Construction Of PcDNA3.1/hVEGF165 And Its Expression In Primary Culture Of Nascent Mice Cardiocytes

Background: Coronary artery disease (CAD) is a common illness among middle-aged and elder people, and is the first cause of death in the Occident. In our country, with the development of people's stantard of living, the incidence of CAD is rising, which is severely threatening people's health. Satisfied result of treatment of CAD has not been acquired in clinic at present, because some of CADs can't be treated by coronary artery bypass graft(CABG); or percutaneous transluminal coronary angioplasty (PTCA); or transmyocardial laser revascularization (TMLR); or other ways, such as diffuse coronary heart disease, and there is no good means for restenosis of coronary artery whose incidence rate is 25%'-50% after operations. Recently a new therapy for CAD was attached importance, and This novel strategy for the treatment of vascular insufficiency has been called "therapeutic angiogenesis" which means that some Angiogenic peptides or encoding genes were administered to ischemic myocardium or limb to induce collateral blood vessel formation and improve perfusion of the ischemic areas. These kinds of factor contain mostly : vascular endothelial growth factor(VEGF) fibroblast growth factor(FGF),angiopoietin(ANG) and so on. Being one of them, VEGF is a primary secreted angiogenic mitogen specific for endothelial cells. Which has some biological functions as follows: (1) promoting angiogenesis and vasculogenesis with the activity of angiogenic mitogen, VEGF can change extracellular matrix and promote neovascularization. (2) augmenting vasopermeability. (3) mataining vascular normal condition and integrity.Besides gene therapy of ischemic heart disease, cell transplantation around local areas can impair sick heart and improve its function. On the condition of myocardial injury, such as myocardial infarction, myocardial cell always be replaced by fibroblastmultiplication to improve the heart function. But no stem cell in grownup in cardiac muscle, the cardiac muscle can't multiply and neogenesis. So it might not be a feasible method at present. With the development of cardiac tissue engineering, it is feasible to repair the injured cardiac muscle by the cell transplantation vivo. Transplanting cells include skeletal muscle celK smooth muscle cell n cardiac muscle cell and others cell. Objective: The content of this study include: construction of eukaryotic expressional plasmid pcDNA3.1/hVEGF165; primary culture of nascent mice cardiocytes; hVEGF165 gene expression in cardiocytes transfected with pcDNA3.1/hVEGF165 to provide the foundation of therapy of CAD with gene and cell transplantation. Methods: Part 1 Construction of eukaryotic expressional plasmid pcDNA3.1/ hVEGF165 Extracting total RNA from fetal human myocardium tissue; constructing the first chain of cDNA by taking the total RNA as template; obtaining hVEGF165 gene by PCR amplifying in light of devised and composed primers; recombining it into T vector (pMD18-T), then identifying it by PCR and sequencing. The results appear to be exact full-length hVEGF165 gene sequence (576bp). The fragment was digested by two kinds of enzyme and inserted into eukaryotic expre- ssional plasmid pcDNA3.1/ myc-hisB to construct pcDNA3.1/hVEGF165. And then COS-7 cells were transfected by the recombined plasmid mediated by liposome, at last the expressed protein was detected with Western Blotting. Part 2 Observation and identification of Primary culture of neonate mice cardiocytes In the condition of sterility, the hearts of Kunming Mice were taken out of, digested by 0.25% trypsase, and cultured with IMDM medium (contains 10% FCS > Penicillin and Streptomycin). The cardiocyte was observed, its motility rate detected by trypan blue stained, detected by immuno -histochemistry with anti-myosin heavy chain. Part 3 Recombined plasmid pcDNA3 l/hVEGF165 trans-fecting the cultured cardiocyte mediated by liposome then detected hVEGF gene expression by RT-PCR, immunohistochemistry and Western blotting. Results: exect gene order of hVEGF165 was obtained from the fetal human myocardium tissue by RT-PCR; pcDNA3.1/VEGF165 was constructed; and transient...