【作者】 魏来；

【导师】 徐军美；

【作者基本信息】 中南大学 ， 麻醉学， 2010， 博士

【摘要】 目的：①构建携带人过氧化物酶体增殖物激活受体γ1基因的重组腺病毒载体(Ad-PPARγ1)、携带增强型绿色荧光蛋白的重组腺病毒载体(Ad-EGFP);②研究冠脉途径转染腺病毒载体到大鼠心肌的可行性,如果可行观察PPARγ1基因转染对缺血再灌注心肌是否有保护作用；③研究心肌缺血再灌注以后PPARγ1表达的改变以及PPARγ1基因保护缺血再灌注心肌的机制。方法：实验分三部分：①设计引物,利用PCR方法从含有目的基因的质粒或者cDNA文库中钓取目的基因,将目的基因和pDC315质粒载体分别酶切。酶切产物经电泳回收后进行定向连接或者交换,其产物转化细菌感受态细胞。对阳性克隆进行菌落PCR鉴定,再对PCR鉴定阳性的克隆进行测序和对比分析,比对正确的即为构建成功的目的质粒。构建成功后行腺病毒包装、扩增、纯化、滴度测定。②通过预实验确认经冠脉转染的可行性。正式实验SD大鼠随机分为4组：Sham组转染Ad-EGFP 3天后,开胸后冠脉左前降支只过线不结扎；IR组转染Ad-EGFP3天后,心肌缺血30min,再灌注120min；IPC组转染Ad-EGFP 3天后,5min缺血,5min再灌注,重复3次后行心肌缺血30min,再灌注120min；PPARγ1组转染Ad-PPARγ1 3天后,行心肌缺血30min,再灌注120min。记录缺血再灌注前后不同时间点心功能指标,检测心律失常发生率并作出评分,测定肌钙蛋白I含量,伊文氏蓝灌注和TTC染色测定危险区面积和心梗面积,光镜和电镜观察心肌组织形态学超微结构变化,TUNEL法测凋亡。③在第二部分的基础上,用Western Blot测定蛋白含量,PT-PCR测定mRNA含量,研究心肌缺血再灌注后以及转染后PPARγ1表达的变化,PPARγ1通路保护心肌作用中是否有Bcl-2、Bax发挥作用,以及激活PPARγ1通路是否能够通过调控选择素表达保护心肌。结果：①成功构建Ad-EGFP和Ad-PPARγ1。②预实验确定经冠脉途径能够转染心肌,且最佳病毒载体滴度为109pfu/ml。再灌注末,与Sham组相比,IR组多数心功能指标明显恶化,而IPC组和PPARγ1组多数心功能指标优于IR组,且心梗面积较小,凋亡指数较低,心肌组织形态学观察也优于IR组。③心肌缺血再灌注后PPARγ1表达下降,IPC和转染PPARγ1基因后表达上调；激活PPARγ1通路能够上调Bcl-2/Bax比率减少凋亡,能抑制选择素E、P表达上调而抑制炎症细胞聚积。结论：本课题成功构建了Ad-PPARγ1和Ad-EGFP,再灌注以后心肌表达PPARγ1下降,转染后PPARγ1表达增加,通过冠脉途径转染人PPARγ1基因能够保护缺血再灌注心肌,激活PPARγ1通路能够通过上调Bcl-2/Bax比率抑制凋亡,下调选择素E、P抑制炎症细胞浸润。更多还原

【Abstract】 Objective:①To construct the replication-deficient recombinant adenovirus vector with enhanced green fluorescence protein (EGFP) and PPARγ1 gene.②To explore the feasibility of transfection of adenovirus via coronary arteria and the protective effect of PPARγ1 against myocardial ischemia reperfusion injury.③To explore the expressive changes of PPARγ1 mRNA and protein in the ischemia-reperfusion heart tissues, and then to explore the protection mechanism against ischemia reperfusion injury.Methods:The experiment consists of three parts:①We designed the primers first, then used the polymerase chian reaction (PCR) method to obtain purpose gene from the plasmid containing purpose gene or cDNA library, thirdly, we used restriction enzyme to digest the purpose gene and pDC315 plasmid respectively.The digested products after recovery of electrophoresis were directional connected or exchanged.The products were transferd to competent cells.Positive colonies were identified by PCR, then positive colonies were sequenced and comparative analysised. And then we have constructed the purpse plasmid successfully if matching correctly. At last we processed adenovirus of packing, amplification, purification and titer determination.②We make sure whether transfection of Ad-PPARγ1 via coronary artery is feasible in pre-experiment.Sprague-Dawley rats were randomly divided into four groups, including group Sham,group IR,group IPC (ischemic pre-comditioning),group PPARγ1.After group Sham were transfected with Ad-EGFP 3 days,rats were opened chest without ligating the left coronary artery;After group IR were transfected with Ad-EGFP 3 days,left coronary artery were legated for 30 min,then reperfusion for 120 min; After group IR were transfected with Ad-EGFP 3 days,rats were subjected to 5min ischemia followed by 5 min reperfusion for 3 cycles before 30 min ischemia and 120 min reperfusion; After group PPARyl were transfected with Ad-PPARγ1 3 days, left coronary anterior descending artery were legated for 30 min,then reperfusion for 120 min.The index of cardiac function were recorded at different time point.The incidence rate of arrhythmia were observed and graded.The level of plasma troponinlin was detected.Myocardial area at risk and infart region was determined by Evan’s blue dye perfusion and triphenyl tetrazolium chloride (TTC) staining. Myocardial tissue morphological ultrastructural changes were observed by light and electron microscope.Myocardial apoptotic cells were examined by the TUNEL assay.③Based on the second part,protein content was analysised by Western Blot and mRNA content was analysised by RT-PCR.Expression changes of PPARγ1 gene were analysised after reperfusion and transfetcion.To explore whether Bcl-2 and Bax play a role in PPARγ1 pathway to protect myocardial tissue,and whether selectin family were regulated by PPARγ1 pathway to protect myocardial tissue.Results:①We constructed Ad-EGFP and Ad-PPARγ1 successfully.②Feasibility of transfection of adenovirus via coronary arteria pre-experiment was ascertained,and the best virus vector titer was 109pfu/ml.At the end of reperfusion,most parameters of myocardial function of group IR were deteriorate than Sham.Most parameters of myocardial function of group IPC and PPARγ1 were better than IR,with less infarcion size,lower apoptsis index and better myocardial tissue morphological ultrastructural changes.③Expression of PPARγ1 after reperfusion downregulated obviously,but upregulated obviously after ischemic precomditioning and transfection. Ratio of Bcl-2/Bax protein expression was upregulated by activation of PPARyl pathway,Also inflammatory cells infiltration were inhibited by the restrained of selectin family.Conclusion Transfection of PPARγ1 gene via via coronary arteria was feasible and demonstrated the protectiv effect against myocardial ischemia reperfusion injury.The expression of PPARγ1 downregulated after reperfusion. Ratio of Bcl-2/Bax protein expression was upregulated by activation of PPARyl pathway to inhibit apoptosis,also inflammatory cells infiltration were inhibited by the restrained of selectin family.更多还原