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）：2.07±0.21比5.00±0.20,t=17.600,P=0.001;Bcl-2 mRNA（A值）：0.71±0.40比0.42±0.26,t=8.126,P=0.002; Bcl-2蛋白（灰度值）：0.57±0.31比0.40±0.06,t=5.091,P=0.007].结论 热打击后HUVEC内ROS爆发性增高在凋亡中发挥了重要作用,其机制与调控凋亡相关基因Bcl-2、Bax的表达有关;血管内皮细胞凋亡可能是重症中暑的发病机制之一.
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 protein （gray value）：4.80 ± 0.20 vs.1.00 ± 0.00,t=32.909,P=0.001 ; Bcl-2 mRNA（A value）：0.42 ± 0.30 vs.1.00 ± 0.00,t=33.072,P=0.001 ; Bcl-2 protein （gray value）：0.39 ± 0.25 vs.1.00 ± 0.00,t=42.212,P=0.001].It was shown that pre-condition with the antioxidant MnTMPyP significantly decreased the heat stress-induced expression of Bax,caspase-3,and apoptosis,and the expression of Bcl-2 was elevated [Bax mRNA （A value）：2.00 ± 0.20 vs.3.33 ±0.25,t=7.184,P=0.002; Bax protein （gray value）：2.03 ±0.25 vs.3.23 ±0.25,t=5.840,P=0.004; caspase-3 protein （gray value）：2.07 ± 0.21 vs.5.00 ± 0.20,t=17.600,P=0.001 ; Bcl-2 mRNA（A value）：0.71 ± 0.40 vs.0.42 ± 0.26,t=8.126,P=0.002; Bcl-2 protein （gray value）：0.57 ± 0.31 vs.0.40 ± 0.06,t=5.091,P=0.007].Conclusions A burst in an increase of ROS plays an important role on heat stress-induced HUVEC apoptosis,and the mechanism is probably related to the expressions of Bcl-2 and Bax.The vascular endothelial cells apoptosis may be one of the pathogenetic factor in severe heat stroke.
出处
《中华危重病急救医学》
CAS
CSCD
北大核心
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）
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
Heat stress
Severe heat stroke
Apoptosis
Reactive oxygen species
Bcl-2
Bax
Human umbilical vein endothelial cell
