| With the use of chemotherapeutic drugs in malignant tumors and autoimmune diseases,the survival of young women was prolonged.But they also face the side effects of premature ovarian failure(POF)caused by chemotherapeutic drugs.This is also a major challenge for the clinical treatment.Previous animal research has shown that mesenchymal stem cells transplantation can partially recover ovarian damage caused by chemotherapy drugs,but the therapeutic effect is not up to expectations.Cell loss after transplantation is the main cause of poor therapeutic efficiency.Therefore,find a way to improve the survival of transplanted cells is the key to improve the therapeutic efficiency.Heat shock pretreatment is an effective way to improve the ability of cell anti-apoptosis.However,there is no consistent conclusion on the condition of heat shock pretreatment.And there is no investigation about therapeutic potential of the heat shocked mesenchymal stem cells to chemotherapy-induced premature ovarian failure.Therefore,four heat shock pretreatment conditions for MSCs were set up in this study,and the optimal heat shock treatment conditions were obtained by detecting cell apoptosis.The changes of anti-apoptotic ability,cell cycle and proliferative ability of MSCs under the optimal heat shock condition were further detected.The animal model of premature ovarian failure induced by chemotherapy was established,and the heat shock preconditioned MSCs transplantation was carried out to evaluate the value of heat shock preconditioned MSCs in treating premature ovarian failure induced by chemotherapy.Methods Isolated,cultured and identified rat bone marrow mesenchymal stem cells.Treated mesenchymal stem cells with heat shock by 42? water bath for Omin?30min?1h?2h?3h.The optimal condition of heat shock pretreatment was obtained by flow cytometry detection after 24?48 and 72 hours after heat shock.Under the optimal condition of heat shock pretreatment,the cell proliferation was detected by CCK-8,cell cycle was detected flow cytometry and apoptosis was detected by flow cytometry at 96,120,180,240 hours after heat shock treatment.125 Wistar female rats were randomly divided into five groups,25 rats in each group:control group;model group;sham-operation group;MSCs group;heat shock MSCs group.The later four groups was established chemotherapy-induced premature ovarian failure model by intraperitoneal injection of cyclophosphamide.The first day after the model was established,each side ovary of MSCs group was injected with 1x1O6 MSCs and heat shock MSCs group was injected with 1x1O6 heat shocked MSCs;sham-operation group were injected with saline.The estrous cycle of rats was observed.The serum estradiol and follicle-stimulating hormone were measured before the injection of cyclophosphamide and on the 1st,15th,30th,45th and 60th day after transplantation.5 rats were killed on the 1st,15th,30th,45th and 60th day after transplantation in each group to observe the ovarian structure,the number of follicles and apoptosis of granulosa cells in ovary.Results After heat shock at 42 ? for 1 hour,the apoptotic rate of MSCs was significantly decreased,and so as determined as the optimal condition of heat shock pretreatment.The apoptosis rate of MSCs was significantly lower than that of untreated MSCs at 48 hours after heat shock,and lasted until 120 hours after treatment.After heat shock treatment,the percentage of S phase cells of MSCs was higher than that of untreated mesenchymal stem cells.The result of CCK-8 suggested that MSCs proliferation was accelerated after heat shock treatment.After cell transplantation,part of rats in heat shock MSCs group began to recovered normal estrous cycle.15 days after cell transplantation,apoptosis of ovarian granulosa cell of MSCs group and heat shock MSCs group were significantly reduced,and the apoptosis of ovarian granular cells of the heat shock MSCs group was lower than that of MSCs group,and the difference was statistically significant;30 days after cell transplantation,the estradiol level of heat shock MSCs group was significantly higher than MSCs group,and follicle-stimulating hormone levels were significantly lower than those of MSCs group,the differences were statistically significant;45 days and 60 days after cell transplantation,the number of primordial follicles,primary follicles,secondary follicles and antral follicles of heat shock MSCs group were higher than those of MSCs group,the differences were statistically significant.Conclusion After heat shock treatment at 42 °C for 1 hour,the anti-apoptotic ability of MSCs was enhanced and continued until 120 hours after treatment,and heat shock treatment at 42 ? for 1 hour could promote the proliferation of MSCs.Heat shocked bone marrow mesenchymal stem cells could have a more effective therapeutic effect on repairing the ovarian construction and function damaged by chemotherapy. |
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