Gene Transfer Mediated By Ultrasonic Destruction Of Gene-loaded Microbubbles And Experimental Study Of Antisense RNA Transfer Treated The Postinfarcation Heart Failure

Part IIn vitro transfer of reporter gene and antisense phospholambanRNA into cardiacmyocytes by ultrasonic destruction ofgene-loaded microbubblesObjectives: Though a few of noviral gene transfer methods were used for myocardiocytes gene transfection, whereas they can achieve very low gene transfer efficiency in cardiac myocytes. Ultrasound exposure (USE) has been used for transfer gene into cell, and early studies also demonstrated that ultrasound can enhance naked plasmid DNA transfection. Although encouraging, the enhancement is still not enough and high-energy ultrasound is necessary in the studies. Recently, gene transfer by ultrasonic destruction of microbubbles has attracted attention due to their high effects, and their site-specific in vivo. However, most of those reports were from a variety of immortalised cell lines and primary cultured noncardiac cells, little had been investigated on the transfer of naked plasmid DNA into primary cultured cardiac myocytes mediated by the USE. In addition, the exact mechanisms that causethis phenomenon are still unclear. Although the rapid collapse of microbubbles is thought to play a major role, whether the characteristic of microbubbles could influence the efficiency of gene transfection was still controversial. Thus, prior to pre-clinical applications, optimization of gene transfer using various microbubbles is required. This study was designed to investigate optimization of gene transfer using various microbubbles and ultrasound exposure condition, which enhance the transfection efficiency of plasmids carried reporter gene and antisense RNA in primary curltured cardiomyocytes. Materials and Methods:1.Cloning of rat PLB cDNA and construction of PLB antisense RNA plasmids vector (PcDNA4-asPLB)The PLB antisense RNA plasmids vector (PcDNA4-asPLB) was sub-cloned from the PAAV-asPLB. The eukaryon vector PcDNA4/myc-HisA (Invitrogen) and PAAV-asPLB were digested by Xho I and EcoR I simultaneously and then the ligation and transfer reaction was porfermed. The PLB antisense RNA plasmids vector was confirmed by restriction enzymes digestion and sequencing. The pSV-P-galactosidase vector is a plasmid carrying a galactosidase reporter gene drived by an SV40 promoter from Promega Corp. 2.Preparation of gene-loaded microbubblesThe air-contained sonicated dextrose albumin (ASDA) microbubbles and perfluoro propane-exposed sonicated dextrose albumin (PESDA) microbubbles were synthesized by sonication. To attach the plasmid to the microbubble, 15μl solution of plasmid (1μg/μl) was added into the microbubble suspension and mixed for 2 hoursat 4℃.3. Hemodynamic measurementsIn order to determine whether the ultrasound targeted destruction gene-loadedmicrobubbles could impair the cardiac function, the hemodynamic measurementswere performed. Rabbits were anesthetized with intravenous doses of pentobarbital sodium (30mg/kg) and spontaneous respiration. A 5F catheter was inserted into the right carotid artery and then advanced into left ventricle of the rabbit to measure L V pressures(LV end-systolic pressure and LV end-diastolic pressure) using Med-Lab system. After intravenous injection of gene-loaded microbubbles (0.1 ml/kg), transthoracic echocardiography was performed to observe the myocardium contrast enhancement, and then the mechanic index was turned to the maximum (MI1.7) to induce microbubbles destruction within the myocardium. 4.Culture of cardiac myocytesThe cardiac myocytes were cultured as the Sampon's method. 5.Cell viability assayIn order to evaluate the influence of ultrasound exposure (USE) and microbubble on cardiomyocytes, the cell viability assay was performed by MTT reaction. The cell activity was determined by ODultrasound/ODcontrol×100%. 6.Gene transfectionIn order to explore the optimal condition of ultrasound destruction of gene-loaded microbubbbles, the gene transfection of pSV-P-galactosidase into cardiomyocytes was firstly performed as previously described methods. The pSV-J3-galactosidase transfer study was divied to seven groups.①control group;...