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
활성양조공Bcl-2、Bax표체삼여열타격후인제정맥내피세포조망적연구
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
  • 万方数据
    中华危重病急救医学 중화위중병급구의학
    Chinese Critical Care Medicine
    2014年 7期 458-463 ,共6页

    李莉%古正涛%刘志锋%苏磊

    리리%고정도%류지봉%소뢰

    热打击%中暑,重症%凋亡%活性氧%Bcl-2%Bax%人脐静脉内皮细胞 熱打擊%中暑,重癥%凋亡%活性氧%Bcl-2%Bax%人臍靜脈內皮細胞
    열타격%중서,중증%조망%활성양%Bcl-2%Bax%인제정맥내피세포
    Heat stress%Severe heat stroke%Apoptosis%Reactive oxygen species%Bcl-2%Bax%Human umbilical vein endothelial cell
    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.
    목적 관찰열타격후인제정맥내피세포(HUVEC)내활성양(ROS)폭발성증고조공Bcl-2、Bax표체대조망적영향,탐토중증중서소치혈관내피손해적발병궤제.방법 건립HUVEC열타격모형.장세포분별치우39、41、43 ℃세포배양상중진행열타격2h,연후치우37 ℃、5％ CO2세포배양상후계속부육24 h;재43 ℃열타격전이10μmol/L ROS특이성청제제MnTMPyP예처리lh진행간예.대조세포부치우37 ℃、5％CO2세포배양상중부육.응용이록이경형광소-을선을산지(DCFH-DA)법급이경을정(DHE)법검측세포내ROS표체;사용Hoechst33258형광염색검측세포조망;채용역전록-취합매련반응(RT-PCR)검측Bcl-2、Bax적mRNA표체;채용단백질면역인적시험(Western Blot)검측Bcl-2、Bax급천동안산특이성반광안산단백매3(caspase-3)적단백표체;동시검측MnTMPyP대열타격후세포조망적영향.결과 여대조조비교,39℃열타격대세포조망무영향,수착열타격온도증가지41℃、43℃시,세포존활솔정진행성하강,ROS폭발성증가,Bax적mRNA화단백표체이급caspase-3단백표체현저증가,Bcl-2적mRNA화단백표체현저강저,차정온도의뢰성,기변화이43℃열타격최위명현[세포존활솔:(46.00±4.00)％비(96.33±1.53)％,t=20.164,P=0.001;ROS(형광상대치):400.67±12.10비99.33±4.04,t=32.909,P=0.001;Bax mRNA(A치):3.03±0.15비1.00±0.00,t=23.056,P=0.001;Bax단백(회도치):3.97±0.21비1.00±0.00,t=24.684,P=0.001;caspase-3단백(회도치):4.80±0.20비1.00±0.00,t=32.909,P=0.001;Bcl-2 mRNA(A치):0.42±0.30비1.00±0.00,t=33.072,P=0.001;Bcl-2단백(회도치):0.39±0.25비1.00±0.00,t=42.212,P=0.001].MnTMPyP예처리가명현역전43℃열타격대HUVEC적손해,가명현억제Bax、caspase-3표체,상조Bcl-2표체[BaxmRNA(A치):2.00±0.20비3.33±0.25,t=7.184,P=0.002;Bax단백(회도치):2.03±0.25비3.23±0.25,t=5.840,P=0.004;caspase-3단백(회도치):2.07±0.21비5.00±0.20,t=17.600,P=0.001;Bcl-2 mRNA(A치):0.71±0.40비0.42±0.26,t=8.126,P=0.002; Bcl-2단백(회도치):0.57±0.31비0.40±0.06,t=5.091,P=0.007].결론 열타격후HUVEC내ROS폭발성증고재조망중발휘료중요작용,기궤제여조공조망상관기인Bcl-2、Bax적표체유관;혈관내피세포조망가능시중증중서적발병궤제지일.
    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.
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