活性氧调控Bcl-2、Bax表达参与热打击后人脐静脉内皮细胞凋亡的研究  

The effect of reactive oxygen species regulation of expression of Bcl-2 and Bax in apoptosis of human umbilical vein endothelial cell induced by heat stress

作　　者：李莉[1,2] 古正涛[3] 刘志锋[1] 苏磊[1] 

机构地区：[1]广州军区广州总医院重症医学科,全军热区创伤救治与组织修复重点实验室,广东510010 [2]南方医科大学 [3]南方医科大学附属第三医院重症医学科

出　　处：《中华危重病急救医学》2014年第7期458-463,共6页Chinese Critical Care Medicine

基　　金：国家自然科学基金（81071529,81101467）;广东省自然科学基金（10151001002000001）;广东省科技计划项目（2012B031800416）;全军医学科技研究＂十二五＂发展计划（BWS12J018）

摘　　要：目的 观察热打击后人脐静脉内皮细胞（HUVEC）内活性氧（ROS）爆发性增高调控Bcl-2、Bax表达对凋亡的影响,探讨重症中暑所致血管内皮损害的发病机制.方法 建立HUVEC热打击模型.将细胞分别置于39、41、43 ℃细胞培养箱中进行热打击2h,然后置于37 ℃、5％ CO2细胞培养箱后继续孵育24 h;在43 ℃热打击前以10μmol/L ROS特异性清除剂MnTMPyP预处理lh进行干预.对照细胞仅置于37 ℃、5％CO2细胞培养箱中孵育.应用二氯二氢荧光素-乙酰乙酸酯（DCFH-DA）法及二氢乙啶（DHE）法检测细胞内ROS表达;使用Hoechst33258荧光染色检测细胞凋亡;采用逆转录-聚合酶链反应（RT-PCR）检测Bcl-2、Bax的mRNA表达;采用蛋白质免疫印迹试验（Western Blot）检测Bcl-2、Bax及天冬氨酸特异性半胱氨酸蛋白酶3（caspase-3）的蛋白表达;同时检测MnTMPyP对热打击后细胞凋亡的影响.结果 与对照组比较,39℃热打击对细胞凋亡无影响,随着热打击温度增加至41℃、43℃时,细胞存活率呈进行性下降,ROS爆发性增加,Bax的mRNA和蛋白表达以及caspase-3蛋白表达显著增加,Bcl-2的mRNA和蛋白表达显著降低,且呈温度依赖性,其变化以43℃热打击最为明显[细胞存活率：（46.00±4.00）％比（96.33±1.53）％,t=20.164,P=0.001;ROS（荧光相对值）：400.67±12.10比99.33±4.04,t=32.909,P=0.001;Bax mRNA（A值）：3.03±0.15比1.00±0.00,t=23.056,P=0.001;Bax蛋白（灰度值）：3.97±0.21比1.00±0.00,t=24.684,P=0.001;caspase-3蛋白（灰度值）：4.80±0.20比1.00±0.00,t=32.909,P=0.001;Bcl-2 mRNA（A值）：0.42±0.30比1.00±0.00,t=33.072,P=0.001;Bcl-2蛋白（灰度值）：0.39±0.25比1.00±0.00,t=42.212,P=0.001].MnTMPyP预处理可明显逆转43℃热打击对HUVEC的损害,可明显抑制Bax、caspase-3表达,上调Bcl-2表达[BaxmRNA（A值）：2.00±0.20比3.33±0.25,t=7.184,P=0.002;Bax蛋白（灰度值）：2.03±0.25比3.23±0.25,t=5.840,P=0.004;caspase-3蛋白（灰度值�Objective To observe the effect of heat stress-induced reactive oxygen species （ROS） burst on the regulation of expression of Bcl-2 and Bax in human umbilical vein endothelial cell （HUVEC） apoptosis induced by heat stress,and explore the pathogenesis of vascular endothelial damage caused by severe heat stroke.Methods HUVEC heat stress model was reproduced.Cells of heat stress group were incubated at either 39,41,or 43 ℃ for 2 hours,then all the cells were further incubated at 37 ℃ and 5％ CO2 for 24 hours.Before heat stress,cells of 43 ℃ heat stress group were pretreated with 10 μmol/L MnTMPyP,which was a specific scavenger of ROS,for 1 hour.Cells of control group were incubated at 37 ℃ and 5％ CO2.The amount of ROS was assayed with 2＇,7＇-dichlorofluorescin diacetate （DCFH-DA） and dihydroethidium （DHE） staining.Apoptosis was determined by using staining with Hoechst33258.The mRNA expressions of Bcl-2 and Bax were determined by reverse transcription-polymerase chain reaction （RT-PCR）.The protein levels of Bcl-2,Bax,caspase-3 were analyzed by Western Blot.In addition,the effect of MnTMPyP on heat stress-induced apoptosis was also studied.Results Compared with control group,there was no obvious change in cells after 39 ℃ heat stress.With the increase in heat stress temperature up to 41 ℃ and 43 ℃,viability of cells showed a lowering trend,with a burst of ROS,and an increase of mRNA and protein of Bax,and the protein of caspase-3 was significantly increased,the mRNA and protein of Bcl-2 were significantly decreased in a temperature-dependent manner.These changes were marked in 43 ℃ heat stress group as compared with those of the control group [cell viability：（46.00 ±4.00）％ vs.（96.33 ± 1.53）％,t=20.164,P=0.001; ROS （fluorescence relative value）：400.67 ± 12.10 vs.99.33 ±4.04,t=32.909,P=0.001; Bax mRNA （A value）：3.03 ±0.15 vs.1.00 ± 0.00,t=23.056,P=0.001; Bax protein （gray value）：3.97 ±0.21 vs.1.00 ± 0.00,t=24.684,P=0.001; caspase-3 protei

关 键 词：热打击 中暑 重症 凋亡 活性氧 BCL-2 BAX 人脐静脉内皮细胞 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]