Effects Of Angiopoietin-1 Gene-Modified Rat Mesenchymal Stem Cells Transplantation On The Improvement Of Heart Function After Myocardial Infarction

Part One:Construction and identification of Ang-1 gene-modified mesenchymal stem cells mediated by lentiviral vectorObjectives:Construction and identification of Ang-1 gene-modified mesenchymal stem cells,provide the foundation of transgene stem cells(Ang-MSC) transplant to treat the ischemic cardiomyopathy.Methods:Transformation of three lentiviral virus plamids into competent cell respectively,using QIAGEN Plasmid kit to exact the plasmids.The lentiviral virus three plasmid system were cotransfected to 293T cells by lipofectamin2000 to packaging angiopoietin-1 gene and the reporter gene enhanced green fluorescent protein(EGFP) gene co-expressing lentivirus,rMSCs were separated,cultured and indetification,rMSCs were infected with the lentivirus produced formerly,Western blotting to dectect the protein Ang-1 expression in transgenic cells.ResuLts:Lentiviral virus three plasmids co-transfecting to the 293T cells,the amount of green fluorescence protein expression in 293T cells was observed with the fluorescent microscope.Concentrated the lentiviral supernatant to infect rMSCs, pNL-MSC and Ang-MSC both express EGFP with the fluorescent microscope. Western boltting analysis showed that Ang-1 protein expressed by Ang-MSC rather than pNL-MSC and MSC.Conclusions:Successful construction of Ang-1 gene-modified rMSCs,which faciliate our next step Ang-MSC transplant to treat the ischemic heart disease. Part Two:Effects of angiopoietin-1 gene-modified rat mesenchymal stem cells transplantation on the improvement of heart function after myocardial infarctionObjectives:Ang-MSC were inject into the center and border zone of infarcted myocardium of the myocardial infarction rat model,then observe the Ang-MSC survival rate,dectec the target gene expression level and angiogenesis density,discuss the effects of Ang-MSC on improving the heart function.Methods:The rat myocardial ischemia model was established.The second thoracotomy was operated after the left anterior descending coronary artery ligation two weeks,rMSCs,Ang-MSC or pNL-MSC were injected in and around the ischemia myocardium.After the cells transplanted 7 days and 28 days,the distribution of transplanted rMSCs were observed with fluorescence microscope,we use RT-PCR to detect the Ang-lmRNA expression level,â…§factor fluorescence immunostaining to detect angiogenesis and echocardiography to evaluate each group's heart function improvement.ResuLts:Ang-MSC and pNL-MSC transplantation groups of rat heart histological sections can observe the EGFP positive cell with the fluorescence microscope,Ang-MSC transplantation group myocardium can long-term express of Ang-lmRNA.Compared with rMSCs and pNL-MSC groups,Ang-MSC group vessel density was significantly greater.Ang-MSC was significantly improved heart function of rat myocardial infarction model.Conclusions:The transplanted cells survival rate of Ang-MSC is higher,it can sustained express the target gene.In the ischemia myocardium area,angiogenesis density is higher and heart function improved significantly,which can promote the theory basement for genetic engineering of stem cell transplant to treat ischemia myocardium.