线粒体凋亡途经参与热应激后人脐静脉内皮细胞凋亡的研究李莉古正涛刘志锋苏磊DOI：10.3760/cma.j.issn.1671-0282.2014.12.006基金项目：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）;军队十二五医学科研基金重点项目（BWS12J018）作者单位：510010广州，南方医科大学第三附属医院重症医学科（李莉、古正涛）;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（刘志锋、苏磊）通信作者：苏磊，Email：sulei_icu@163.com【摘要】目的观察细胞色素C、caspase-9和caspase-3在热应激后人脐静脉内皮细胞的表达及相互关系，阐明热应激后人脐静脉内皮细胞凋亡的信号转导机制，探讨重症中暑所致血管内皮损害的发病机理。方法建立人脐静脉内皮细胞热应激模型，对照组将细胞置于标准37℃、5%CO₂细胞培养箱，热应激组将细胞置于39℃、41℃、43℃细胞培养箱中进行热应激2h，热应激后继续在细胞培养箱孵育24h。电子显微镜观察人脐静脉内皮细胞（37℃、43℃）线粒体改变，应用AnnexinV-FITC/PI双染色方法检测不同温度热应激后细胞凋亡率、Westernblot检测细胞色素C、caspase-9、caspase-3蛋白表达。结果电镜观察对照组（37℃）HUVEC细胞线粒体结构基本正常。热应激后（43℃）HUVEC细胞线粒体肿胀并出现结构改变;与对照组比较，39℃热应激对内皮细胞凋亡无明显影响（P>0.05），随着热应激温度的增加人脐静脉内皮细胞凋亡明显增多（41℃17.8%，43℃25.6%）并且细胞色素C、caspase-9、caspase-3蛋白表达明显增加（P<0.05）。结论线粒体通路的激活参与了热应激后人脐静脉内皮细胞凋亡的调控;血管内皮细胞凋亡可能与重症中暑的发病存在相关性。【关键词】人脐静脉内皮细胞;凋亡;线粒体;热应激;重症中暑;细胞色素C;caspase-9、caspase-3MitochondrialapoptosispathwayinvolvedinapoptosisofhumanumbilicalveinendothelialcellafterheatstressLiLi*，GuZhengtao，LiuZhifeng，SuLei.DepartmentofIntensiveCareUnit，TheThirdAffiliatedHospitalofSouthernMedicalUniversity，Guangzhou510010，ChinaCorrespondingauthors：SuLei，Email：sulei_icu@163.com【Abstract】ObjectiveToobservetheexpressionsofcytochromeC，Caspase-9，Caspase-3andtheirrelationships，andinvestigateapoptosissignaltransductionmechanismafterheatstress-inductioninhumanumbilicalveinendothelialcell（HUVEC），andexplorepathogenesisofvascularendothelialdamageinthewakeofsevereheatstroke.MethodsHumanumbilicalveinendothelialcellheatstressmodelwassetup.Controlgroupwereincubatedat37°C，whileheatstressgroupofcellswereincubatedat39°C，41°C，and43°Cfor2h，thenallthecellswerefurtherincubatedat37°Cfor24h.Mitochondriaofhumanumbilicalveinendothelialcellwereexaminedwithelectronmicroscopy.ApoptosiswasanalyzedbyflowcytometryusingAnnexinV-FITC/PIstaining，andproteinlevelsofcytochromeC，caspase-9，caspase-3wereanalyzedbywesternblot.ResultsInthecontrolgroup（37°C），thestructureofmitochondrialwasintactinHUVECundertransmissionelectronmicroscope.Incontrast，mitochondrialswellingwasfoundinthegroupof43°Cheatstress.Comparedwithcontrolgroup，asincreasinginheatstresstemperature，theratesofinducedapoptosiswere17.8%at41℃and25.6%at43℃，andthelevelsofcytochromeC，Caspase-9，andcaspase-3weresignificantlyincreased（P<0.05）.Therewasnoobviouschangeat39℃heatstress（P>0.05）.ConclusionsMitochondrialapoptosispathwayisinvolvedinapoptosisofhumanumbilicalveinendothelialcellsinthewakeofheatstress.Thevascularendothelialcellsapoptosismaybeassociatedwiththeoccurrenceofsevereheatstroke.【Keywords】Humanumbilicalveinendothelialcell;Apoptosis;Mitochondrial;Heatstress;Severeheatstroke;CytochromeC;Caspase-9;Caspase-3重症中...