ABSTRACT
ObjectiveBone marrow-derived mesenchymal stem cells (MSCs) can promote ovarian angiogenesis, improve ovarian insufficiency caused by chemotherapy, and repair ovarian function, while heat shock pretreatment can reduce the apoptosis rate of stem cells and improve the therapeutic effect of stem cells. This study aims to investigate the effect of heat shock pretreatment on MSCs, and further study the effect of heat shock pretreated mesenchymal stem cells on chemotherapy-induced apoptosis of ovarian granulosa cells.Methods1. The bone marrow-derived MSCs of rats were isolated, cultured and identified, and pretreated within a 42 °C water bath for one hour. 2. Cisplatin (5 mg/L) was added to MSCs to simulate the local microenvironment of chemotherapy. MSCs were divided into four groups: blank control group, heat shock control group, model group, and heat shock model group. The effects of heat shock pretreatment on the proliferation, apoptosis and survival rate of MSCs were investigated by CCK-8 method, Hoechst33342/PI, and flow cytometry. 3. We isolate and culture rat ovarian granulosa cells (GCs) to establish an in vitro model of GCs injury under the induction of cisplatin (5 mg/L). The experiment was carried out in four groups: a control group, model group, MSCs model group, HS-MSCs model group. The apoptosis and survival rate were detected by Hoechst33342/PI and flow cytometry, respectively.Results1. The proliferation level and survival rate of MSCs in the heat shock control group were significantly higher than those in the other three groups, and the apoptosis rate was significantly lower than the other three groups (P<0.05). Compared to the model group, the proliferation level of the heat shock model group was significantly increased, and the apoptosis rate was significantly decreased (P<0.05), and the cell survival rate increased; 2. The apoptosis rate of GCs in the HS-MSCs model group was significantly lower than that in the other three groups. Compared to the MSCs model group, the apoptosis rate of GCs in the HS-MSCs model group was significantly decreased (P<0.05).ConclusionHeat shock pretreatment can increase the proliferation level and survival rate of MSCs, and reduce its apoptosis rate. Heat shock pretreated stem cells can effectively inhibit chemotherapy-induced apoptosis of ovarian granulosa cells.
ABSTRACT
[Abstract] Objective To explore the repair effects of bone marrow mesenchymal stem cells (BMSCs) over-expressing miR-21 on chemotherapeutic premature ovarian failure (POF) in rats. Methods The expression vector of lentivirus-mediated miR-21 (LV-miR-21) was constructed, and BMSCs were transfected with LV-miR-21 to construct miR-21-BMSCs. A hundred rats were divided into 5 groups (20 each): normal group, model group, miR-21 group, BMSCs group and miR-21-BMSCs group. The chemotherapeutic POF model was established by intraperitoneal injection of cyclophosphamide (CTX) in rats of the latter 4 groups. Then LV-miR-21, BMSCs and miR-21-BMSCs were injected respectively into the bilateral ovaries of the rats in the latter 3 groups, and physiological saline was injected into the rats of normal group and model group. At 15, 30, 45 and 60 days after injection, the estrus cycle, estradiol (E2) and follicle stimulating hormone (FSH) level, ovarian weight, follicle count at all levels, and apoptotic rate of granulosa cells in each group were detected. Results After injection of CTX, the estrus cycle of rats remained regular in normal group, and fell into disorder in the other four groups, of which the number of rats with recovered estrus cycle in the miR-21-BMSCs group was more than the other three groups; The ovarian weight and follicle count at all levels were significantly higher in miR-21-BMSCs group than in model group, miR-21 group and BMSCs group 30, 45 and 60 days after CTX injection (P<0.05); The basal levels of E2 and FSH showed no significant difference in each group during the experiment, while the E2 concentration decreased and FSH concentration increased in all the groups except the normal group. The hormone levels of E2 and FSH in miR-21-BMSCs group (F30d=43.10 and F30d=14.71) were significantly different when compared to those in miR-21 group and BMSCs group (F45d=43.57 and F45d=13.16, P<0.05; and F60d=44.98 and F60d=15.20, P<0.05); The apoptotic rate of ovarian granular cells in miR-21-BMSCs group was significantly lower (F60d=21.20) than that in model group, miR-21 group and BMSCs group (F15d=27.20, F30d=23.60 and F45d=21.80, P<0.05). Conclusion miR-21 may enhance the therapeutic effect of BMMSCs on chemotherapeutic premature ovarian failure, and significantly improve the ovarian structure and function.