The Transfection Of ApMI Gene Into Rabbits Epicardial Adipose Tissue And Its Effect On The Coronary Therosclerosis Formation

Part one:The construction and evaluation of apMl eukaryotic expression recombinantObjective:To construct an eukaryotic expression vector plasmid containing human apMl gene, and evaluate its expression efficiency in the adipose cells and HUVEC cells which had been transfected with adiponectin.Methods:The apMl mRNA extracted from human epicardial adipose tissue was amplified by RT-PCR, and cloned into plasmid pcDNA3.1 in order to formulate a recombinant plasmid (pcDNA3.1-apMI). Restriction enzyme digestion and electrophoresis were used to identify the products. The pcDNA3.1-apMl was transient transfected into adipose cells and HUVEC cells, and its expression was assessed by Western Blot.Results:The result of DNA sequencing demonstrated that the pcDNA3.1-apMl recombinant sequence was exactly correct. The expression of the apMl gene can be detected in adipose cells and HUVEC cells 48 hours after the transient transfection(p<0.01). Conclusion:The eukaryotic expression recombinant containing apMl gene was successfully constructed. Ttransfected apMl gene can be effectively expressed in adipose cells and HUVEC cells, which provide an efficient method for the further study.Part two:The transfection of apMl gene into epicardial adipose tissue and its effect of the coronary therosclerosis formation in rabbitsObjective:To investigate the effect of human apMl gene transfection into epicardial adipose tissue on the formation of coronary atherosclerosis in high-cholesterol fed rabbit.Methods:50μl mixture of pEGFP-apMl and liposome was injected into rabbit pericardial cavity. The epicardial adipose tissue was harvested at 2,7 and 28 days after injection respectively, and the fast frozen sections of these samples were examed. The fluorescence microscopy and immunohistochemical method were adopted to identify the transfection efficiency and expression of apMl.24 male New Zealand White rabbits were randomly divided into 4 groups:normal control group, high-fat group, high-fat plus pEGFP injection group, high-fat plus pEGFP-apMl injection group. After 4 weeks, the expression of apMl in epicardial adipose tissue was detected by Western blot analysis. The concentration of TC, HDL-C and LDL-C in peripheral blood was measured by automatic biochemical analyzer. The levels of adiponectin and TNF-a in peripheral blood and pericardial fluid was measured by ELISA. The ratio of intima/media chickness of the left coronary artery was measured in HE staining.Results:In rabbit epicardial adipose tissue, a high expression of apMl was detected at 2 days and 7 days after the injection and the expression lasted for 28 days. No effective transfection was detected in epicardial connective tissues and myocardial cell. No significant changes of adiponectin, TNF-a, TC, HDL-C and LDL-C in peripheral blood were observed after the injection of pEGFP-apMl. In the pericardial cavity fluid of the animals in the high-fat plus pEGFP-apMl injection group, the distinct increase of adiponectin and decrease of TNF-a (p<0.01) were observed. Compared with the high-fat group, the ratio of intima/media chickness of left coronary artery could decrease to 40.66%after the injection of pEGFP-apMl.Conclusion:The apMl gene could be effectively transfectd into epicardial adipose tissue through the pericardial cavity injection. The increase of adiponectin expression in the epicardial adipose tissue could regulate the release of local inflammatory cytokines which were induced by high-fat feeding and so inhibit the formation of coronary atherosclerosis.Part three:Delivery and expression of human apMl gene into aorta of rabbits by using ultrasound with SonoVueObjective:To deliver apMl gene into aorta of rabbits by using ultrasound with SonoVue. To provides a non-invasivenon-toxic transfection technique, and explore new areas for clinical research and treatment for the adiponectin used in vivo intervention in atherosclerosisMethods:While the mixture of SonoVue and pcDNA3.1-apMl was injected though helix vein, the thoracoabdominal aorta of rabbits were sonicated by ultrasound for 3min. The arterial vessels and blood serum of rabbits were harvested at 2,7and 14 days respectively after delivery to detect apMl expression by Western blot analyses and to detect TNF-a, TC, HDL-C, LDL-C by ELISA.Results:After mixture of pcDNA3.1-apMl and SonoVue was injected into the rabbit ear vein, the high expression of apMl gene was detected at 2 days and 7 days after delivery and at lasts to 14 days. ELISA results showed that the concentration of apMl protein in serum of rabbit could be significantly increased by delivery of apMl gene using ultrasound with SonoVue(p<0.01). There was no significant effect to the expression of TNF-a, total cholesterol (TC), high-density lipoprotein-colesterol (HDL-C) or low-density lipoprotein cholesterol (LDL-C).Conclusion:ApMl gene can be transferred into the deep area of rabbit aorta vessel wall by Ultrasound with SonoVue and to be expressed and secreted effectively without obvious side effects, which established the new in vivo non-invasive drug-free apMl gene transmission technology and provides a new technique for the study of intervention of atherosclerosis in vivo.