治疗性低温对缺氧期心肌细胞自噬的影响及机制  

Effect and mechanism of therapeutic hypothermia on autophagy in hypoxia cardiomyocytes

作　　者：杨羽菲 赵丛[1] 杨平珍[1] 王先宝[1] 邓意[1] 陈爱华[1] 

机构地区：[1]南方医科大学珠江医院,广州510280

出　　处：《山东医药》2016年第22期1-4,共4页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(81400190;81270218);广东省自然科学基金资助项目(2015A030310478);广东省科技计划项目(2014A020212191)

摘　　要：目的观察治疗性低温(TH)在缺氧期对心肌细胞自噬活性及自噬流的影响,并探讨其作用机制。方法取大鼠心肌细胞株H9c2,分为对照组、缺氧组、TH干预组、雷帕霉素干预组、雷帕霉素+TH干预组,对照组置于37℃培养箱中培养,不作处理;缺氧组和TH干预组构建缺氧模型后,分别置于37、32℃培养箱中培养;雷帕霉素干预组和雷帕霉素+TH干预组于构建模型前2 h加入雷帕霉素0.1μmol/L,构建模型后分别置于37、32℃培养箱中培养。采用Western blot法检测对照组、缺氧组和TH干预组自噬相关蛋白自噬微管相关蛋白轻链3B(LC3B)、p62、Beclin-1蛋白表达,以及应用雷帕霉素前后哺乳动物雷帕霉素靶蛋白(m TOR)、核糖体蛋白S6(S6)的表达差异;采用腺病毒GFP-mRFP-LC3荧光瞬时转染技术检测自噬流。结果 1对照组、缺氧组和TH干预组LC3B、p62、Beclin-1蛋白表达:缺氧组和TH干预组LC3B、Beclin-1高于对照组(P均<0.05),缺氧组p62降低(P<0.05)。TH干预组LC3B明显低于缺氧组,p62增高(P均<0.05)。缺氧组与TH干预组Beclin-1无统计学差异。2雷帕霉素处理后自噬蛋白LC3B、p62及m TOR通路相关蛋白p-m TOR、p-S6表达:TH干预组与缺氧组比较LC3B降低,而p62、p-m TOR和p-S6均升高(P均<0.05);雷帕霉素干预组与缺氧组比较LC3B升高,p62降低(P均<0.05),而pm TOR和p-S6差异无统计学意义;雷帕霉素+TH干预组与TH干预组比较LC3B升高,p62、p-m TOR和p-S6均降低,结果均有统计学差异(P均<0.05);雷帕霉素+TH干预组与雷帕霉素干预组比较LC3B降低,p62升高(P均<0.05),而p-m TOR和p-S6差异无统计学意义。3双荧光自噬流:缺氧组和TH干预组的黄点和红点数多于对照组(P均<0.05),TH干预组的黄点与红点数少于缺氧组(P均<0.05)。结论 TH能够抑制缺氧期心肌细胞自噬,其机制可能与促进m TOR信号通路相关蛋白表达有关。Objective To investigate the effects of therapeutic hypothermia（ TH） on autophagy and autophagic flux of cardiomyocytes in ischemia and to explore the underlying mechanism. Methods H9c2 rat cardiomyocytes were divided into the control group,hypoxia group,hypoxia ＋ TH group,rapamycin ＋ hypoxia group and rapamycin ＋ hypoxia ＋ TH group. Cells in the control group were normally cultured at 37 ℃ and received no further treatments. Cells with or without TH treatment in the other four groups were subjected to hypoxia in a hypoxic chamber at 32 ℃ or 37 ℃ respectively. Then,0. 1 μmol / L rapamycin was added 2 h before cells were subjected to hypoxia at 32 ℃ or 37 ℃. Western blotting was performed to detect protein expression of LC3 B,p62,Beclin-1,phospho-m TOR（ p-m TOR） and phospho-S6（ p-S6）. The autophagic flux was detected under adenovirus GFP-mRFP-LC3 confocal laser scanning microscopy. Results 1The protein expression of LC3 B,p62 and Beclin-1 in the control group,hypoxia group and hypoxia ＋ TH group： compared with the control group,LC3 B and Beclin-1 expression was significantly increased in the hypoxia group and hypoxia ＋ TH group（ all P 0. 05）,while p62 expression was significantly decreased in the hypoxia group（ P 0. 05）. Hypoxia ＋ TH group exhibited lower LC3 B expression as well as higher p62 expression as compared with that of the hypoxia group（ P 0. 05）.There was no significant difference in Beclin-1 expression between the hypoxia group and hypoxia ＋ TH group. 2 The expression of autophagy-related proteins LC3 B,p62 and m TOR pathway-related proteins p-m TOR and p-S6 after rapamycin treatment： compared with the hypoxia group,hypoxia ＋ TH group showed decreased expression of LC3 B and increased expression of p62,p-m TOR and p-S6（ all P 0. 05）. Compared with the hypoxia group,rapamycin ＋ hypoxia group showed higher expression of LC3 B and lower expression of p62（ all P 0. 05）,while there were no significant differences in pm TOR and p-S6. Rapymyci

关 键 词：治疗性低温 心肌细胞 缺氧 自噬 雷帕霉素 

分 类 号：R541.2[医药卫生—心血管疾病]