Energizing mesenchymal stem cell, energizing mesenchymal stem cell composition and application of energizing mesenchymal stem cell composition in regenerative medicine field

Abstract

The invention belongs to the technical field of biomedicine, and particularly discloses an endowed mesenchymal stem cell, an endowed mesenchymal stem cell composition and application of the endowed mesenchymal stem cell composition to preparation of a medicine for treating organ insufficiency, and the endowed mesenchymal stem cell is obtained by treating human mesenchymal stem cells with ingenol HEP14. The energizing mesenchymal stem cell composition comprises energizing mesenchymal stem cells and HEP14 / PLGA microspheres which are prepared by adopting a polylactic acid-glycolic acid copolymer (PLGA) as a carrier and have high HEP14 loading capacity and long-acting HEP14 slow release. The energized mesenchymal stem cell has the effects of enhancing the survival activity and dryness of the mesenchymal stem cell, resisting tissue fibrosis, promoting tissue angiogenesis, and promoting tissue regeneration and functional recovery; the endowed mesenchymal stem cells and the HEP14 / PLGA microspheres are used in a combined manner, and the retention and transdifferentiation effects of the endowed mesenchymal stem cells in tissues are enhanced, so that the functions of the endowed mesenchymal stem cells and the application of the endowed mesenchymal stem cells in the field of regenerative medicine are further enhanced.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly relates to a mesenchymal stem cell empowered, a mesenchymal stem cell composition empowered, and their application in the preparation of drugs for treating ovarian insufficiency or other tissue and organ insufficiency. Background Art

[0002] Ovarian insufficiency is caused by ovarian follicle development disorders and dysfunction resulting from the reduction or exhaustion of the ovarian follicle pool, including premature ovarian insufficiency (POI) and age-related ovarian aging, that is, ovarian senescence. Premature ovarian insufficiency refers to the functional failure caused by ovarian damage in women of childbearing age due to various reasons, mainly chemotherapy or / and radiotherapy, that is, the phenomenon of amenorrhea occurs in women before the age of 40, which is the main cause of female infertility. Ovarian aging is a natural law, that is, normal ovarian function gradually declines with age until ovarian function is exhausted. The female menopausal syndrome and related degenerative diseases caused by ovarian insufficiency seriously damage women's health. 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 relieve the symptoms of low estrogen levels, and has large side effects, 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 curative effects and there is a problem of lack of ovarian tissue resources. Therefore, there is an urgent need in this field to find / explore a method that can more effectively treat ovarian insufficiency, which has always been one of the most concerned research hotspots in the world.

[0003] Stem cells, especially various types of mesenchymal stem cells (MSCs), are a current research hotspot in the application research 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 for treating ovarian insufficiency. By promoting ovarian tissue regeneration and ovarian follicle development, the reproductive and fertility functions of the ovary can be promoted, that is, ovarian insufficiency can be cured or improved. Compared with (mesenchymal) stem cells from other sources, adipose-derived mesenchymal stem cells, namely adipose stem cells (ADSCs), have many significant advantages and characteristics, including that human adipose stem cells (hADSCs) are easy to obtain, cause little damage to patients, there is enough adipose tissue, rapid in vitro expansion, high biological safety, making autologous stem cell transplantation possible, etc. It has become one of the stem cell sources with the most promising application prospects and has attracted more and more attention in recent years. The results of animal transplantation experiments and a recent preliminary clinical trial show that ADSCs are effective in treating premature ovarian failure, but the therapeutic effect is insufficient and cannot meet the purpose of clinical translation for treatment. The main limitations of using ADSCs or mesenchymal stem cells from other sources to treat ovarian or other tissue and organ insufficiency are (ovarian) tissue fibrosis, ischemia in the injection area, and the differentiation or aging of "transplanted" stem cells. To address these obstacles, improved stem cell-based treatment regimens have been in the process of research and development, including pre-treating stem cells with cytokines, combining stem cells with collagen scaffolds, etc. to enhance the therapeutic effect. Unfortunately, the research and development regimens reported so far have not made breakthrough progress in promoting the recovery of ovarian and / or other tissue and organ functions or / and clarifying the treatment mechanism. To address this clinical challenge, basic research and clinical trials on the application of stem cells in regenerative medicine are still ongoing.

[0004] HEP14 is a 5β-O-angeloyl-20-deoxyphorbol compound extracted from the whole herb of Euphorbia peplus Linn., and is a novel activator of the protein kinase C (PKC) pathway. Currently, except for the reported role in regulating lysosome biogenesis, other pathways of HEP14 have not been reported. In the process of exploring the therapeutic effects and potential mechanisms of HEP14, to overcome the poor water solubility of HEP14 and obtain a sustained and stable release of HEP14 in the body circulation, we used poly(lactic-co-glycolic acid) (PLGA) as a drug carrier to prepare PLGA microspheres loaded with HEP14, 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, controllable 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, and this property makes it particularly important in the biomedical field.

[0005] PLGA is a drug carrier approved by the US Food and Drug Administration (FDA). Summary of the Invention

[0006] In view of the situation that there has been no breakthrough in the promotion of ovarian function or / and the elucidation of the treatment mechanisms of other tissues and organs by existing mesenchymal stem cell single treatment or combined treatment, the empowered mesenchymal stem cells of the present invention have significantly enhanced viability and stemness, and have the effects of anti-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 function recovery, and having the effect of treating organ dysfunction of ovaries, liver, lung, brain, heart, muscle, bone, kidney, skin, etc.; the combined application of the empowered mesenchymal stem cells and the HEP14 / PLGA microsphere composition further strengthens the retention and transdifferentiation of the empowered mesenchymal stem cells in organ tissues, so the combined application of the composition further strengthens the anti-tissue fibrosis and tissue angiogenesis-promoting effects of the empowered mesenchymal stem cells, further strengthens the effects of promoting tissue regeneration and function recovery, and the effect of treating organ dysfunction.

[0007] Particularly in the ovary, the empowered mesenchymal stem cells have significantly enhanced viability and stemness, and have the effects of anti-ovarian tissue fibrosis, promoting angiogenesis of ovarian tissue, promoting ovarian regeneration, follicular development, and improving ovarian endocrine function and / or fertility function (including improving the endocrine function and fertility function of the reproductive-age ovary, and improving the endocrine function of the senescent ovary); the combined application of the empowered mesenchymal stem cells and the HEP14 / PLGA microsphere composition further strengthens the retention of the empowered mesenchymal stem cells in the ovarian tissue and their transdifferentiation into follicular granulosa cells and theca cells, and thus the combined application of the composition further strengthens the effects of the empowered mesenchymal stem cells on anti-ovarian tissue fibrosis, promoting angiogenesis of ovarian tissue, ovarian regeneration, follicular development, and improving ovarian endocrine function and / or fertility function.

[0008] The present invention solves the technical problems by adopting the following technical solutions:

[0009] On the one hand, the present invention provides an empowered mesenchymal stem cell, which is obtained by treating human mesenchymal stem cells with HEP14, and the HEP14 is a 5β-O-angeloyl-20-deoxyingol compound with a molecular structure as shown in the following structural formula:

[0010]

[0011] Preferably, the treatment of human mesenchymal stem cells with HEP14 includes the following steps:

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

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

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

[0015] (4) Washing the human mesenchymal single cells with physiological saline to obtain the empowered mesenchymal stem cells suspended in physiological saline.

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

[0017] Preferably, the source of the human mesenchymal stem cells includes mesenchymal stem cells from different human tissues such as bone marrow, adipose tissue, placenta, umbilical cord, skin, amnion, and dental pulp.

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

[0019] On the other hand, the present invention provides an empowered mesenchymal stem cell composition, which comprises:

[0020] Empowered mesenchymal stem cells, which are obtained by treating human mesenchymal stem cells with HEP14.

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

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

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

[0024] (2) Adding HEP14 with a concentration of 1.25 - 20 μM to the culture medium to treat human mesenchymal stem cells for 12 hours - 48 hours;

[0025] (3) Digesting human mesenchymal stem cells to prepare a single-cell suspension;

[0026] (4) Washing the human mesenchymal single cells with physiological saline to obtain the empowered mesenchymal stem cells suspended in physiological saline.

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

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

[0029] In a dark environment, poly(lactic-co-glycolic acid) (PLGA) and HEP14 are dissolved in dichloromethane at a mass ratio of 5 - 10:1, and stirred with a magnetic stirrer 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 stirred with a magnetic stirrer at a speed of 300 - 500 rpm at 25°C to volatilize dichloromethane for at least 12 hours. Then, the microspheres are collected by centrifugation, and repeatedly washed with distilled water to completely remove the residual polyvinyl alcohol. Finally, the microspheres are placed in a freeze dryer for freeze-drying treatment to obtain HEP14 / PLGA microspheres.

[0030] More preferably, poly(lactic-co-glycolic acid) copolymer and HEP14 are dissolved in dichloromethane at a mass ratio of 5:1.

[0031] Preferably, the human mesenchymal stem cell source includes mesenchymal stem cells from different human tissue sources such as bone marrow, adipose tissue, placenta, umbilical cord, skin, amnion, and dental pulp.

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

[0033] On the other hand, the present invention provides the use of the empowered mesenchymal stem cells in the preparation of a drug for anti-tissue fibrosis.

[0034] On the other hand, the present invention provides the use of the empowered mesenchymal stem cells in the preparation of a drug for promoting tissue angiogenesis.

[0035] On the other hand, the present invention provides the use of the empowered mesenchymal stem cells in the preparation of a drug 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, and skin tissue.

[0037] On the other hand, the present invention provides the use of the empowered mesenchymal stem cells in the preparation of a drug for treating organ dysfunction.

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

[0039] More preferably, the organ is the ovary.

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

[0041] (1) Application in the preparation of a drug for treating premature ovarian failure in reproductive-aged women;

[0042] (2) Application in the preparation of a drug for treating ovarian function decline in elderly women and its resulting complications;

[0043] (3) Application in the preparation of a drug for restoring ovarian endocrine and / or reproductive function.

[0044] On the other hand, the present invention provides the use of the empowered mesenchymal stem cell composition in the preparation of a drug for anti-tissue fibrosis.

[0045] On the other hand, the present invention provides the use of the empowered mesenchymal stem cell composition in the preparation of a drug for promoting tissue angiogenesis.

[0046] On the other hand, the present invention provides the use of the empowered mesenchymal stem cell composition in the preparation of a drug 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] On the other hand, the present invention provides the use of the empowered mesenchymal stem cell composition in the preparation of a drug for treating organ dysfunction.

[0049] Preferably, the organ dysfunction is caused by damage or aging of the regeneration function of the organ, and the organ is one of ovary, liver, lung, brain, heart, muscle, bone, kidney, and skin.

[0050] More preferably, the organ is the ovary.

[0051] Preferably, a physiological saline solution containing the empowered mesenchymal stem cells is locally injected into the ovary of a subject in need, and a physiological saline solution containing the HE P14 / PLGA microspheres is injected into the subject in need, including any one of the following applications:

[0052] (1) Use in the preparation of a drug for treating premature ovarian failure in women of childbearing age;

[0053] (2) Use in the preparation of a drug for treating ovarian function decline in elderly women and its resulting complications;

[0054] (3) Use in the preparation of a drug for restoring ovarian endocrine and / or reproductive functions.

[0055] Fibrosis and ischemia of ovarian tissue or other organ tissues are important reasons leading to the loss of transplanted stem cells and the failure of cell therapy. The number of transplanted stem cells and the cytokines they secrete significantly affect the treatment effect. Locally injecting a physiological saline solution containing the empowered mesenchymal stem cells into the ovary and simultaneously injecting a physiological saline solution containing the HEP 14 / PLGA microspheres into the subject in need, this separate and combined administration method has the following advantages: First, using different administration routes helps to provide more accommodation space for locally injecting empowered mesenchymal stem cells. Second, this administration route is more conducive to the more direct, timely and effective interaction between empowered mesenchymal stem cells, follicular cells, cytokines and the extracellular matrix. Therefore, it promotes the retention and viability of transplanted empowered mesenchymal stem cells in the ovary and / or the corresponding treated organ tissue. Third, this administration method can achieve multiple administrations. Coupled with the long-term slow-release characteristics of the HEP 14 / PLGA microspheres, it can maintain a stable and continuous HEP 14 blood drug concentration, ultimately improving the treatment effect of the empowered mesenchymal stem cell composition on ovarian insufficiency and / or the insufficiency of other organ tissues, especially improving the treatment effect of severe ovarian insufficiency.

[0056] More preferably, the injection route of the physiological saline solution containing the HEP 14 / PLGA microspheres includes any one of intraperitoneal injection, subcutaneous injection, and intramuscular injection.

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

[0058] The ovarian follicle regeneration and development include the development of ovarian mesenchymal tissue and follicles at all levels and / or the development of mature follicles.

[0059] The research of the present invention finds that: HEP14 activates the PKC-ERK1 / 2 signaling pathway, stimulates a variety of secreted factors, particularly including promoting the secretion of STC1, MMP1 and PDGFD. These factors act synergistically to strengthen the anti-tissue fibrosis and pro-angiogenic effects of transplanted empowered mesenchymal stem cells through MMP1 and PDGFD, and improve the microenvironment of organ tissues; in the ovary, STC1 stimulates the secretion of CYP19A1, and synergistically stimulates the growth of ovarian follicular granulosa and foam cells, thus promoting follicle development and improving the secretion and endocrine function of ovarian hormones. In summary, these results indicate that the empowered mesenchymal stem cells and the empowered mesenchymal stem cell composition of the present invention have excellent effects in promoting the regeneration and functional recovery of damaged organ tissues.

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

[0061] The empowered mesenchymal stem cells of the present invention have significantly enhanced viability and stemness, and have the effects of anti-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 function recovery, and have the effect of treating organ dysfunction of ovaries, liver, lung, brain, heart, muscle, bone, kidney, skin, etc.; the combined application of empowered mesenchymal stem cells and HE P14 / PLGA microsphere composition further strengthens the retention and transdifferentiation effects of empowered mesenchymal stem cells in organ tissues. Therefore, the combined application of the composition further strengthens the anti-tissue fibrosis and tissue angiogenesis-promoting effects of empowered mesenchymal stem cells, further strengthens the effects of promoting tissue regeneration and function recovery, and the effect of treating organ dysfunction.

[0062] Especially in the ovaries, the empowered 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, follicular development, and improving ovarian endocrine function and / or fertility function (including improving the endocrine function and fertility function of reproductive-age ovaries, and improving the endocrine function of senescent ovaries); the combined application of empowered mesenchymal stem cells and HE P14 / PLGA microsphere composition further strengthens the retention of empowered mesenchymal stem cells in ovarian tissues and their transdifferentiation into follicular granulosa cells and theca cells. Therefore, the combined application of the composition further strengthens the anti-ovarian tissue fibrosis, ovarian tissue angiogenesis, ovarian regeneration, follicular development, and improvement of ovarian endocrine function and / or fertility function of empowered mesenchymal stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 HEP14 treatment enhanced the stemness as well as the proliferation and survival ability of hADSCs.

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

[0065] Figure 3 For the morphological and property analysis of HE P14 / PLGA microspheres.

[0066] Figure 4To establish a mouse model of adriamycin-induced ovarian insufficiency, the results showed that single intraperitoneal injection of adriamycin at a dose of 12 mg / kg could cause obvious damage to the ovarian structure and function of mice.

[0067] Figure 5 To empower adipose stem cells and adipose stem cell compositions to treat and restore premature ovarian failure in women of childbearing age, both the treatment with empowered adipose stem cells alone or in combination with the HE P14 / PLGA microsphere composition have significant effects in reducing ovarian tissue fibrosis, promoting angiogenesis in ovarian tissue, promoting ovarian follicle development in PO l mice, and restoring reproductive and endocrine functions. The combined treatment with the composition is superior to the treatment effect of using empowered hADSCs alone.

[0068] Figure 6 To empower adipose stem cells and adipose stem cell compositions to treat senile ovarian function decline and its resulting complications, both the treatment with empowered adipose stem cells alone or in combination with the adipose stem cell / PLGA microsphere composition have significant effects in reducing ovarian tissue fibrosis, promoting angiogenesis in ovarian tissue, and promoting ovarian follicle development and regeneration in senile mice. The effect of the combined application of the composition is significantly better than that of using empowered hADSCs alone.

[0069] Figure 7 For the mechanism study of empowering adipose stem cells and adipose stem cell compositions to treat premature ovarian failure in women of childbearing age, HE P14 can stimulate the secretion of multiple factors, particularly including STC1, CYP19A1, MMP1, and PDGFD. These factors can resist ovarian tissue fibrosis and promote ovarian angiogenesis through synergistic paracrine and endocrine mechanisms, improve the ovarian tissue microenvironment, stimulate the growth of ovarian follicular granulosa and foam cells, and thus promote follicle development and improve ovarian hormone secretion. Detailed implementation manners

[0070] The following combines specific embodiments to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

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

[0072] Example 1: Preparation and identification of empowered mesenchymal stem cells

[0073] The preparation method of empowered mesenchymal stem cells includes the following steps:

[0074] (1) Cultivate and expand human mesenchymal stem cells;

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

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

[0077] (4) Wash the human mesenchymal cells with physiological saline to obtain the empowered mesenchymal stem cells suspended in physiological saline.

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

[0079] The specific preparation method and identification of the empowered mesenchymal stem cells are as follows:

[0080] Under the supervision of the medical ethics committee, human mesenchymal stem cells are isolated and extracted from 5 - 10 ml of fresh adipose tissue from healthy donors, and human adipose-derived stem cells (hADSCs) are obtained. Primary cell culture and expansion are carried out in a serum-free culture system, and the serum-free culture system is: Ultraculture (Lonza) + commercially available growth factors. Place it in an incubator at 37 °C containing 5% carbon dioxide. Replace the culture medium after 24 hours, aspirate the suspended dead cells or cell debris, and then add physiological saline to wash the cells once and change the medium. When the cells reach 90% confluence, subculture and expansion can be carried out. The primary cells of passage 2 (P2) are cryopreserved as seed cells. In this specification, the P2 generation refers to the passage number of primary cells in the culture flask after reaching 90% confluence twice and being digested and reseeded into the culture flask. hADSCs from the 3rd to 5th passages are used, preferably the 4th passage hADSCs. Morphologically, the isolated primary hADSCs have a fibroblast-like appearance ( Figure 1 A). Flow cytometry analysis shows that more than 95% of hADSCs are negative for CD31 and HLA-DR and positive for CD73, CD90, CD105, and CD29 ( Figure 1B). The results of alizarin red and oil red staining showed that hADSCs had osteogenic Figure 1 C) and adipogenic differentiation ability Figure 1 D). These results indicated that the isolated hADSCs were highly pure 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 treating hADSCs with 2.5 μM HEP14 for 32 hours, the viability Figure 1 E) and proliferation ability of hADSCs were significantly increased Figure 1 F). This proliferation was manifested as an increase in hADSCs in the S phase and a decrease in G0 / G1 determined by flow cytometry Figure 1 G and 1H). The results of real-time quantitative PCR showed that compared with control group hADSCs (hADSCs not treated with HEP14, denoted as c-hADSCs), the expression of the pluripotent stem cell marker NANOG in HEP14-treated hADSCs was significantly increased, and the expression of the mesenchymal stem cell proliferation marker protein NUCLEOSTEMlN was slightly increased Figure 1 l). Notably, the Ct values of the real-time qPCR reaction showed that both nuclear NUCLEOSTEMIN mRNA in HEP14-treated and control group hADSCs presented high expression levels. The results of cell immunofluorescence staining and Western blot further confirmed that HEP14 treatment significantly increased the expression of NANOG and NUCLEOSTEMIN in hADSCs Figure 1 J and 1K). These results indicated that HEP14 treatment enhanced the stemness, proliferation and viability of hADSCs. Therefore, we treated hADSCs with 2.5 μM HEP14 for 32 hours, then digested the HEP14-treated hADSCs with digestive enzymes to make a single-cell suspension, and finally washed the single cells of HEP14-treated hADSCs with physiological saline to obtain HEP14-treated hADSCs, that is, empowered mesenchymal stem cells, also known as empowered adipose stem cells (denoted as h-hADSCs). The empowered mesenchymal stem cells were suspended in physiological saline for standby.

[0081] Example 2: The change in the gene expression profile of hADSCs induced by HEP14 contributes to tissue remodeling and regeneration

[0082] To deeply understand the molecular biological characteristics of h-hADSCs, we performed RNA sequencing on h-hADSCs and c-hADSCs. The results showed that a total of 1003 differentially expressed genes (DEGs) were found in h-hADSCs, including 573 up-regulated genes and 430 down-regulated genes Figure 2A and 2B). Gene Ontology analysis showed that these DEGs were enriched in biological function processes such as extracellular matrix organization and tissue remodeling, angiogenesis, cell fate determination, ERK1 and ERK2 cascades, and response to estradiol ( Figure 2 C), which was closely related to the regeneration and functional recovery of organ tissues, especially ovarian tissues. We reconstructed the sub-thermogram of key genes enriched in these biological processes ( Figure 2 D). Real-time quantitative PCR confirmed that the expression levels of these genes in h-hADSCs were significantly altered compared with c-hADSCs ( Figure 2 E and 2F). Among them, STC1 was the DEG with the highest up-regulated expression by HEP14. It has been reported that STC1 is expressed in various tissues such as mammalian 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 tissues, and the latter is a marker gene of ovarian granulosa cells, which promotes the production of estrogen. MMP1 (Matrix metalloproteinase 1) can degrade type 1, type 2 and type 3 collagens in the extracellular matrix, promote the remodeling of the extracellular matrix, and thus lead to changes in the cell microenvironment. Platelet-Derived Growth Factor D (PDGFD) is a powerful angiogenic growth factor. 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 exerted its biological functions by regulating signal pathways such as extracellular matrix (ECM) receptor interaction, cell fate determination, angiogenesis, ovarian steroid hormone production and MAPK activity activation ( Figure 2 G-2K). And these biological functions are crucial for the regeneration and functional recovery of organ tissues, especially ovarian tissues. In summary, RNA transcriptomics analysis showed that HEP14-empowered hADSCs contribute to tissue remodeling, regeneration and functional recovery.

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

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

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

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

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

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

[0089] To conduct a detailed morphological and property analysis of the microspheres prepared in Example 3, Example 4, and the experimental controls, we used a scanning electron microscope to detect the above-prepared samples. Meanwhile, we used Image J and Origin software to perform precise quantitative analysis of the particle size of the microspheres. In addition, we also used an FTIR system to characterize the chemical structure of the samples. The detection was carried out under the conditions of a scanning range of 400 to 4000 cm-1, a resolution of 4 cm-1, and a 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 patterns of the samples were collected by a Rigaku Smartlab X-ray diffractometer. During the experiment, we used a CuKα radiation source (45 kV, 120 mA), and set the 2θ angle range to 5° to 100°. The obtained data were collected in the form of transmittance values (%) and processed and analyzed by Origin software.

[0091] Through SEM analysis, we found that both PLGA and HEP14 / PLGA microspheres exhibited a uniform size distribution, with a spherical shape and a smooth surface ( Figure 3 A and 3B). Due to the large loading of HEP14, the particle sizes of the 5:1 HEP14 / PLGA microspheres (prepared in Example 3) (0.1688 μm ± 0.0685) and the 10:1 HEP14 / PLGA microspheres (prepared in Example 4) (0.100 μm ± 0.0735) were significantly increased compared to the pure PLGA microspheres (0.052 μm ± 0.028). To verify whether the HEP14 drug was successfully encapsulated in PLGA, we analyzed the drug-loaded and unloaded microspheres. From the infrared spectrum of the HEP14 / PLGA sample, we did not observe the vibration peak of HEP14 ( Figure 3 C). This indicates that HEP14 may be completely encapsulated in the PLGA matrix, or the vibration peak of the PLGA matrix is similar to that of HEP14, thus 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 during the loading process, the crystallinity of HEP14 decreased ( Figure 3 D). This change enables HEP14 to be gradually released from the PLGA microspheres, making it easier to be absorbed and utilized by organisms.

[0092] To determine the drug loading rate, encapsulation efficiency and in vitro release of HEP14 / PLGA microspheres, we measured the drug loading amount and encapsulation efficiency of HEP14 / PLGA microspheres using an ultraviolet spectrophotometer, and plotted the standard curve of the 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 formulas respectively, where the theoretical drug loading of the microspheres refers to the initial amount of drug used to prepare the microspheres. Drug loading rate (%) = (weight of drug in microspheres) / (weight of microspheres) × 100%; Entrapment rate (%) = (actual drug loading amount in microspheres) / (theoretical drug loading amount of microspheres) × 100%. The in vitro release rate of HEP14 / PLGA / microspheres was measured at 201 nm using an ultraviolet spectrophotometer. The HEP14 / PLGA microspheres were placed in PBS solution at pH 7.4, incubated at 37 °C and 100 rpm in a shaker, and the microspheres were precipitated by centrifugation (5000 × g, 3 min), and the supernatant was collected on days 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 for detection.

[0093] To study the drug loading rate, encapsulation rate and release degree 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 amount and content of microspheres. The drug loading rate and encapsulation rate of HEP14 in the HEP14 / PLGA microspheres prepared in Example 3 were 3.10% and 18.04% respectively. The drug loading rate and encapsulation rate 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 ultraviolet spectrophotometry, as shown in ( Figure 3 E). Initially, rapid release occurred within the first day, and then sustained and controlled release occurred from day 1 to day 9. Subsequently, additional burst release was observed from day 9 to day 17, ultimately resulting in gradual release until equilibrium was reached on day 17. At the end of the study, the cumulative release amount of HEP14 / PLGA microspheres was approximately 9.73%.

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

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

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

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

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

[0099] Female C57BL / 6N mice at 6-7 weeks of age were weighed and randomly divided into two groups (n = 18 per group). Then, they were intraperitoneally injected with different doses (8 mg / kg, 12 mg / kg, and 16 mg / kg) of doxorubicin (Sigma) (n = 18 / group) or an equal volume of saline solution as a control. At 1, 2, and 4 weeks after treatment, the mice were sacrificed by cervical dislocation to collect samples. Body weight, ovarian size, estrous cycle, serum hormone levels, follicle count and morphology were examined, and the POI model was evaluated. The results showed that among the 6 mice injected with 16 mg / kg doxorubicin intraperitoneally, 2 mice showed continuous weight loss within 4 weeks and very poor health conditions. Therefore, according to the national regulations for the use of experimental animals, they were euthanized (data not shown), but this did not occur in mice with 8 mg / kg or 12 mg / kg doses of doxorubicin. Hematoxylin and eosin (H&E) staining of ovarian tissues showed that the ovarian size and follicle structure in mice treated with 8 mg / kg doxorubicin changed less, while compared with the ovaries of mice treated with 12 mg / kg doxorubicin, the ovaries of mice treated with 16 mg / kg doxorubicin showed irreparable ovarian atrophy and follicular atresia. Therefore, we chose to prepare a mouse POI model with a 12 mg / kg dose of doxorubicin ( Figure 4 A). At 1, 2, and 4 weeks after doxorubicin induction, the results of H&E staining of tissues showed that the ovarian structure of control mice was normal, with multiple follicles and corpora lutea at all developmental stages, and all follicles were surrounded by flattened stromal cells, while the ovaries of doxorubicin-treated mice showed degenerative changes in follicles and mesenchyme, and up to 80% of the follicles showed atretic follicles ( Figure 4 B). At 1 and 2 weeks after doxorubicin induction, the proportion of growing follicles in the total number of follicles in the damaged ovaries increased slightly. By the 4th week, in sharp contrast to the control ovaries, the total number of follicles in the ovaries of doxorubicin-treated mice further decreased significantly ( Figure 4 C). Immunofluorescence chemistry (IF) staining showed that the number of follicles with positive Ki67 and negative TUNEL expression in the control ovaries was more than that in the doxorubicin-treated ovaries ( Figure 4 D-4E). These results indicate that doxorubicin treatment leads to a significant loss of ovarian follicle reserve.

[0100] Hormone analysis showed that within 1 - 4 weeks after induction, compared with the control group, the serum FSH level in doxorubicin - treated mice was significantly increased statistically ( Figure 4 F), but the E2 and Amh levels were significantly decreased ( Figure 4 G and 4H). The results of vaginal smears showed that the control group mice had a normal estrous cycle of 4 - 5 days, while the doxorubicin - treated mice presented an irregular estrous cycle, mainly stopping at estrus ( Figure 4 I). The results indicated that a single intraperitoneal injection of doxorubicin at a dose of 12 mg / kg could cause obvious damage to the ovarian structure and function in mice. Here, we successfully established a doxorubicin - induced POI mouse model (abbreviated as POI mice).

[0101] Example 8: Empowering adipose - derived stem cells and an empowering adipose - derived stem cell composition for treating premature ovarian failure in women of childbearing age

[0102] To study the effect of empowering adipose - derived stem cells and an empowering adipose - derived stem cell composition on treating premature ovarian failure in women of childbearing age, the empowering adipose - derived stem cells prepared in Example 1 and the empowering adipose - derived stem cell composition prepared in Example 6 were used to conduct a study on treating premature ovarian failure in POI model mice. Specifically, the empowering adipose - derived stem cell composition used in this example was: the empowering adipose - derived stem cells prepared in Example 1 + the HEP14 / PLGA microspheres prepared in Example 3.

[0103] I. Treatment of POI model mice

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

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

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

[0107] h-hADSCs mice: Inject 15 μl of normal saline containing empowered h-hADSCs-CM-DIL into the ovaries of POI mice and inject 100 μl of normal saline intraperitoneally (i.p.); the density of empowered h-hADSCs is 5×10 4 cells / μl.

[0108] h-hADSCs / HEP14 mice: Treat POI mice by injecting 15 μl of normal saline containing empowered h-hADSCs-CM-DIL into the ovaries and injecting 100 μl of normal saline containing HEP14 / PLGA microspheres intraperitoneally (i.p.); the density of empowered h-hADSCs is 5×10 4 cells / μl, and the content of HEP14 / PLGA microspheres is 10 μg / μl.

[0109] To shorten the operation time and avoid inter-group deviation caused by the decrease of cell viability over time, only the right ovarian tissue of the four groups of mice was injected. At 2 weeks and 4 weeks after treatment, 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 fertile males at a ratio of 1:2 for 2 weeks. Since mating, the vaginal plugs were checked every morning at 9:00 the next day for 2 weeks. At 18 to 19 days of pregnancy in the mice, the fetuses were removed by cesarean section. The number of pregnant mice, the total number of fetuses produced, and the average number of offspring per mouse in each group were recorded to evaluate their fertility.

[0110] II. Promote the restoration of ovarian tissue structure and follicle regeneration in premature ovarian failure during the reproductive age

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

[0112] These results indicate that the treatment with empowered adipose stem cells alone or in combination with the HE P14 / PLGA microsphere composition can significantly promote follicular regeneration and development in PO l mice, and the combined treatment with the composition is superior to the treatment with empowered hADSCs alone.

[0113] III. Promotion of the restoration of endocrine function in premature ovaries of women of childbearing age

[0114] Four weeks after treatment, the estrous cycles, serum hormone levels and fertility of the four groups of mice were further evaluated. Compared with the normal 4-5-day estrous cycle presented by the control group mice, 5 out of 6 h-hADSCs / HEPl 4 mice restored normal estrous cycles, 4 out of 6 h-hADSCs mice restored normal estrous cycles, while the estrous cycles of PO l mice were prolonged ( Figure 5 J). The results of serum ovarian hormone detection showed that compared with POI mice, the h-hADSCs / HEP14 mice and h-hADSCs mice presented higher levels of E2 (estradiol) and AM H (anti-Müllerian hormone) ( Figure 5 K-5L), but lower levels of FSH (follicle-stimulating hormone) ( Figure 5 M). The levels of these hormones in the serum of h-hADSCs / HEP14 mice were restored to near normal control.

[0115] Starting from the 4th week after treatment, the four groups of mice were subjected to mating experiments at a ratio of 1:2 (male: female) for 2 weeks. To obtain reliable data for evaluating the fertility of each group of mice, the fetal mice were removed by cesarean section at 18 - 19 days of pregnancy. The pregnancy rate of POI mice was (2 / 10), which was significantly lower compared to that of h-hADSCs mice (7 / 10), h-hADSCs / HEP14 mice (10 / 10), and normal control mice (10 / 10). The average number of fetuses / litters (0 / 2) and the number of healthy fetuses / litters (1 / 0) in POI mice were significantly lower than those of the empowered h-hADSCs mice (2.4 / 2.0), h-hADSCs / HEP14 mice (4.6 / 3.5), and control group mice (5.7 / 5.5) ( Figure 5 N and 5O). There was 1 pair of identical twins in the h-hADSCs / HEP14 group ( Figure 5 P). These results indicate that h-hADSCs / HEP14 mice and h-hADSCs mice have better fertility than POI mice. Treatment with empowered h-hADSCs alone and the composition (empowered h-hADSCs combined with HEP14 / PLGA microspheres) both have 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 is better than that of using empowered adipose stem cells alone (i.e., h-hADSCs).

[0116] Example 9: Treatment of senile ovarian function decline and its resulting complications with empowered adipose stem cells and empowered adipose stem cell compositions

[0117] To study the effect of treating senile premature ovarian function decline with empowered adipose stem cells and empowered adipose stem cell compositions, 73 - 74-week-old female C57BL / 6N senile mice were used as model animals and randomly divided into three groups: a control group treated with normal saline (CTL mice), a group treated with the empowered mesenchymal stem cells prepared in Example 1 alone (h-hADSCs mice), and a group treated with the empowered mesenchymal stem cell composition prepared in Example 6 (h-hADSCs / HEP14 mice). Specifically, the empowered adipose stem cell composition used in this example was: the empowered adipose stem cells prepared in Example 1 + the HEP14 / PLGA microspheres prepared in Example 3.

[0118] The specific grouping and treatment are as follows′

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

[0120] h-hADSCs mice were treated with 15 μl of saline solution containing energized h-hADSCs-CM-DIL intra-ovarian injection and 100 μl of saline solution intraperitoneally (ip). The density of energized h-hADSCs was 5×10 4 cells / μl.

[0121] Mice were treated with the empowered adipose-derived stem cell composition (h-hADSCs / HEP14 mice): 15 μl of saline solution containing empowered h-hADSCs-CM-DIL was injected into the ovaries of the mice and 100 μl of saline solution containing HEP14 / PLGA microspheres was injected intraperitoneally (ip). The density of the empowered h-hADSCs was 5×10 4 cells / μl, and the content of HEP14 / PLGA microspheres was 10 μg / μl.

[0122] 1. Anti-ovarian tissue fibrosis and promote ovarian blood vessel proliferation

[0123] After 4 weeks of treatment, the ovaries of the three groups of mice (CTL-, h-hADSCs-, and h-hADSCs / HEP14 mice) were collected for histopathological examination and the proliferation and number of follicles at each developmental stage. The results showed that the ovaries of aged h-hADSCs / HEP14 mice and h-hADSCs mice were significantly larger than those of untreated aged mice. The ovaries of h-hADSCs / HEP14 mice recovered better than those of h-hADSCs mice ( Figure 6 A). Sirius red (PSR) staining results showed that the collagen fibers in the ovaries of untreated aged mice were significantly more than those in the ovaries of h-hADSCs mice and h-hADSCs / HEP14 mice, and the collagen fibers in the ovaries of h-hADSCs mice were significantly more than those in the ovaries of h-hADSCs / HEP14 mice ( Figure 6 B). Immunohistochemistry (IHC) staining results showed that compared with the ovaries of h-hADSCs mice and CTL mice, the myofibroblast (MFB) marker protein α-SMA in the ovaries of h-hADSCs / HEP14 mice was significantly reduced, and the endothelial cell-specific marker protein CD31 and signal were significantly increased. The expression of α-SMA and CD31 in the ovaries of h-hADSCs mice was between that in the ovaries of h-hADSCs / HEP14 mice and CTL mice ( Figure 6 C and 6D). The above results indicate that empowered adipose-derived stem cells alone or in combination with the HEP14 / PLGA microsphere composition can reduce ovarian tissue fibrosis and promote ovarian tissue angiogenesis.

[0124] 2. Promote the development and regeneration of ovarian follicles in aged mice

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

[0126] III. Promote the restoration of endocrine function of senescent ovaries

[0127] Furthermore, the restoration of serum hormone levels in the three groups of old mice was evaluated 4 weeks after treatment. Serum ovarian hormone detection results showed that compared with the old control group mice, h-hADSCs / HEP14 mice and h-hADSCs mice showed significantly increased levels of E2 (estradiol), AMH (anti-Müllerian hormone), INHA and INHB ( Figure 6 J, 6, K, 6L and 6M) and decreased levels of FSH (follicle-stimulating hormone) and T (testosterone) ( Figure 6 N and 6O). The improvement of serum levels in h-hADSCs / HEP14 mice was better than that in h-hADSCs mice. These results indicate that both the sole empowerment of h-hADSCs and the empowered h-hADSCs composition have a significant effect on promoting the regeneration of old ovarian tissue and endocrine function.

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

[0129] Example 10: Mechanism study on the treatment of premature ovarian failure in women of childbearing age with empowered adipose stem cells and empowered adipose stem cell compositions

[0130] I. Empowered adipose stem cells alone or in combination with HEP14 / PLGA microsphere composition reduce ovarian tissue fibrosis and promote ovarian angiogenesis by enhancing the secretion of MMP1 and PDGFD

[0131] Follicular development is characterized by angiogenesis and extracellular matrix remodeling. Therefore, we evaluated the degree of ovarian tissue fibrosis and angiogenesis in four groups of mice 4 weeks after treatment. Sirius red (PSR) staining was used to determine the collagen content, and polarized light microscopy was used to distinguish collagen I (red) and collagen III (green). The results showed that the collagen fibers in the normal ovary were thinner and evenly distributed, while the collagen fibers in the ovaries of POI mice were significantly increased, relatively thicker, and clustered. 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 ( Figure 7 A 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 ( Figure 7 A and 7B). Further immunohistochemistry and immunocytofluorescence (IHC and IF) staining results showed that the expression of the myofibroblast (MFB) marker protein α-SMA ( Figure 7 C) in the ovaries of h-hADSCs / HEP14 mice was significantly reduced, and the signal of the endothelial cell-specific marker protein CD31 was increased ( Figure 7 D), which was close to the expression level in the normal control group, but was in sharp contrast to the significant increase in the expression of α-SMA and the significant decrease in the expression of CD31 in the ovaries of POI mice ( Figure 7 D). The expression of α-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 7 D). The above results demonstrate that empowered adipose stem cells alone or in combination with HEP14 / PLGA microsphere composition can reduce ovarian tissue fibrosis and promote ovarian tissue angiogenesis.

[0132] To gain an in-depth understanding of the molecular mechanisms underlying the anti-ovarian tissue fibrosis and pro-angiogenic effects of hADSCs alone or in combination with the HEP14 / PLGA microsphere composition, we examined the expression of three key differential genes, MMP1, PDGFD, and PDGFR, in the ovarian tissues of mice in each group. MMP1 is the most abundant matrix metalloproteinase and can degrade type I, II, and III collagens. PDGFD is a potent angiogenic growth factor that reportedly promotes angiogenesis by binding to its cognate receptor, PDGFRβ. Western blot confirmed that HEP14 significantly increased the expression of MMP1, PDGFD, and PDGFRβ in h-hADSCs compared with that in the c-hADSCs ( Figure 7 E). Results of immunohistochemistry (IHC) and immunocytochemistry (ICC) staining of ovarian tissue sections showed that the expression of MMP1, PDGFD, and PDGFRβ in the ovaries of h-hADSCs / HEP14 mice was significantly higher than that in the ovarian tissues of h-hADSCs mice and POI mice ( Figure 7 F-7G). The expression levels of MMP1, PDGFD, and PDGFRβ in the ovaries of h-hADSCs mice were significantly higher than those in POI mice ( Figure 7 F-7G). To confirm the enhanced anti-fibrotic and angiogenic capabilities observed above, conditioned media, including c-hADSCs-CM and h-hADSCs / HEP14-CM, were collected. Consistent with the results of real-time qPCR and Western blot analysis ( Figure 2 F and Figure 7 E), results of enzyme-linked immunosorbent assay (ELISA) confirmed that treatment with HEP14 did increase the secretion level of MMP1 in hADSCs ( Figure 7 H). Meanwhile, human umbilical vein endothelial cells (HUVECs) were cultured using the above conditioned media and a specific PDGFD neutralizing antibody (PDGFDNab) for tube formation assays. Results of the tube formation assays based on the total length and the number of branch points showed that the h-hADSCs / HEP14-CM medium significantly increased the tube formation ability of HUVECs compared with the c-hADSCs-CM conditioned medium ( Figure 7 I). IF staining of CD31 confirmed the formation of these tubes ( Figure 7 J). These results strongly supported that HEP14 enhanced the angiogenic ability of hADSCs. In summary, these findings indicated that HEP14-empowered hADSCs significantly enhanced the anti-fibrotic and pro-angiogenic effects of h-hADSCs.

[0133] The above results indicate that the empowered adipose stem cells alone or the combination of empowered adipose stem cells and HEP14 / PLGA microspheres both stimulate the secretion of MMP1, PDGFD, and PDGFRβ, and thus have the effects of reducing ovarian tissue fibrosis and promoting ovarian angiogenesis. The anti-fibrosis and angiogenesis-promoting effects of the combination of empowered adipose stem cells and HEP14 / PLGA microspheres are stronger than those of the treatment with empowered stem cells alone.

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

[0135] The anti-Vimentin antibody (Ab) has been confirmed to be a human cell-specific antibody and does not react with mouse tissue cells (certificate of analysis, ab8069, Abcam). The results of immunohistochemical staining showed that the Vimentin expression signal overlapped with the CM-DIL signal labeling the empowered adipose stem cells. Therefore, Vimentin-positive adipose stem cells (hADSCs Vimentin+ ) represent the empowered h-hADSCs transplanted in the ovary ( Figure 7 K). Four weeks after the transplantation of empowered h-hADSCs, the empowered h-hADSCs detected in the ovaries of h-hADSCs / HEP14 mice Vimentin+ were significantly more than those of the h-hADSCs in the ovaries of mice transplanted with empowered h-hADSCs alone Vimentin+ . Eight weeks after transplantation, the remaining empowered hADSCs in the ovaries of h-hADSCs / HEP14 mice Vimentin+ were significantly more than those of the empowered hADSCs in the ovaries of h-hADSCs mice Vimentin+ ( Figure 7 K). In addition, these cells were found to be involved in the granulosa cell layer or theca cell layer of follicles in the ovaries of h-hADSCs / HEP14 mice ( Figure 7 K). The hADSCs detected in the ovaries of h-hADSCs mice Vimentin+ were mainly distributed in the ovarian stromal tissue or around growing follicles ( Figure 7 K). These results indicate that HEP14 promotes the retention and proliferation of h-hADSCs in the ovary. More importantly, the Vimentin signal in these hADSCs Vimentin+ overlaps with the FSHR or CYP19A1 expression signal in the ovarian granulosa layer ( Figure 7 L); it overlaps with the CYP17A1 expression signal in the theca cell layer ( Figure 7M). FSHR and CYP19A1 are markers of granulosa cells in ovarian tissue, and CYP17A1 is a marker of follicular cells in ovarian tissue. These results indicate that the combined application of HEP14 / PLGA microspheres and empowered hADSCs promotes the retention and survival of transplanted empowered hADSCs and induces their transdifferentiation into functional granulosa-like cells or follicle-like cells. Therefore, the composition of HEP14 / PLGA microspheres and empowered hADSCs has a stronger effect on promoting follicular hyperplasia, development, and ovarian function recovery.

[0136] Consistent with the RNA-seq detection results ( Figure 2 E), the Western blot results showed that HEP14 significantly promoted the secretion of cytokines such as STC1 and CYP9A by empowered adipose stem cells. Figure 7 N). Immunofluorescence staining detection found that in the ovaries of h-hADSCs / HEP14 mice, the expression of STC1 was significantly higher than that in the ovaries of h-hADSCs mice and POI mice. The expression level in h-hADSCs mice was significantly higher than that in POI mice. Figure 7 O). Further Western blot results showed that knocking down the expression of STC1 in ovarian granulosa cell lines with siRNA led to a decrease in CYP19A1 expression, indicating that STC1 promotes the expression of CYP19A1 in granulosa cells in ovarian tissue. Figure 7 P). The main function of CYP19A1 is to stimulate granulosa cells to produce estradiol. At the same time, granulosa cells are also the target of action of estradiol in ovarian tissue, thus promoting the regeneration and development of ovarian follicles.

[0137] In summary, the research of this invention found that HEP14 can stimulate the secretion of multiple factors, particularly including STCl, CYP19A1, MMP1, and PDGFD. These factors act synergistically. On the one hand, MMP1 and PDGFD strengthen the anti-ovarian tissue fibrosis and promote ovarian angiogenesis of transplanted empowered mesenchymal stem cells, improving the ovarian tissue microenvironment; on the other hand, STC1 and CYP19A are synergistically stimulating the growth of ovarian follicular granulosa cells, thus promoting follicular development and improving the secretion and endocrine function of ovarian hormones. In short, these results indicate that the empowered mesenchymal stem cells and the empowered mesenchymal stem cell composition of this invention have excellent effects on promoting the regeneration of damaged ovarian tissue and the recovery of function.

[0138] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above-described exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be embraced within the present invention.

[0139] In addition, it should be understood that although this specification is described in terms of embodiments, not every embodiment only contains an independent technical solution. This narrative manner of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

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

2. The empowered mesenchymal stem cells according to claim 1, characterized in that The method of treating human mesenchymal stem cells with HEP14 comprises the following steps: (1) Cultivate and expand human mesenchymal stem cells; (2) adding HEP14 at a concentration of 1.25 to 20 μM to the culture medium and treating human mesenchymal stem cells for 12 to 48 hours; (3) Digest human mesenchymal stem cells to prepare single-cell suspension; (4) Eluting the human mesenchymal single cells with physiological saline to obtain the empowered mesenchymal stem cells suspended in physiological saline.

3. The empowered mesenchymal stem cells according to claim 2, characterized in that The step (2) of treating human mesenchymal stem cells with HEP14 is to add HEP14 at a concentration of 2.5 μM to the culture medium to treat the human mesenchymal stem cells for 32 hours.

4. The empowered mesenchymal stem cells according to claim 1, characterized in that The human mesenchymal stem cells are derived from various human tissues including bone marrow, fat, placenta, umbilical cord, skin, amniotic membrane, and dental pulp.

5. The empowered mesenchymal stem cells according to claim 4, characterized in that The human mesenchymal stem cells are mesenchymal stem cells derived from adipose tissue.

6. A composition for empowering mesenchymal stem cells, characterized in that: The composition comprises: Empowered mesenchymal stem cells, wherein the empowered mesenchymal stem cells are the empowered mesenchymal stem cells according to claims 1-5; HEP14 / PLGA microspheres are HEP14 / PLGA microspheres prepared using poly(lactic acid-co-glycolic acid) (PLGA) as a carrier and have a high HEP14 loading capacity and long-term HEP14 sustained release.

7. The empowered mesenchymal stem cell composition according to claim 6, characterized in that The HEP14 / PLGA microspheres with high HEP14 loading and long-term HEP14 sustained release are prepared by the following method: In a dark environment, polylactic acid-co-glycolic acid (PLGA) and HEP14 were dissolved in dichloromethane at a mass ratio of 5-10:1 and stirred at 400-500 rpm using a magnetic stirrer. Then, the obtained solution was added to an aqueous solution containing 1-2% (w / v) polyvinyl alcohol, and the dichloromethane was evaporated by stirring at 300-500 rpm using a magnetic stirrer at 25°C for at least 12 hours. Then, the microspheres were collected by centrifugation and repeatedly rinsed with distilled water to completely remove residual polyvinyl alcohol. Finally, the microspheres were placed in a freeze dryer and freeze-dried to obtain HEP14 / PLGA microspheres.

8. The empowered mesenchymal stem cell composition according to claim 7, characterized in that The polylactic acid-glycolic acid copolymer and HEP14 were dissolved in dichloromethane at a mass ratio of 5:

1.

9. Use of the empowered mesenchymal stem cells according to any one of claims 1 to 5 in the preparation of anti-tissue fibrosis drugs.

10. Use of the empowered mesenchymal stem cells according to any one of claims 1 to 5 in the preparation of a drug for promoting tissue angiogenesis.

11. Use of the empowered mesenchymal stem cells according to any one of claims 1 to 5 in the preparation of a drug for promoting tissue regeneration and functional recovery.

12. The use according to any one of claims 9 to 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 empowered mesenchymal stem cells according to any one of claims 1 to 5 in the preparation of a medicament for treating organ dysfunction.

14. Use of the empowered mesenchymal stem cells according to claim 13 in preparing a medicament for treating organ dysfunction, characterized in that: The organ dysfunction is caused by damage to the regenerative function of the organ or aging, and the organ is one of ovary, liver, lung, brain, heart, muscle, bone, kidney, and skin.

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

16. Use of the empowered mesenchymal stem cells in preparing a medicament for organ dysfunction according to claim 15, characterized in that: Local injection of a physiological saline solution containing the empowered mesenchymal stem cells into the ovaries of a subject in need thereof includes any of the following applications: (1) Application in the preparation of drugs for the treatment of premature ovarian failure and its resulting complications in women of childbearing age; (2) Application in the preparation of drugs for the treatment of ovarian dysfunction in elderly women and its resulting complications; (3) Use in the preparation of drugs for restoring ovarian endocrine and / or reproductive function.

17. Use of the empowered mesenchymal stem cell composition according to any one of claims 6 to 8 in the preparation of an anti-tissue fibrosis drug.

18. Use of the empowered mesenchymal stem cell composition according to any one of claims 6 to 8 in the preparation of a drug for promoting tissue angiogenesis.

19. Use of the empowered mesenchymal stem cell composition according to any one of claims 6 to 8 in the preparation of a drug for promoting tissue regeneration and functional recovery.

20. The use according to any one of claims 17 to 19, 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.

21. Use of the empowered mesenchymal stem cell composition according to any one of claims 6 to 8 in the preparation of a medicament for treating organ dysfunction.

22. Use of the empowered mesenchymal stem cell composition according to claim 21 in preparing a medicament for treating organ dysfunction, characterized in that: The organ dysfunction is caused by damage to the regenerative function of the organ or aging, and the organ is one of ovary, liver, lung, brain, heart, muscle, bone, kidney, and skin.

23. The use of empowered mesenchymal stem cells in the preparation of a medicament for organ dysfunction according to claim 22, characterized in that: The organ is an ovary.

24. The use of the empowered mesenchymal stem cells in preparing a medicament for organ dysfunction according to claim 23, characterized in that: Local injection of a physiological saline solution containing the empowered mesenchymal stem cells into the ovaries of a subject in need, combined with injection of a physiological saline solution containing the HEP14 / PLGA microspheres into a subject in need, including any of the following applications: (1) Application in the preparation of drugs for treating premature ovarian failure in women of childbearing age; (2) Application in the preparation of drugs for treating ovarian dysfunction in elderly women and its resulting complications; (3) Use in the preparation of drugs for restoring ovarian endocrine and / or reproductive function.

25. Use of the empowered mesenchymal stem cell composition according to claim 24 in preparing a drug for treating organ dysfunction, characterized in that: The injection route of the physiological saline solution containing the HEP14 / PLGA microspheres includes any one of intraperitoneal injection, subcutaneous injection, and intramuscular injection.

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