| BackgroundWith the continuous improvement in cancer diagnosis and treatment,the long-term survival rate of patients with cancer has increased significantly.The quality of life and fertility maintenance of young cancer survivors is of increasing concern.Adjuvant chemotherapy may lead to ovarian dysfunction and infertility.Stem cell therapy has shown potential in the treatment of various diseases.Different types of stem cells have been used in the treatment of ovarian dysfunction.Menstrual blood-derived endometrial stem cells（Men SCs）can be noninvasively and periodically obtained from discarded menstrual blood samples and exhibit high proliferative capacity,low immunogenicity and autologous transplantation.Objective1.The aim of this study was to evaluate the effect of Men SCs treatment on the improvement of cisplatin-injured human ovarian granulosa cells（GCs）.2.To further explore the mechanism of the improvement effect of Men SCs treatment on cisplatin-injured human ovarian granulosa cells（GCs）.Methods1.The sex hormone test reports of 10 female breast cancer patients younger than 45 years old were collected to analyze the effect of chemotherapy on female hormone levels.2.CCK8 was used to detect the inhibitory effect of different concentrations of cisplatin on the proliferation of human ovarian GCs,to determine the optimal concentration,and to establish a model of cisplatin-induced injury to human ovarian GCs.3.Observe the effect of different times of cisplatin treatment on the apoptosis of human ovarian GCs by apoptosis assay and vital staining.4.Prepare Men SC-CM,and establish Men SC-CM non-contact co-culture system.5.The effect of Men SC-CM on the activity of cisplatin-treated human ovarian GCs was detected by CCK8;changes in the apoptosis rate of cisplatin-injured human ovarian GCs were detected by flow cytometry after the non-contact co-culture intervention of Men SC-CM and Men SCs,and the changes in the expression levels of apoptosis-related proteins as well as autophagy-related proteins in cisplatin-injured human ovarian GCs were detected by Western blot changes.6.After Men SC-CM intervention with the addition of autophagy inhibitors MHY1485 and Spautin-1,changes in the activity of cisplatin-injured human ovarian GCs were detected by CCK8,changes in the apoptosis rate were detected by flow cytometry,and changes in the expression levels of apoptosis-related proteins as well as autophagy-related proteins were detected by Western blot.Results1.Men SCs and Men SC-derived conditional medium（Men SC-CM）treatment effectively improved the viability of cisplatin-injured ovarian GCs and significantly upregulated their antiapoptotic capacity,and direct Men SC-CM treatment exhibited a superior improvement effect.2.Men SCs treatment significantly upregulated autophagy activity in cisplatin-injured ovarian GCs,and the degree of autophagy activation was positively correlated with the viability improvement of ovarian GCs,while autophagy inhibitors significantly impaired Men SC-promoted viability improvement of cisplatin-injured ovarian GCs.3.Men SCs treatment can also significantly promote the proliferation of normal GCs by activating the PI3K/Akt signaling pathway.ConclusionMen SCs treatment not only enhanced the anti-apoptotic ability and survival of cisplatin-injured ovarian GCs through upregulation of autophagic activity,but also improved the survival of normal ovarian GCs through activation of PI3K/Akt signaling path.These results provide a theoretical and experiment basis for the clinical application of Men SCs in improving chemotherapy-induced ovarian damage and delaying ovarian aging. |
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