【作者】 王清；

【导师】 付霞霏；

【作者基本信息】 南方医科大学 ， 妇产科学（专业学位）， 2019， 硕士

【摘要】 背景随着肿瘤发病的年轻化,化疗引起的卵巢功能不全的发病率逐年上升。因此,在不影响癌症治疗的同时,保护卵巢的功能、预防不孕不育以及提高卵巢功能不全患者的生存质量成为我们急需解决的问题。卵巢功能不全作为一种严重并发症,目前尚缺乏有效的治疗方法。我们实验前期研究发现干细胞能在体内外抑制化疗诱导的颗粒细胞凋亡,部分修复化疗损伤的卵巢结构和功能。但治疗效果未达我们的预期。移植后干细胞的存活率低下可能是主要原因。热休克预处理是一种简单易行的增强细胞存活力的方法。本实验研究了热休克预处理对化疗局部微环境下干细胞生物学特性的影响以及热休克预处理干细胞对化疗药物诱导的卵巢颗粒细胞凋亡的影响。并进一步探索热休克预处理抑制干细胞凋亡的机制。方法1.分离、培养大鼠骨髓间充质干细胞(MSCs),向MSCs培养基中加入顺铂诱导细胞损伤模型。实验分为4组:空白对照组、热休克对照组、模型组、热休克模型组。通过CCK-8检测各组MSCs增殖能力、Hoechst33342/PI测定MSCs存活率,流式检测MSCs凋亡率。探究热休克预处理是否抑制顺铂作用下MSCs凋亡,提高MSCs的增殖能力及存活率。2.分离、培养大鼠卵巢颗粒细胞(GCs),建立顺铂诱导卵巢颗粒细胞损伤的体外模型,实验设为6组:(1)空白对照组;(2)实验对照组;(3)MSC治疗组;(4)HS-MSC治疗组;(5)MSC预防组;(6)HS-MSC预防组。通过流式检测各组颗粒细胞的凋亡率。Hoechst33342/PI测定细胞存活率。探究热休克预处理MSCs是否具有预防卵巢颗粒细胞的损伤及发挥更强的抗卵巢颗粒细胞凋亡的作用。3.进一步从热休克蛋白及自噬角度探究热休克预处理抑制干细胞凋亡、增强存活力的相关机制。Western-blot检测间充质干细胞经过热休克预处理后胞内HSP70和HSP90的表达。自噬实验分为6组:(1)对照组;(2)热休克组;(3)模型组;(4)热休克模型组;(5)3-MA组;(6)Rapamycin组。通过透射电镜观察各组细胞内自噬体的数量。Western-blot检测各组自噬相关抗体Beclin1的表达,LC3Ⅰ/Ⅱ比值变化。结果在顺铂模拟化疗卵巢局部微环境中,模型组的凋亡率为14.30%±0.80%,热休克模型组凋亡率为:11.94%±0.63%,低于模型组(P<0.05)。在顺铂诱导卵巢颗粒细胞损伤的体外模型中,实验对照组的凋亡率为53.81%±1.89%;HS-MSC预防组凋亡率为:39.88%±1.65%,HS-MSC治疗组凋亡率为:36.72±0.96%;HS-MSC治疗组及预防组凋亡率均显著低于实验对照组(P<0.05)。但HS-MSC治疗组及预防组两组间凋亡率无统计学意义(P>0.05)。Western-blot结果示MSCs经热休克预处理后,随着时间的推移,热休克蛋白70、90的表达明显上调,并在48小时后达到峰值。透射电镜观察各组自噬小体的数量发现经热休克处理后,细胞内的自噬小体减少。自噬相关抗体Beclin1的表达降低,LC3Ⅰ/Ⅱ比值下降。结论热休克预处理能够提高化疗微环境下MSCs的抗凋亡能力,HS-MSCs能够更明显地抑制顺铂诱导的卵巢颗粒细胞凋亡,其预防及治疗作用可能与热休克预处理诱导热休克蛋白HSP70及90的表达增加及减弱自噬有关。更多还原

【Abstract】 Background:With the the younger onset of tumor,the incidence of chemotherapy-induced ovarian failure is increasing year by year.Therefore,the protection of ovarian function to prevent infertility and improve the quality of life of patients with ovarian dysfunction has become a problem that we urgently need to solve.As a serious complication,chemotherapy-induced ovarian failure is still lack of effective treatment.Previously,we identified that bone marrow mesenchymal stem cells(BMSCs)could inhibit ovarian granulosa cell apoptosis(GCs)induced by chemotherapeutic drugs in vivo and in vitro,and partially repair the ovarian structure and function by chemotherapeutic drugs.However,the rapid death loss of MSCs after transplantation resulted in poor therapeutic effect.Heat shock was used as a pretreatment method to enhance cell viability.Here,we further investigated the effect of heat shock on apoptosis of MSCs in local microenvironment and the effect of MSCs pretreatment on apoptosis of ovarian granulosa cells induced by cisplatin.Moreover,we further explore the possible mechanism of heat shock stress to inhibit the apoptosis of MSCs.Method:1.Rat BSCs were isolated,cultured and identified.cisplatin was added to MSCs medium to simulate the local microenvironment of ovary.The experiment was divided into 4 groups:blank control group,HS-MSCs group,model group,HS-MSCs model group.the optimal concentration of cisplatin(5mg/L)was added to the culture medium to induce the apoptosis of MSCs.The proliferative ability of MSCs was detected by CCK-8 assay,the cell survival rate was measured by Hoechst33342/PI,and the apoptosis rate was detected by flow cytometry.The first experiment was to determine whether heat shock pretreatment could inhibit the apoptosis of MSCs and increase the proliferation and survival rate of MSCs.2.The rat GCs were isolated,cultured and identified,cisplatin was added to MSCs medium to s establish a GCs apoptosis model in vitro.The experiments were divided into 6 groups:Group 1(blank control group),Group 2(experimental control group),Group 3(the treatment experimental group of MSCs),Group 4(the treatment experimental group of HS-MSCs),Group 5(the preventive experimental group of MSCs),Group 6(the preventive experimental group of HS-MSCs).the GCs survival rate was measured by Hoechst33342/PI,and the apoptosis rate was detected by flow cytometry.To explore whether HS-MSCs can prevent the injury of ovarian granulosa cells and play a stronger role in anti-apoptosis of ovarian granulosa cells.3.To further explore the mechanism of heat shock pretreatment inhibiting apoptosis and enhancing viability of stem cells from the perspective of heat shock protein and autophagy.The expressions of HSP70 and HSP90 in mesenchymal stem cells pretreated with heat shock were detected by Western-blot.The autophagy experiments were divided into 6 groups:Group 1(blank control group),Group 2(HS-MSCs group),Group 3(model group),Group 4(HS-MSCs model group),Group 5(3-MA group),Group 5(Rapamycin group).the number of autophagy in the cells was observed by transmission electron microscopy.The expression of autophagy-related antibody Beclinl,and the ratio of LC3Ⅰ/Ⅱ were detected by using Western-blot.Results:The apoptosis rate in the model group was 14.30%±0.80%,and that in the heat shock model group was 11.94%±0.63%,lower than that in the model group(P<0.05).In the cisplatin induced ovarian granulosa cell injury model in vitro,the apoptosis rate in the experimental control group was53.81%± 1.89%.The apoptosis rate of HS-MSCs prevention group was 39.88%±1.65%,and that of HS-MSCs treatment group was 36.72±0.96%.GCs apoptosis rate in treatment group and prevention group were significantly lower than that of control group(P<0.05),but the GCs apoptosis rate between the two groups have no statistical significance(P>0.05).After heat shock treatment,the expression of heat shock protein 70 and 90 was upregulated over time after treatment with 42℃ water bath,and reached its peak 48 hours later.the number of autophagy bodies in cells was decreased by transmission electron microscope.and autophagy-related antibody Beclinl,and the ratio of LC3Ⅰ/Ⅱ were decreased.Conclusion:Heat shock pretreatment could improve the anti-apoptosis ability of MS Cs,and the co-culture of HS-MSCs and GCs could obviously inhibit the apoptosis of GCs in chemotherapeutic microenvironment,It prevention and treatment effect may be related to the high expression of heat shock protein HSP70 and 90 and autophagy was inhibited by heat shock treatment.更多还原