0070] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with specific embodiments. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0071] The experimental methods used in the following embodiments are conventional methods unless otherwise specified. The materials, reagents and the like used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0072] Embodiment 1: Preparation and identification of enabled mesenchymal stem cells

[0073] The preparation method of the enabled mesenchymal stem cells comprises the following steps:

[0074] (1) Culturing and expanding human mesenchymal stem cells;

[0075] (2) Adding HEP14 with a concentration of 1.25-20 μM to the culture medium to treat human mesenchymal stem cells for 12-48 hours.

[0076] (3) Digesting human mesenchymal stem cells to prepare a single cell suspension;

[0077] (4) Eluting the human mesenchymal stem cells with normal saline to obtain the enabled mesenchymal stem cells suspended in normal saline.

[0078] HEP14 is extracted from dried whole grass of Euphorbia peplus Linn. (Takahisa Nakane, Arai, Kazuo Masuda, et al. Fern constituents: six new triterpenoid alcohols from Adiantum capillus-veneris. Chem. Pharm. Bull. [J]. 1999, 47(4): 543-547.).

[0079] A specific method of preparing and identifying the enabled mesenchymal stem cells is as follows:

[0080] Under the supervision of the Medical Ethics Committee, human mesenchymal stem cells were isolated from 5-10 ml fresh adipose tissue of healthy donors, i.e. human adipose-derived stem cells (hADSCs) were obtained, primary cell culture and expansion were carried out in a serum-free culture system, i.e. Ultraculture (Lonza) + commercially available growth factors. Put in a carbon dioxide incubator containing 5% carbon dioxide at 37°C. Replace the culture medium after 24 hours, aspirate the suspended dead cells or cell fragments, then wash the cells with normal saline once, and replace the medium. When the cells reach 90% confluence, passaging and expansion can be carried out. P2 generation of primary cells is frozen as seed cells. In this specification, the P2 generation refers to the generation of primary cells in a culture flask, which reaches 90% confluence twice and is digested and reseeded into a culture flask. hADSCs from the 3rd to 5th generation were used, preferably the 4th generation of hADSCs. Morphologically, the isolated primary hADSCs have a fibroblast-like appearance (Figure 1A). Flow cytometry analysis showed that more than 95% of hADSCs were negative for CD31 and HLA-DR, and positive for CD73, CD90, CD105 and CD29 (Figure 1B). Alizarin red and oil red staining results showed that hADSCs had osteogenic (Figure 1C) and adipogenic differentiation ability (Figure 1D). These results indicate that the isolated hADSCs are high-purity undifferentiated adipose stem cells. hADSCs were treated with HEP14 in a concentration-dependent manner (1.25-20 μM) and time-dependent manner (12 h-48 h). After 32 hours of treatment of hADSCs at a concentration of 2.5 μM HEP14, the survival viability (Figure 1E-4) and proliferation ability of hADSCs were significantly improved (Figure 1F). This proliferation is manifested as an increase in S-phase hADSCs, and flow cytometry indicates a decrease in G0 / G1 (Figure 1G and 1H). Real-time quantitative PCR results showed that, compared with control hADSCs (hADSCs not treated with HEP14, represented by c-hADSCs), the expression of pluripotent stem cell marker NANOG in HEP14-treated hADSCs was significantly increased, and the expression of mesenchymal stem cell proliferation marker protein NUCLEOSTEMIN was slightly increased (Figure 1l), and it is worth noting that the Ct value of the real-time qPCR reaction showed that HEP14 treatment and control hADSCs both showed high expression levels of NUCLEOSTEMIN mRNA. Cell immunofluorescence staining and Western blot results further confirmed that HEP14 treatment significantly increased the expression of NANOG and NUCLEOSTEMIN in hADSCs (Figure 1J and 1K). These results indicate that HEP14 treatment enhances the stemness and proliferation and survival ability of hADSCs.Therefore, we used 2.5 μΜ HEP14 to treat hADSCs for 32 hours, then used digestive enzyme to digest the HEP14-treated hADSCs to make a single cell suspension, and finally used physiological saline to elute the HEP14-treated hADSCs single cells to obtain HEP14-treated hADSCs, i.e. the enabled mesenchymal stem cells, also known as enabled adipose-derived stem cells (denoted as h-hADSCs). The enabled mesenchymal stem cells were suspended in physiological saline for standby.

[0081] Example 2: HEP14-induced gene expression profile changes of hADSCs help tissue remodeling and regeneration

[0082] To further understand the molecular biological characteristics of h-hADSCs, we performed RNA sequencing on h-hADSCs and c-hADSCs, and the results showed that 1003 differentially expressed genes (DEGs) were found in h-hADSCs, including 573 up-regulated genes and 430 down-regulated genes (Figures 2A and 2B). Gene Ontology analysis showed that these DEGs were enriched in biological functional processes such as extracellular matrix organization and tissue remodeling, angiogenesis, cell fate determination, ERK1 and ERK2 cascade, and response to estradiol (Figure 2C), which are closely related to organ tissue, especially ovarian tissue regeneration and functional recovery. We reconstructed the subheatmaps of key genes enriched in these biological processes (Figure 2D). Real-time quantitative PCR confirmed that the expression levels of these genes in h-hADSCs were significantly changed compared with c-hADSCs (Figures 2E and 2F). Among them, STC1 is the DEG with the highest up-regulated expression by HEP14. It has been reported that STC1 is expressed in various tissues of mammals such as liver, kidney and ovary, and can promote the growth and migration of mesenchymal stem cells. It specifically promotes the production of granulosa cells and the expression of CYP19A1 gene in ovarian tissue, which is a marker gene of ovarian granulosa cells and promotes the production of estrogen. MMP1 (Matrix metalloproteinase 1) can degrade type 1, type 2 and type 3 collagen in the extracellular matrix, promote extracellular matrix remodeling, and thus lead to changes in the cellular microenvironment. Platelet-Derived Growth Factor D (PDGFD) is a powerful angiogenic growth factor, and it has been reported that it can stimulate angiogenesis by binding to its homologous receptor PDGFRβ (Platelet-Derived Growth Factor receptor β). The results of gene set enrichment analysis (GSEA) showed that HEP14 exerts its biological functions by regulating the interaction of extracellular matrix (ECM) receptors, cell fate determination, blood vessel formation, ovarian steroid hormone production, and MAPK activity activation, etc. (Figures 2G-2K). And these biological functions are crucial for organ tissue, especially ovarian tissue, regeneration and functional recovery. In summary, RNA transcriptomic analysis showed that HEP14-enabled hADSCs contribute to tissue remodeling, regeneration and functional recovery.

[0083] Example 3: Preparation of HEP14 / PLGA microspheres

[0084] HEP14 / PLGA microspheres were prepared by single emulsion solvent evaporation technique. Poly(lactic-co-glycolic acid) (PLGA) and HEP14 were dissolved in dichloromethane at a mass ratio of 5:1 in the dark, and stirred at a speed of 400 rpm using a magnetic stirrer. Next, the resulting solution was added to an aqueous solution containing 1% (w / v) polyvinyl alcohol (PVA), and dichloromethane was allowed to evaporate at 25°C by magnetic stirring (300 rpm) for at least 12 hours. Then, the microspheres were collected by centrifugation (2000 x g, 5 minutes), and repeatedly rinsed with distilled water to completely remove residual PVA. Finally, the microspheres were placed in a freeze dryer, and subjected to a freeze-drying process for 24 hours. HEP14 / PLGA microspheres formed by PLGA encapsulating HEP14 were finally prepared.

[0085] Example 4: Preparation of HEP14 / PLGA microspheres

[0086] HEP14 / PLGA microspheres were prepared by single emulsion solvent evaporation technique. Poly(lactic-co-glycolic acid) (PLGA) and HEP14 were dissolved in dichloromethane at a mass ratio of 10:1 in the dark, and stirred at a speed of 500 rpm using a magnetic stirrer. Next, the resulting solution was added to an aqueous solution containing 2% (w / v) polyvinyl alcohol (PVA), and dichloromethane was allowed to evaporate at 25°C by magnetic stirring (500 rpm) for at least 12 hours. Then, the microspheres were collected by centrifugation (2000 x g, 5 minutes), and repeatedly rinsed with distilled water to completely remove residual PVA. Finally, the microspheres were placed in a freeze dryer, and subjected to a freeze-drying process for 24 hours. HEP14 / PLGA microspheres formed by PLGA encapsulating HEP14 were finally prepared.

[0087] Example 5: Identification of HEP14 / PLGA microspheres

[0088] For comparative analysis, we also prepared pure PLGA microspheres (PLGA microspheres without HEP14) as experimental control group using the same method as in Example 3.

[0089] To analyze the morphology and properties of the microspheres prepared in Example 3, Example 4, and the experimental control, we used a scanning electron microscope to detect the samples prepared above. At the same time, we used image J and Origin software to accurately quantify the particle size of the microspheres. In addition, we used the FTIR system to characterize the chemical structure of the samples. The detection was carried out under the conditions of scanning range of 400 to 4000 cm-1, resolution of 4 cm-1, and scanning speed of 64 times / min, and a total of 1867 data points were obtained.

[0090] To further study the crystal structure characteristics of the samples, we used X-ray diffraction (XRD) technology to analyze the microspheres. The XRD pattern of the sample was collected by a Rigaku Smartlab X-ray diffractometer. During the experiment, we used a Cu Ka radiation source (45 kV, 120 mA), and set the 2θ angle range to 5°-100°. The obtained data was collected in the form of transmittance value (%), and processed and analyzed by Oringin software.

[0091] Through SEM analysis, we found that both PLGA and HEP14 / PLGA microspheres exhibited uniform size distribution, spherical morphology, and smooth surface (Figures 3A and 3B). Due to the large loading of HEP14, the particle size of 5:1 HEP14 / PLGA microspheres (prepared in Example 3) (0.1688 μm ± 0.0685) and 10:1 HEP14 / PLGA microspheres (prepared in Example 4) (0.100 μm ± 0.0735) increased significantly compared to pure PLGA microspheres (0.052 μm ± 0.028). To verify whether HEP14 drugs were successfully encapsulated in PLGA, we analyzed the drug-loaded and non-drug-loaded microspheres. From the infrared spectrum of HEP14 / PLGA samples, we did not observe the vibration peak of HEP14 (Figure 3C). This indicates that HEP14 may be completely encapsulated in the PLGA matrix, or the vibration peak of the PLGA matrix is similar to HEP14, masking the signal of HEP14. X-ray diffraction analysis found that there were multiple diffraction peaks in HEP14, which showed its inherent crystal properties. In HEP14 / PLGA, the diffraction peaks of HEP14 disappeared, indicating that the crystallinity of HEP14 decreased during the loading process (Figure 3D). This change allows HEP14 to be gradually released from the PLGA microspheres, making it easier to be absorbed and utilized by the organism.

[0092] To determine the drug loading and encapsulation efficiency of HEP14 / PLGA microspheres, we measured the drug loading and encapsulation efficiency of HEP14 / PLGA microspheres by UV spectrophotometry and plotted the standard curve of OD value (Y) and concentration (c) of HEP14 / PLGA microspheres. The detection wavelength was 201 nm, and the sample volume was 1 mL. The drug loading (%) and encapsulation efficiency (%) were calculated using the following formula, where the theoretical drug loading of the microspheres refers to the initial drug amount used to prepare the microspheres. Drug loading (%) = (weight of drug in microspheres) / (weight of microspheres) x 100%; entrapment efficiency (%) = (actual drug loading in microspheres) / (theoretical drug loading of microspheres) x 100%. The in vitro release rate of HEP14 / PLGA / microspheres was measured by UV spectrophotometry at 201 nm. HEP14 / PLGA microspheres were incubated in PBS solution at pH 7.4 at 37°C and 100 rpm in a shaking flask, and the microspheres were precipitated by centrifugation (5000 x g, 3 min), and the supernatant was collected at 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 days for detection.

[0093] To study the drug loading, encapsulation efficiency and release of HEP14, the standard curve measured at the maximum absorption peak of 201 nm was y = 0.0785c + 0.1555 (R2= 0.9966, n = 6). The linear correlation coefficient R2 reached 0.99853, fully meeting the requirements for determining the drug loading and content of the microspheres. The drug loading and encapsulation efficiency of HEP14 in the HEP14 / PLGA microspheres prepared in Example 3 were 3.10% and 18.04%, respectively. The drug loading and encapsulation efficiency of HEP14 in the HEP14 / PLGA microspheres prepared in Example 4 were 2.1% and 26.02%, respectively. The in vitro release kinetics of HEP14 / PLGA microspheres was determined by UV spectrophotometry, as shown in (Figure 3E). Initially, there was a rapid release within the first day, followed by a sustained and controlled release from day 1 to day 9. Subsequently, an additional burst release was observed from day 9 to day 17, ultimately leading to a gradual release until day 17, when equilibrium was reached. At the end of the study, the cumulative release of HEP14 / PLGA microspheres was approximately 9.73%.

[0094] In summary, the HEP14 / PLGA microspheres prepared in Example 3 and Example 4 have high HEP14 drug loading, encapsulation efficiency, and long-acting HEP14 sustained release functions.

[0095] Example 6: Preparation of an enabled mesenchymal stem cell composition

[0096] The enabled mesenchymal stem cells prepared in Example 1 and the HEP14 / PLGA microspheres prepared in Example 3 or Example 4 were combined to obtain an enabled mesenchymal stem cell composition.

[0097] Example 7: Establishment of doxorubicin-induced premature ovarian insufficiency (POI) mouse model

[0098] Doxorubicin (also known as Adriamycin) is a first-line clinical anticancer chemotherapy drug with non-specific cycle and strong anti-tumor effect. We established a POI mouse model induced by doxorubicin with significant reduction of follicles at different developmental stages. In the pre-experiment, we detected the effects of three different concentrations of doxorubicin on the ovarian function of 6-7-week-old mice by single intraperitoneal injection.

[0099] The 6-7-week-old female C57BL / 6N mice were weighed and randomly divided into two groups (n = 18 per group), then intraperitoneally injected with different doses (8 mg / kg, 12 mg / kg and 16 mg / kg) of doxorubicin (Sigma) (n = 18 / group) or the same volume of saline solution as control. At 1, 2, 4 weeks after treatment, the mice were sacrificed by cervical dislocation to collect samples. Body weight, ovarian size, estrus cycle, serum hormone levels, follicle count and morphology were examined, and the POI model was evaluated. The results showed that among the 6 mice intraperitoneally injected with 16 mg / kg of doxorubicin, 2 mice had a persistent decrease in body weight within 4 weeks and were in very poor health, so they were euthanized according to the national experimental animal use operation regulations (data not shown), but this did not occur in mice treated with 8 mg / kg or 12 mg / kg of doxorubicin. Hematoxylin and eosin (H&E) staining of ovarian tissue showed that the ovarian size and follicle structure changed less in mice treated with 8 mg / kg of doxorubicin, while the ovaries of mice treated with 16 mg / kg of doxorubicin showed irreversible atrophy and follicle atresia compared to the ovaries of mice treated with 12 mg / kg of doxorubicin. Therefore, we chose to use 12 mg / kg of doxorubicin to prepare the POI mouse model (Figure 4A). At 1, 2, 4 weeks after doxorubicin induction, H&E staining of the tissue showed that the ovarian structure of the control mice was normal, with multiple follicles and corpora lutea at all stages of development, and all follicles were surrounded by flat interstitial cells, while the ovaries of doxorubicin-treated mice showed regressive changes in follicles and mesenchyme, with up to 80% of follicles showing atresia (Figure 4B). At 1 and 2 weeks after doxorubicin induction, the number of growing follicles accounted for a slightly increased proportion of the total number of follicles in the damaged ovaries. By the fourth week, in sharp contrast to the control ovaries, the total number of follicles in the ovaries of doxorubicin-treated mice was further significantly reduced (Figure 4C). Immunofluorescence chemical (IF) staining showed that the number of Ki67-positive and TUNEL-negative follicles in the control ovaries was greater than that in the doxorubicin-treated ovaries (Figures 4D-4E). These results indicate that doxorubicin treatment leads to a significant loss of ovarian follicle reserve.

[0100] Hormone analysis showed that the serum FSH level of mice treated with doxorubicin was statistically increased compared with the control group within 1-4 weeks after induction (Figure 4F), but the levels of E2 and Amh were significantly reduced (Figures 4G and 4H). The results of vaginal smear showed that the control group mice had a normal estrous cycle of 4-5 days, while the doxorubicin-treated mice showed irregular estrous cycles, mainly stopped at the estrus stage (Figure 4I). The results showed that a single intraperitoneal injection of doxorubicin at a dose of 12 mg / kg could cause significant damage to the structure and function of mouse ovaries, and here we successfully established a doxorubicin-induced POI mouse model (POI mouse for short).

[0101] Example 8: Treatment of Premature Ovarian Failure in Reproductive Age by Enabling Adipose Stem Cells and Enabling Adipose Stem Cell Compositions

[0102] To study the effect of enabling adipose stem cells and enabling adipose stem cell compositions on the treatment of premature ovarian failure in reproductive age, the enabling adipose stem cells prepared in Example 1 and the enabling adipose stem cell compositions prepared in Example 6 were used to treat the POI model mice to study the treatment of premature ovarian failure in reproductive age. Specifically, the enabling adipose stem cell composition used in this embodiment is: enabling adipose stem cells prepared in Example 1 + HEP14 / PLGA microspheres prepared in Example 3.

[0103] I. Treatment of POI model mice

[0104] The treatment of POI model mice, sample collection and fertility were evaluated (Figure 5A). To track the transplanted human adipose stem cells (hADSCs) in vivo, the enabling adipose stem cells were pre-labeled with 5 μM CellTracker TM , i.e. CM-DIL for 30 minutes (defined as hADSCs-CM-DIL here), and observed under a fluorescence microscope (Leica). After washing twice with saline, the labeled cells were kept at 4°C. 7-8-week-old female C57BL / 6N POI mice were used for the experiment, and were divided into four groups (n = 22 for each group): normal control (CTL mice); POI mice were further divided into three groups, including POI mice, h-hADSCs mice and h-hADSCs / HEP14 mice. The specific treatments are as follows:

[0105] Normal control mice: i.o. injection of 15 μl of normal saline and i.p. injection of 100 μl of normal saline.

[0106] POI mice: i.o. injection of 15 μl of normal saline and i.p. injection of 100 μl of normal saline.

[0107] h-hADSCs mice: POI mice were injected with 15 μΐ of saline containing the enablers h-hADSCs-CM-DIL in the ovary and 100 μΐ of saline i.p.; the density of enablers h-hADSCs was 5 x 10 4

[0108] h-hADSCs / HEP14 mice: POI mice were injected with 15 μΐ of saline containing the enablers h-hADSCs-CM-DIL in the ovary and 100 μΐ of saline i.p. containing HEP14 / PLGA microspheres; the density of enablers h-hADSCs was 5 x 10 4

[0109] To shorten the operation time and avoid the bias between groups caused by the cell viability over time, only the right ovary tissue of the four groups of mice was injected. At 2 and 4 weeks after treatment, the mouse tissues and blood samples (n = 6 for each) were collected to evaluate the changes in ovarian structure and function. At 4 weeks after treatment, the mice (n = 10 / group) were further mated with confirmed fertile males at a ratio of 1 :2 for 2 weeks. From the day after mating, the vaginal plugs were checked every morning at 9:00, and the checking time was 2 weeks. The fetuses were removed by cesarean section at 18 to 19 days of pregnancy in mice. The number of pregnant mice, the total number of fetuses produced, and the average number of pups per mouse in each of the above groups were recorded to evaluate their fertility.

[0110] II. Promoting the recovery of the structure of the early-aging ovary and the regeneration of the follicle in the reproductive age

[0111] ​​The histology of the ovaries of the four groups of mice (CTL-, POI-, h-hADSCs-, and h-hADSCs / HEP14 mice) was examined at 2 and 4 weeks after treatment, and the follicle development and number at each stage were detected by histopathological methods. The results showed that the body weight and ovary size of the h-hADSCs / HEP14 mice and the h-hADSCs mice were significantly restored compared with the POI mice. The restoration of the h-hADSCs / HEP14 mice was better than that of the h-hADSCs mice (Figures 5A and 5B). Morphologically, at 2 and 4 weeks after treatment, almost all the follicles in the POI mice were atretic (Figure 5B), while the number of healthy follicles at different stages of development in the ovaries of the h-hADSCs mice and the h-hADSCs / HEP14 mice was significantly increased (Figure 5C). Detailed follicle counting showed that the total number of follicles and the number of primordial follicles in the ovaries of the h-hADSCs / HEP14 mice were higher than those in the h-hADSCs mice (Figure 5C). However, there was no statistical difference in the number of growing follicles and antral follicles between the two groups (Figures 5D and 5E). Immunofluorescence chemical (IF) staining showed that the number of follicles with positive Ki67 expression and negative TUNEL expression in the control ovaries was higher in the h-hADSCs / HEP14 mice than in the POI and h-hADSCs mice, and the number of follicles in the h-hADSCs mice was between that in the h-hADSCs / HEP14 mice and that in the POI mice, which was significantly higher than that in the POI mice (Figures 5F-5G). Immunohistochemical staining showed that the expression of FSHR, ERβ, and AMH in the ovaries of the h-hADSCs / HEP14 mice was significantly higher than that in the h-hADSCs mice and the POI mice. The level in the h-hADSCs mice was between that in the h-hADSCs / HEP14 mice and that in the POI mice (Figures 5H and 5I).

[0112] These results show that the treatment of the engrafted adipose stem cells alone or in combination with the HEP14 / PLGA microsphere composition has a significant effect on promoting the follicle regeneration and development in the POI mice, and the combined treatment of the composition is superior to the treatment of the engrafted hADSCs alone.

[0113] III. Promoting the restoration of endocrine function in the early-aging ovary

[0114] The estrous cycle, serum hormone levels, and fertility of the four groups of mice were evaluated further after 4 weeks post-treatment. Compared with the normal 4-5 day estrous cycle presented by the control mice, 5 out of 6 h-hADSCs / HEP14 mice and 4 out of 6 h-hADSCs mice restored normal estrous cycle, while the estrous cycle of POI mice was prolonged (Figure 5J). The results of serum ovarian hormone detection showed that h-hADSCs / HEP14 mice and h-hADSCs mice presented higher E2 (estradiol) and AMH (anti-Mullerian hormone) levels compared with POI mice (Figures 5K-5L), but lower FSH (follicle-stimulating hormone) levels (Figure 5M). The hormone levels in the serum of h-hADSCs / HEP14 mice returned to near normal controls.

[0115] Starting from the 4th week post-treatment, the four groups of mice were mated in a 1 :2 (male:female) ratio for 2 weeks. To obtain reliable data for evaluating the fertility of mice in each group, the mouse fetuses were removed by cesarean section at 18-19 days of gestation. The pregnancy rate of POI mice was (2 / 10), which was significantly lower than that of h-hADSCs mice (7 / 10), h-hADSCs / HEP14 mice (10 / 10), and normal control mice (10 / 10). The average number of fetuses / litter (0 / 2) and the number of healthy fetuses / litter (1 / 0) of POI mice were significantly lower than the average number of fetuses / litter (2.4 / 2.0) of h-hADSCs mice, the average number of fetuses / litter (4.6 / 3.5) of h-hADSCs / HEP14 mice, and the average number of fetuses / litter (5.7 / 5.5) of control mice (Figures 5N and 50). There was 1 monozygotic twin in the h-hADSCs / HEP14 group (Figure 5P). These results indicated that h-hADSCs / HEP14 mice and h-hADSCs mice had better fertility than POI mice. Both treatment with h-hADSCs alone and the combination (h-hADSCs combined with HEP14 / PLGA microspheres) had the effect of promoting the regeneration of ovarian follicles and the recovery of reproductive endocrine function in POI mice. The effect of h-hADSCs / HEP14 treatment was better than that of h-hADSCs alone.

[0116] Example 9: Treatment of senescent ovarian decline and its resulting complications with h-hADSCs, h-hADSCs combination

[0117] To investigate the effect of the h-hADSCs and the h-hADSCs composition in treating the early decline of ovarian function in the elderly, 73-74 week old female C57BL / 6N elderly mice were used as model animals and randomly divided into three groups: a control group (CTL mice) treated with normal saline, a h-hADSCs treatment group (h-hADSCs mice) treated with the h-hADSCs prepared in Example 1, and a h-hADSCs composition treatment group (h-hADSCs / HEP14 mice) treated with the h-hADSCs composition prepared in Example 6. Specifically, the h-hADSCs composition used in this embodiment was: the h-hADSCs prepared in Example 1 + the HEP14 / PLGA microspheres prepared in Example 3.

[0118] The specific grouping and treatment were as follows:

[0119] Normal elderly control mice (CTL mice): 15 μl of normal saline was injected into the ovaries of the mice (i.o.) and 100 μl of normal saline was injected intraperitoneally (i.p).

[0120] h-hADSCs treatment mice (h-hADSCs mice): 15 μl of normal saline containing h-hADSCs-CM-DIL was injected into the ovaries of the mice and 100 μl of normal saline was injected intraperitoneally (i.p). The density of the h-hADSCs was 5 x 10 4 cells / μl.

[0121] h-hADSCs composition treatment mice (h-hADSCs / HEP14 mice): 15 μl of normal saline containing h-hADSCs-CM-DIL was injected into the ovaries of the mice and 100 μl of normal saline containing HEP14 / PLGA microspheres was injected intraperitoneally (i.p). The density of the h-hADSCs was 5 x 10 4 cells / μl and the content of the HEP14 / PLGA microspheres was 10 μg / μl.

[0122] I. Anti-ovarian tissue fibrosis and promotion of ovarian angiogenesis

[0123] The ovaries of three groups of mice (CTL-, h-hADSCs-, and h-hADSCs / HEP14 mice) were collected after 4 weeks of treatment for histopathological examination and the proliferation and number of follicles at each developmental stage. The results showed that the ovaries of the aged h-hADSCs / HEP14 mice and the h-hADSCs mice were significantly larger than those of the untreated aged mice. The recovery of the ovaries of the h-hADSCs / HEP14 mice was better than that of the h-hADSCs mice (Figure 6A). The results of the picosirius red (PSR) staining showed that the collagen fibers in the ovaries of the untreated aged mice were significantly more than those in the ovaries of the h-hADSCs mice and the h-hADSCs / HEP14 mice, and the collagen fibers in the ovaries of the h-hADSCs mice were significantly more than those in the ovaries of the h-hADSCs / HEP14 mice (Figure 6B). The results of the immunohistochemical (IHC) staining showed that, compared with the ovaries of the h-hADSCs mice and the CTL mice, the expression of the myofibroblast (MFB) marker protein a-SMA in the ovaries of the h-hADSCs / HEP14 mice was significantly reduced, and the expression of the endothelial cell-specific marker protein CD31 was significantly increased, and the expression of a-SMA and CD31 in the ovaries of the h-hADSCs mice was between that in the ovaries of the h-hADSCs / HEP14 mice and the CTL mice (Figures 6C and 6D). The above results showed that the engrafted adipose stem cells alone or in combination with the HEP14 / PLGA microsphere composition had the effects of reducing the fibrosis of the ovarian tissue and promoting the angiogenesis of the ovarian tissue.

[0124] II. Promoting the development and regeneration of ovarian follicles in aged mice

[0125] H&E staining results showed that morphologically, the number of healthy follicles at different developmental stages in the h-hADSCs / HEP14 mouse ovaries was more than that in the h-hADSCs mice, and the number of healthy follicles in the h-hADSCs mice was more than that in the control aged mice (Figure 6E). Detailed follicle counting showed that the total number of follicles and the number of primordial follicles in the h-hADSCs / HEP14 mouse ovaries were significantly higher than those in the h-hADSCs mice, and the number of follicles in the h-hADSCs mice was significantly higher than that in the CTL mice (Figure 6F). Immunofluorescence staining showed that the number of follicles with PCNA positive (cell proliferation marker protein) and TUNEL negative (cell apoptosis marker protein) expression in the ovaries of the h-hADSCs / HEP14 mice was significantly more than that in the ovaries of the h-hADSCs mice and the CTL mice, and the expression of PCNA positive and TUNEL negative in the ovaries of the h-hADSCs mice was between that in the ovaries of the h-hADSCs / HEP14 mice and the CTL mice (Figure 5G), which was significantly higher than that in the CTL mice. Immunohistochemical staining results showed that the expression of Stc1 and CYP26B1 in the ovaries of the h-hADSCs / HEP14 mice was significantly higher than that in the ovaries of the h-hADSCs mice and the CTL mice, and the expression in the ovaries of the h-hADSCs mice was significantly higher than that in the ovaries of the CTL mice (Figure 6H and 6I). These results showed that the application of the enabled adipose stem cells alone or in combination with the HEP14 / PLGA microsphere composition in the promotion of the development and regeneration of the ovarian follicles in the aged mice had a significant effect, and the combined application of the composition had a significantly better effect than the application of the enabled hADSCs alone.

[0126] III. Promoting the recovery of endocrine function in the aged atrophic ovary

[0127] Further, the recovery of serum hormone levels in the three groups of aged mice was evaluated 4 weeks after treatment. The results of serum ovarian hormone detection showed that compared with the aged control group of mice, the h-hADSCs / HEP14 mice and the h-hADSCs mice showed significantly increased E2 (estradiol), AMH (anti-Mullerian hormone), INHA and INHB levels (Figures 6J, 6K, 6L and 6M) and decreased FSH (follicle-stimulating hormone) and T (testosterone) levels (Figures 6N and 6O). The improvement of the serum levels of the h-hADSCs / HEP14 mice was better than that of the h-hADSCs mice. These results showed that both the enabled h-hADSCs alone and the enabled h-hADSCs composition had a significant effect on promoting the regeneration of the aged ovarian tissue and the endocrine function.

[0128] In summary, both treatment with h-hADSCs alone and treatment with h-hADSCs composition (h-hADSCs combined with HEP14 / PLGA microspheres) have the effects of anti-ovarian fibrosis, promoting follicular development and regeneration, and restoring endocrine function in aged mice. The effect of treatment with h-hADSCs / HEP14 composition is better than that of h-hADSCs alone.

[0129] Example 10: Mechanism of treatment of h-hADSCs, h-hADSCs composition for premature ovarian failure in women of childbearing age

[0130] I. h-hADSCs alone or in combination with HEP14 / PLGA microsphere composition reduces ovarian fibrosis and promotes ovarian angiogenesis by enhancing the secretion of MMP1 and PDGFD

[0131] Follicular development is characterized by vascular regeneration and extracellular matrix remodeling. Therefore, we evaluated the degree of fibrosis and angiogenesis in the ovarian tissues of the four groups of mice 4 weeks after treatment. Collagen content was determined by picrosirius red (PSR) staining, and polarized light microscopy was used to distinguish collagen I (red) and collagen III (green). The results showed that the collagen fibers in normal ovaries were thin and evenly distributed, while the collagen fibers in the ovaries of POI mice were significantly increased and relatively thick, forming clusters. The collagen fibers in the ovaries of POI mice were significantly more than those in the ovaries of h-hADSCs / HEP14 mice and HEP14-hADSC mice (Figures 7A and 7B). The collagen content in the ovaries of h-hADSCs mice was between that in the ovaries of h-hADSCs / HEP14 mice and POI mice (Figures 7A and 7B). Further immunohistochemical and immunofluorescence (IHC and IF) staining results showed that the expression of the myofibroblast marker protein a-SMA (Figure 7C) in the ovaries of h-hADSCs / HEP14 mice was significantly reduced, and the expression of the endothelial cell-specific marker protein CD31 was increased (Figure 7D), which was close to the expression level in the normal control group, but was in sharp contrast to the significant increase in a-SMA expression and significant decrease in CD31 expression in the ovaries of POI mice (Figure 7D). The expression of a-SMA and CD31 in the ovaries of h-hADSCs mice was between that in the ovaries of h-hADSCs / HEP14 mice and POI mice (Figure 7D). The above results demonstrate that both h-hADSCs alone and h-hADSCs combined with HEP14 / PLGA microsphere composition have the effects of reducing ovarian fibrosis and promoting ovarian angiogenesis.

[0132] To further understand the molecular mechanisms of the anti-ovarian fibrosis and pro-angiogenic effects of the empowered hADSCs alone or in combination with the HEP14 / PLGA microsphere composition, we examined the expression of three key differentially expressed genes, MMP1, PDGFD, and PDGFR, in the ovarian tissues of the mice in each group. MMP1 is the most abundant matrix metalloproteinase that can degrade collagen types I, II, and III. PDGFD is a potent angiogenic growth factor that has been reported to promote angiogenesis by binding to its cognate receptor, PDGFRbeta. Western blot confirmed that HEP14 significantly increased the expression of MMP1, PDGFD, and PDGFRbeta in h-hADSCs compared to the c-hADSCs (Figure 7E). IHC and ICC staining of the ovarian tissue sections showed that the expression of MMP1, PDGFD, and PDGFRbeta in the ovarian tissues of the h-hADSCs / HEP14 mice was significantly higher than that in the ovarian tissues of the h-hADSCs mice and the POI mice (Figures 7F-7G). The expression levels of MMP1, PDGFD, and PDGFRbeta in the ovarian tissues of the h-hADSCs mice were significantly higher than those in the POI mice (Figures 7F-7G). To confirm the enhanced anti-fibrotic and pro-angiogenic capabilities observed above, the conditioned media, including c-hADSCs-CM and h-hADSCs / HEP14-CM, were collected. Consistent with the results of the real-time qPCR and Western blot analyses (Figures 2F and 7E), the enzyme-linked immunosorbent assay (ELISA) detection results confirmed that the treatment of HEP14 did indeed increase the secretion level of MMP1 by hADSCs (Figure 7H). At the same time, human umbilical vein endothelial cells (HUVECs) were cultured with the above-mentioned conditioned media and a specific PDGFD neutralizing antibody (PDGFD Nab) for tube formation experiments. The tube formation experiment results based on the total length and the number of branch points showed that the h-hADSCs / HEP14-CM medium significantly increased the tube formation capability of HUVECs compared to the c-hADSCs-CM conditioned medium (Figure 71). IF staining of CD31 confirmed the formation of these tubes (Figure 7J). These results strongly support that HEP14 enhances the pro-angiogenic capability of hADSCs. In summary, these findings indicate that the HEP14-empowered hADSCs significantly enhance the anti-fibrotic and pro-angiogenic effects of h-hADSCs.

[0133] The above results show that both the engrafted adipose stem cells alone or the engrafted adipose stem cells in combination with the HEP14 / PLGA microspheres stimulate the secretion of MMP1, PDGFD and PDGFRβ, thus having the effects of reducing the fibrosis of the ovarian tissue and promoting the angiogenesis of the ovary. The anti-fibrosis effect and the effect of promoting the angiogenesis of the engrafted adipose stem cells in combination with the HEP14 / PLGA microspheres are stronger than the therapeutic effect of the engrafted adipose stem cells alone.

[0134] II. STC1 secreted by HEP14-stimulated hADSCs specifically promotes the development and functional recovery of ovarian follicles

[0135] Anti-Vimentin antibody (Ab) was confirmed to be a human cell-specific antibody that does not react with mouse tissue cells (analysis certificate, ab8069, Abeam). The results of immunohistochemical staining showed that the Vimentin expression signal overlaps with the CM-DIL signal that labels the engrafted adipose stem cells, and thus the Vimentin-positive adipose stem cells (hADSCs Vimentin+ ) represent the engrafted h-hADSCs transplanted into the ovary (Fig. 7K). The engrafted h-hADSCs detected in the ovary of the h-hADSCs / HEP14 mice were significantly more than the h-hADSCs Vimentin+ in the ovary of the h-hADSCs mice transplanted with the engrafted h-hADSCs alone Vimentin+ after 4 weeks of transplantation of the engrafted h-hADSCs. Eight weeks after transplantation, the engrafted hADSCs V i mentin+ in the ovary of the h-hADSCs / HEP14 mice were significantly more than the engrafted hADSCs Vimentin+ in the ovary of the h-hADSCs mice (Fig. 7K). In addition, these cells were found to be involved in the granulosa cell layer or theca cell layer of the follicle in the ovary of the h-hADSCs / HEP14 mice (Fig. 7K). In contrast, the hADSCs Vimentin+ detected in the ovary of the h-hADSCs mice were mainly distributed in the interstitial tissue of the ovary or around the growing follicle (Fig. 7K). These results show that HEP14 promotes the survival and proliferation of h-hADSCs in the ovary. More importantly, these hADSCs Vimentin+The Vimentin signal in the HEP14 / PLGA microspheres overlaps with the FSHR or CYP19A1 expression signal in the granulosa layer of the ovary (Fig. 7L); and overlaps with the CYP17A1 expression signal in the theca layer of the ovary (Fig. 7M). FSHR and CYP19A1 are markers of granulosa cells in the ovarian tissue, and CYP17A1 is a marker of theca cells in the ovarian tissue. These results indicate that the HEP14 / PLGA microspheres in combination with the engrafted hADSCs promote the survival and transdifferentiation of the engrafted hADSCs into functional granulosa-like cells or theca-like cells, and thus the HEP14 / PLGA microspheres in combination with the engrafted hADSCs have a stronger effect of promoting the growth and development of the ovarian follicle and the recovery of the ovarian function.

[0136] Consistent with the results of the RNA-seq detection (Fig. 2E), the Western blot results showed that HEP14 significantly promoted the secretion of STC1 and CYP9A and other cytokines by the engrafted adipose stem cells (Fig. 7N). Immunofluorescence staining detection found that the expression of STC1 in the ovary of the h-hADSCs / HEP14 mice was significantly higher than that in the ovary of the h-hADSCs mice and the POI mice. The expression level in the h-hADSCs mice was significantly higher than that in the POI mice (Fig. 7O). Further Western blot results showed that the knockdown of STC1 expression in the granulosa cell line of the ovary by siRNA led to a decrease in the expression of CYP19A1, indicating that STC1 promotes the expression of CYP19A1 by the granulosa cells in the ovarian tissue (Fig. 7P). The main function of CYP19A1 is to stimulate the production of estradiol by the granulosa cells, and the granulosa cells are also the target of estradiol in the ovarian tissue, thus promoting the regeneration and development of the ovarian follicle.

[0137] In summary, the present application has found that HEP14 can stimulate the secretion of a plurality of factors, particularly including STC1, CYP19A1, MMP1 and PDGFD, which synergistically act to, on the one hand, strengthen the anti-fibrosis and pro-angiogenesis of the engrafted mesenchymal stem cells on the ovarian tissue, and improve the microenvironment of the ovarian tissue; and on the other hand, STC1 and CYP19A1 synergistically stimulate the growth of the granulosa cells of the ovarian follicle, thus promoting the development of the ovarian follicle and improving the secretion and endocrine function of the ovarian hormones. In summary, these results show that the engrafted mesenchymal stem cells and the engrafted mesenchymal stem cell composition of the present application have an excellent effect of promoting the regeneration and functional recovery of the damaged ovarian tissue.

[0138] It will be obvious to a person skilled in the art that the application is not limited to the details of the above-described exemplary embodiments, but that the application can be implemented in other embodiments without departing from the scope of the application. The embodiments are therefore to be seen as exemplary and in no way restrictive, the scope of the application being defined by the claims below rather than by the above description, and all variations falling within the meaning and range of equivalency of the essential characteristics of the claims are therefore intended to be embraced therein.

[0139] Furthermore, it should be understood that although the description is made according to embodiments, not every embodiment contains only one independent technical solution, and the description is made in this way only for the sake of clarity, and a person skilled in the art should consider the description as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by a person skilled in the art.

Claims

1. An empowered mesenchymal stem cell, characterized in that, The enabled mesenchymal stem cells are obtained by treating human mesenchymal stem cells with HEP14, wherein the HEP14 is a compound with a molecular structure as shown in the following structural formula:

2. The empowered mesenchymal stem cell of claim 1, wherein, The treatment of human mesenchymal stem cells with HEP14 comprises the following steps: (1) Culturing and expanding human mesenchymal stem cells; (2) Treating human mesenchymal stem cells with HEP14 at a concentration of 1.25-20 μM in the culture medium for 12-48 hours; (3) Digesting human mesenchymal stem cells into single cell suspension; (4) Eluting the human mesenchymal stem cells with normal saline to obtain the enabled mesenchymal stem cells suspended in normal saline.

3. The empowered mesenchymal stem cell of claim 2, wherein, The step (2) of the treatment of human mesenchymal stem cells with HEP14 is treating human mesenchymal stem cells with HEP14 at a concentration of 2.5 μM in the culture medium for 32 hours.

4. The empowered mesenchymal stem cell of claim 1, wherein, The source of the human mesenchymal stem cells comprises mesenchymal stem cells from different human tissues such as bone marrow, fat, placenta, umbilical cord, skin, amniotic membrane and dental pulp.

5. The empowered mesenchymal stem cell of claim 4, wherein, The human mesenchymal stem cells are fat tissue-derived mesenchymal stem cells.

6. A potentiated mesenchymal stem cell composition, characterized in that, The composition comprises: enabled mesenchymal stem cells as claimed in claims 1-5; and HEP14 / PLGA microspheres prepared by using poly-lactic-co-glycolic acid (PLGA) as a carrier, which have high HEP14 loading capacity and long-term HEP14 sustained release.

7. The empowered mesenchymal stem cell composition of claim 6, wherein, The HEP14 / PLGA microspheres having high HEP14 loading capacity and long-term HEP14 sustained release are prepared by the following method: poly-lactic-co-glycolic acid (PLGA) and HEP14 are dissolved in dichloromethane at a mass ratio of 5-10:1 in a dark environment, and a magnetic stirrer is used to stir at a speed of 400-500 rpm, then the obtained solution is added into an aqueous solution containing 1-2% (w / v) polyvinyl alcohol, dichloromethane is volatilized by stirring at a speed of 300-500 rpm with a magnetic stirrer at 25°C for at least 12 hours, then the microspheres are collected by centrifugation, and repeatedly washed with distilled water to completely remove residual polyvinyl alcohol, finally the microspheres are placed in a freeze dryer for freeze-drying treatment to obtain HEP14 / PLGA microspheres.

8. The empowered mesenchymal stem cell composition of claim 7, wherein The poly-lactic-co-glycolic acid and HEP14 are dissolved in dichloromethane at a mass ratio of 5:

1.

9. Use of the enabled mesenchymal stem cells as claimed in any one of claims 1-5 in the preparation of a drug for resisting tissue fibrosis.

10. Use of the enabled mesenchymal stem cells as claimed in any one of claims 1-5 in the preparation of a drug for promoting tissue angiogenesis.

11. Use of the enabled mesenchymal stem cells as claimed in any one of claims 1-5 in the preparation of a drug for promoting tissue regeneration and functional recovery.

12. The use according to any one of claims 9-11, wherein the tissue is one of ovarian tissue, liver tissue, lung tissue, brain tissue, heart tissue, muscle tissue, bone tissue, kidney tissue and skin tissue.

13. Use of the enabled mesenchymal stem cells as claimed in any one of claims 1-5 in the preparation of a drug for treating organ dysfunction.

14. Use of the empowered mesenchymal stem cell according to claim 13 for the preparation of a medicament for the treatment of organ dysfunction, characterized in that, The organ is ovary.

15. Use of the empowered mesenchymal stem cell according to claim 14 for the preparation of a medicament for organ dysfunction, characterized in that, The organ is ovary.

16. Use of the empowered mesenchymal stem cell according to claim 15 for the preparation of a medicament for organ dysfunction, characterized in that, The application of the physiological saline solution containing the enabled mesenchymal stem cells to the ovary of the subject in need thereof includes any one of the following applications: (1) the application in the preparation of a drug for treating premature ovarian failure in women of childbearing age and its complications; (2) the application in the preparation of a drug for treating ovarian function decline in elderly women and its complications; (3) the application in the preparation of a drug for restoring the endocrine and / or reproductive function of the ovary.

17. The application of the enabled mesenchymal stem cell composition of any one of claims 6-8 in the preparation of a drug for resisting tissue fibrosis.

18. The application of the enabled mesenchymal stem cell composition of any one of claims 6-8 in the preparation of a drug for promoting tissue angiogenesis.

19. The application of the enabled mesenchymal stem cell composition of any one of claims 6-8 in the preparation of a drug for promoting tissue regeneration and functional recovery.

20. The application of any one of claims 17-19, wherein the tissue is one of ovary tissue, liver tissue, lung tissue, brain tissue, heart tissue, muscle tissue, bone tissue, kidney tissue, and skin tissue.

21. The application of the enabled mesenchymal stem cell composition of any one of claims 6-8 in the preparation of a drug for treating organ dysfunction.

22. Use of the empowered mesenchymal stem cell composition of claim 21 for the manufacture of a medicament for treating organ dysfunction. The organ is ovary.

23. Use of the empowered mesenchymal stem cell according to claim 22 for the preparation of a medicament for organ dysfunction, characterized in that, The application of the physiological saline solution containing the enabled mesenchymal stem cells to the ovary of the subject in need thereof, combined with the injection of the physiological saline solution containing the HEP14 / PLGA microspheres to the subject in need thereof, includes any one of the following applications:

24. The use of the empowered mesenchymal stem cell according to claim 23 for the preparation of a medicament for organ dysfunction, characterized in that, (1) the application in the preparation of a drug for treating premature ovarian failure in women of childbearing age; (2) the application in the preparation of a drug for treating ovarian function decline in elderly women and its complications; (3) the application in the preparation of a drug for restoring the endocrine and / or reproductive function of the ovary. The injection route of the physiological saline solution containing the HEP14 / PLGA microspheres includes any one of intraperitoneal injection, subcutaneous injection, and intramuscular injection.

25. Use of the empowered mesenchymal stem cell composition of claim 24 for the manufacture of a medicament for treating organ dysfunction. ​

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