hHSP70基因冠脉转染对大鼠心肌缺血再灌注损伤的保护作用

hHSP70基因冠脉转染对大鼠心肌缺血再灌注损伤的保护作用毕业论文【摘要】目的：研究hHSP70基因经冠脉转染对大鼠心肌抗缺血再灌注损伤的效应，并探讨其可能机制。（2）选择50只S-D大鼠随机分成7组（n=10）。大鼠左侧开胸，暴露心脏及升主动脉和肺动脉根部，用无损伤血管钳钳夹于主、肺动脉根部，阻断循环10秒钟，同时用27号针头于左心尖部注射各组相应试剂至左心室腔，使之在密闭的冠脉循环内分布。第Ⅰ、Ⅱ、Ⅲ组仅注射生理盐水；第Ⅳ、Ⅴ组分别注射含Adv-EGFP、adv-Hhsp70（1×109pfu)的生理盐水；关胸苏醒后，稳定4天，使经冠脉转染的基因充分表达于心肌。（3）冠脉转染4天后，再次暴露胸腔，在动脉圆锥与左心耳根部间结扎冠状动脉左前降支(LAD)。结扎45min后,松开缝线使冠状动脉再通180min。Ⅰ组只穿线不结扎。Ⅲ组在LAD结扎5min后开放5min，并重复3次，然后再行45min缺血、180min再灌注。（4）采用Evan’sBlue和TTC双染色法后利用 image-pro 测定心肌梗死面积;光学显微镜下观察心肌的炎症改变； WesternBlotting 免疫印迹测定心肌基因表达的改变并利用 bandscan进行灰度测量进而统计计算。 （2）hHSP70过量表达能够增加Bcl-2表达及减少caspase-3活化态的生成进。（3）hHSP70过量表达能减少 NF-κB的活化。（4）心肌表达的蛋白发生相应的变化，hHSP70转染组hHSP70过量表达，，Bcl-2表达增加，活化的 caspase-3减少。（2）hHSP70转染能通过抑制 NF-κB的表达及活化及其他机制起到抑制炎症作用。（3）在HSP70过表达的情况下对心肌梗死与缺血预处理组没有统计学差别， 可以起到与缺血预处理急性期同等程度的心肌的保护作用。关键词 缺血再灌注损伤，腺病毒载体， hHSP70，凋亡，炎症ABSTRACTObjectives ：Toobservetheprotective effect against myocardial ischemiareperfusion injury after transfer ofhumanHSP70geneviacoronaryarteryandtoinvestigatethepotentialmechanisms.Methods：(2)50S-Dratswererandomlydividedinto5groups(n=10).Theleftthoracotomywasperformedandthepericardiumopenedwiththeexposureoftheheartandthebaseofpulmonaryartery andascendingaorta.Pulmonaryarteryandascendingaortawereoccludedbyapplyinganontraumatic vascular clamptoachieveplete cessation ofoutflow, atthesametimeadenovirus particles (×109pfu)inml of%saline solution wereinjectedintotheleftventriclecavityusinga1-mlsyringewitha27-gaugeneedle.After10secondsofpleteoutflowocclusion,theclampwasremoved.salinesolutionalonewasinjectedintotheratheartinGroupⅠⅡⅢ.InGroupⅣAdv-EGFPwasinjected. All ofthemwereclosedandrecovered,andthenstabilizedwithoutoperativeinterventionfor4day.After4days,arepeatthoracotomywasperformedtoexposetheanteriorsurfaceoftheheart.Theproximalleftanteriordescendingcoronaryartery(LAD)wasidentifiedanda6/0suture(Ethicon)wasplacedaroundthearterybelowtheleftatrialappendageandthesurroudingmyocardium.Regionalleftventricularischemiawasestablishedfor45minbyligationofLAD.Ischemiawasconfirmedbydiscolorationofmyocardiumandbychangesincardiacrhythm.Attheendoftheischemiaperiod,theligationwasloosenedandreperfusionwasachievedfor180min.GroupⅠ(sham-operatedgroup)servedassurgicalcontrolsandweresubjectedtothesamesurgicalproceduresasthetheexperimentalgroup,withtheexceptionthattheLADwasnotligated.GroupⅢweresubjectedtoischemicpreconditioning(IPC)beforeI/Rbyrepeatedischemiaandreperfusionthrough3cyclesofinducedLADischemiafor5minfollowedby5minofreperfusion.(2)GenehHSP70overexpressioncanraiseBcl-2production、restraincaspase-3activated.(3)GenehHSP70canrestrainNF-κBactive.Theexpressionofproteininratheartaltered.InAdv-HSP70grouptheheexpressionofHSP70andBcl-2wereraised,activatedcaspase-3decreased.Conclusions：(1)TransfectionofHSP70inhibitsinflammationthroughNF-κBdownregulation.TransfectionofHSP70suppressesapoptosisthroughincreasingBcl-2productionanddecreasingCaspase-3activation.TransfectionofHSP70demonstratessynergisticeffectsofmyocardialtolerance toI/R injury, whichcananaloguetheprotective effect ofIPCtoalargerdegree.KEYWORDSischemiareperfusioninjury,adenovirusvector,hHSP70,apoptosis,inflammation4519915151619220HSP70222323HSP7024NF-B2626HSP70Caspase-327283294325333648AdAdenovirusAdvAdenoviralvectorAdv-EGFPAdvencodingEGFPBcl-2B-cellleukemia/lymphoma-2B细胞淋巴瘤因子-2EGFPenhancedgreenfluorescenceprotein增强型绿色荧光蛋白CPBcardiopulmonarybypass心肺转流术IPCIschemicPreconditioning缺血预处理SODsuperoxidedismutase超氧化物歧化酶HGFhepatocytegrowthfactor肝细胞生长因子HSPheatshockprotein热休克蛋白IGFinsulin-likegrowthfactor胰岛素样生长因子IL-10interleukin10白细胞介素-10I/RIschemiaandreperfusion缺血再灌注LUCluciferase荧光素酶MLCmyosinlightchain肌球蛋白轻链mRNAmessengerribonucleicacid信息核糖核酸OFROxygenfreeradical氧自由基AIFApoptosisinducingfactor凋亡诱导因子Pfuplaqueformingunit斑块形成单位FGFFibroblastgrowthfactor成纤维细胞生长因子TNFTumornecrosisfactor肿瘤坏死因子TUNELTdT-mediateddUTPdigoxigeninnickendlabeling原位缺口末端标记技术VEGFvascularendothelialgrowth