同型半胱氨酸对乳鼠心肌细胞自噬水平及mTOR信号通路的影响  

Effects of homocysteine on autophagy level and mTOR signaling pathway in neonatal rat cardiomyocytes

作　　者：徐嘉惠[1,2] 周艺 王雯[1,2] XU Jiahui;ZHOU Yi;WANG Wen(Department of Physiology and Pathophysiology,School of Basic Medical Sciences,Capital Medical University,Beijing 100069,China.)

机构地区：[1]首都医科大学基础医学院生理学与病理生理学系,北京100069 [2]代谢紊乱相关心血管疾病北京市重点实验室

出　　处：《中南医学科学杂志》2018年第1期56-60,共5页Medical Science Journal of Central South China

基　　金：国家自然科学基金(编号81370450;81671382)

摘　　要：高同型半胱氨酸血症(HHcy)是心血管疾病的一个独立的危险因素,但其对心肌的损伤机制尚不清楚。细胞自噬是近几年研究比较热门的细胞自身保护机制,有研究发现同型半胱氨酸(Hcy)可抑制神经细胞的自噬水平,但其对心肌细胞自噬水平的作用尚不清楚。本研究利用体外细胞实验初步探讨Hcy对心肌细胞自噬水平及自噬调节信号通路mTOR通路的影响。通过原代乳鼠心肌细胞体外培养,给予Hcy共孵育刺激,利用western blot检测心肌细胞自噬相关蛋白LC3表达水平的改变以及mTOR信号通路蛋白磷酸化水平的改变;给予LC3双标签腺病毒Ad-GFP-RFP-LC3感染细胞24 h后,进行Hcy刺激,利用激光共聚焦显微镜观察自噬流的改变;对心肌细胞进行Annexin V/PI双标记染色,利用流式细胞仪计数检测细胞凋亡情况。结果发现,给予Hcy刺激后,乳鼠心肌细胞凋亡率升高,自噬水平降低,自噬调节信号通路mTOR及其下游p70 S6K磷酸化水平升高。这些结果提示Hcy可降低心肌细胞自噬水平,mTOR信号通路可能参与其中。Hyperhomocysteinemia is an independent risk factor for cardiovascular disease,but its mechanism of myocardial damage is unclear.Autophagy is the most popular cell self-protection mechanism in recent years.It has been found that homocysteine can reduce the autophagy level of nerve cells,but its role in cardiomyocyte autophagy is unclear.This study investigated the effect of homocysteine on autophagy level and mTOR signaling pathway in cardiomyocytes by in vitro cell experiments.The expression of LC3 and the phosphorylation of mTOR signal pathway protein in myocardium were detected by Western blot.The expression of LC3 double-labeled adenovirus Ad-GFP-RFP-LC3 cells were cultured for 24 hours.Hcy stimulation was performed.The changes of autophagy were observed by laser confocal microscopy.Annexin V/PI double staining was used to detect the apoptosis of cardiomyocytes by flow cytometry.The results showed that the apoptosis rate of myocardium was increased and the level of autophagy was decreased,and the phosphorylation level of mTOR and downstream p70 S6K were increased.These results suggest that Hcy can reduce autophagy level of cardiomyocytes,and mTOR signaling pathway may be involved in.

关 键 词：同型半胱氨酸 乳鼠心肌细胞 自噬 哺乳动物雷帕霉素靶蛋白 

分 类 号：R363[医药卫生—病理学]