雷帕霉素促进胚胎干细胞向心肌细胞分化  

Rapamycin facilitates cardiac differentiation of embryonic stem cells

作　　者：汪萍萍 李涛 翁晓菲 李贤慧[2] 李彦明 程冠昌 Wang Pingping;Li Tao;Weng Xiaofei;Li Xianhui;Li Yanming;Cheng Guanchang(School of Medicine,Hunan Normal University,Changsha 410013,China;Department of Health Service,Logistics College of People’s Armed Police Force,Tianjin 300309,China;Department of Cardiology,Huaihe Hospital of HenanUniversity,Kaifeng 475000,China)

机构地区：[1]湖南师范大学医学院,长沙410013 [2]武警医学院卫生勤务系人体机能教研室,天津300309 [3]河南大学淮河医院心内科,开封475000

出　　处：《中国组织化学与细胞化学杂志》2021年第3期213-220,共8页Chinese Journal of Histochemistry and Cytochemistry

基　　金：国家自然科学基金(81770296,81870201)。

摘　　要：目的雷帕霉素是小分子mTOR抑制剂,可激活细胞自噬。本研究检测了雷帕霉素对胚胎干细胞(ES细胞)向心肌分化的影响。方法采用拟胚体(embryoid body,EB)加抗坏血酸诱导ES细胞向心肌分化。悬浮诱导阶段添加自噬激动剂雷帕霉素或抑制剂羟氯喹,通过免疫印迹检测LC3蛋白剪切以监测细胞自噬水平,通过检测EB球的心肌搏动百分比以及心肌分化标志物troponin-T和α-actinin的表达来评估细胞分化效率。采用real-time PCR检测心肌分化转录因子Mef2c和Isl1 mRNA表达,免疫印迹法检测Oct4表达,进一步阐释雷帕霉素的效应机制。结果雷帕霉素能够诱导自噬,促进LC3剪切,促进EB球产生自发搏动,增强心肌分化标志基因表达。机制研究发现,雷帕霉素促进心肌转录因子Mef2c和Isl1 mRNA的表达和Oct4在分化细胞的下调。而自噬抑制剂羟氯喹能拮抗雷帕霉素的上述效应。结论雷帕霉素诱导自噬可以促进心肌转录因子表达和促进干性因子Oct4水平下调,从而促进心肌分化。Objective To examine the effect of rapamycin on the differentiation of embryonic stem cells(ES cells)into cardiomyocytes.Methods ES cells were allowed to form embryoid bodies(EBs)under the induction of ascorbic acid.In this way,ES cells differentiated into spontaneously beating cardiomyocytes.Cells were exposed to autophagy inducer rapamycin or inhibitor hydroxychloroquine during the suspension induction stage.LC3 cleavage was monitored to exhibit cell autophagy.The percentage of beating EBs was calculated.The expression of troponin-T andα-actinin,both myocardial differentiation markers,was detected by Western blotting and immunofluorescence.Real-time PCR was used to detect the mRNA expression of cardiac transcription factors Mef2c and Isl1 in myocardial differentiation.Pluripotent factor Oct4 was detected by Western blotting.Results Rapamycin induced autophagy,facilitated LC3 cleavage,enhanced the efficiency of spontaneously beating EBs,and improved the expression of myocardial differentiation marker genes.Experiments on mechanism showed rapamycin promoted the mRNA expression of Mef2c and Isl1,simultaneously down-regulated the Oct4 expression.Hydroxychloroquine antagonized the above rapamycin-induced effects.Conclusion Rapamycin can induce autophagy and promote the expression of myocardial transcription factors and the depletion of Oct4 protein,thereby promoting the cardiac differentiation of ES cells.

关 键 词：胚胎干细胞 心肌分化 自噬 雷帕霉素 

分 类 号：Q254[生物学—细胞生物学]