| 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.
|
PDF Full Text Request