热休克预处理对化疗环境下骨髓间充质干细胞存活率的影响及机制

Effect and mechanisms of heat shock pretreatment on bone marrow mesenchymal stem cells viability in chemotherapy environment

目的:探索热休克预处理(heat shock pretreatment,HSP)对化疗微环境下骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)的影响及其潜在机制。方法:探索HSP最佳条件,往热休克预处理BMSCs(heat shock pretreated bone marrow mesenchymal stem cells,HS-MSCs)培养基中加入顺铂,通过CCK-8检测细胞增殖率,Hoechst33342/PI测定存活率,流式细胞仪检测凋亡率。使用Western blot检测不同时间点HS-MSCs热休克蛋白70(heat shock protein 70,Hsp70)、Hsp90、自噬相关蛋白Beclin1和LC3B的表达。通过透射电镜观察自噬小体数量。结果:加入顺铂后,热休克模型组凋亡率明显低于模型组。此外,热休克模型组增殖速率较模型组增加,而表达亮蓝色/暗红色荧光的比例则低于模型组。HS-MSCs内Hsp70和Hsp90的表达随时间推移逐渐上调,并在48 h后达峰值。同时,细胞内自噬小体减少、Beclin1表达降低、LC3BⅡ/LC3BⅠ比值下降。结论:HSP能够提高化疗微环境下BMSCs的存活与抗凋亡能力,其作用可能与HSP增强Hsp70及Hsp90的表达、减弱自噬水平有关。

Objective:This study aims to explore the potential effects and mechanisms of heat shock pretreatment(HSP)on bone marrow mesenchymal stem cells(BMSCs) in chemotherapy microenvironment. Methods:The optimal condition of HSP was identified.Cisplatin was added to the heat shock pretreated bone marrow mesenchymal stem cells(HS-MSCs)medium to simulate the chemotherapy microenvironment. The effect of HSP on the biological characteristics of HS-MSCs was explored. The proliferation of BMSCs was detected by CCK-8 assay,the viability was measured by Hoechst33342/PI,and the apoptosis was detected by flow cytometry. The potential mechanisms of HSP were further explored from the perspective of heat shock proteins and autophagy. The expression of heat shock protein 70(Hsp70),Hsp90,Beclin1 and LC3B after HSP were detected by Western blot. The number of autophagosomes was observed by transmission electron microscopy. Results:In simulated chemotherapy microenvironment,the apoptosis rate of heat shock model group was lower than that of model group. In addition,the proliferation rate of the heat shock model group was higher than that of the model group,while the ratio of bright blue/dark red fluorescence cells was lower than that of the model group. The expressions of Hsp70 and Hsp90 were significantly up-regulated with time and reached the peak at 48 hours after HSP. Meanwhile,the number of autophagosomes,the expression of Beclin1 and the ratio of LC3 BⅡ/LC3 BⅠ were all decreased after HSP. Conclusion:HSP can improve the viability and anti-apoptosis ability of BMSCs under the chemotherapy microenvironment,and its effect may be related to the enhancement of Hsp70 and Hsp90 expressions and the weakening of autophagy level.