| BackgroundCoronary heart disease (CHD) has become more and more danger for human being. Because medical therapy can't get rid of the cause of CHD-coronary artery stenosis, coronary revascularization by percutaneous coronary angioplasty (PTCA) or coronary artery bypass grafting (CABG) has become common treatment options for patients with advanced ischemic heart disease. However, a significant number of patients (for example, patients with 2- and 3-vessel or long and diffuse disease) are not candidates for standard revascularization procedures or have incomplete revascularization with these procedures. For these patients, gene therapy is thought to promote the development of supplemental collateral blood vessels that will constitute endogenous bypass conduits around occludednative arteries. Vascular endothelial growth factor (VEGF) is one of the most important genes in this stratefy because of its traits in angiogenesis. Although a series of animal experiments and clinical studies have demonstrated that VEGF gene therapy results in significant and physiologically relevant improvement, some fundamental questions have yet to be answered by the reported data: (1)Are proposed gene therapy safe? (2)Are new blood vessels stable and available? (3)How to choose the vascular growth factor, carrier and transfer system? Our study is designed to discuss some of these questions.Aim1. To construct a eukaryotic expression vector of human VEGF 165gene and enhanced green fluorescent protein (EGFP) gene, transfer it into endothelial cell and myocardial cell in vitro and vivo, detect the expression of VEGF 165 gene and EGFP gene.2. To mensurate the accuracy of NOGA electromechanical mappingsystem measurement (location measurement, distance measurement and volume measurement) in vitro and vivo, analyse the relationship between NOGA electromechanical mapping system values and other methods values.3. To distinguish infarcted from healthy myocardium using NOGAelectromechanical mapping system.4. To evaluate the security and efficacy of catheter-basedtransendocardial eukaryotic expression vector transfer of pIRES2-EGFP-hVEGF 165.Methods1. pIRES2-EGFP-hVEGF165 eukaryotic expression plasmid was constructed by inserting the VEGF165 cDNA into pIRES2-EGFP, and new plasmid was confirmed by digested with gene enzyme. Rat primary cultured myocardial cell and human endothelial cell were transfected with Lipofect AMINE2000 in vitro, and we did direct gene transfer of naked DNA encoding VEGF165 into rabbit myocardium. Finally, RT-PCR, fluoroscope, western-blotting, and immunohistochemical staining were used to detect the expression of VEGF gene and EGFP gene.2. Using NOGA electromechanical mapping system to measure known spots, distance and cubage in vitro, we detected its veracity and repeatability by statistical analysis. Using ventriculography and NOGA electromechanical mapping system to measure heart functional index simultaneously, we analysed their relationship by statistical analysis, we got the porcine model of acute myocardial infarction (AMI) by coronary artery angioplasty balloon, then analysed the NOGA mapping values before and after AMI by statistical analysis.3. We transfered eukaryotic expression vector into porcine myocardium by catheter-based transendocardial injection through NOGA electromechanical mapping system, and observed changes of haematological index, ultrasonogram and NOGA mapping values before and after gene transfer.Three months after gene transfer, we did coronary angiography, HE stainning and electronography to evaluate collateral circulation of the occluded artery, number of new blood vessel, functional and structural status of local myocardium respectively.Results1. New plasmid was found to contain the hVEGF165 cDNA sequence by agarose gel electrophoresis analysis. We transfected pIRES2-EGFP-hVEGF165 or pIRES2-EGFP into myocardial cells and endothelial cells in vitro and vivo, then found that all cells expressed EGFP (9.48% in endothelial cell, 3.43% in myo... |
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