| A series of experimental studies had been done to investigate an improved avenue for therapeutic gene delivery by ultrasound contrast.Part I Organic Imaging with a New Ultrasound Contrast Agent: Experimental Studies in DogsObjective To test the effects and safety of a new ultrasound contrast agent (JD-95) . Methods The new ultrasound contrast agent (JD-95) in doses ranging from 0.1ml/ (kg-min) to 0.3ml/ (kg.min) were injected to 6 dogs intravenously by injection pump. The real-time perfusion imaging were performed, and furthermore the heart rate ,left ventricular systolic function, arterial blood gas were supervised. Results The real-time perfusion images were perfect and the heart rate, left ventricular systolic function and arterial blood gas did not show distinctly change (p>0.05) . Conclusion The new ultrasound contrast agent (JD-95 ) can give a perfect real-time images and is relatively safe.Part II Construction of VEGF Eukaryotic Expression Vector and itsExpression in the Endothelial Cells and Cardiac Myocytes in vitro Objective To study the feasibility of VEGF165 gene transfection to the endothelial cells and cardiac rnyocytes in vitro. Methods The VEGF165 DNA fragment was inserted into a green fluorescent protein fusion vector, and it was transfected to the endothelial cells and cardiac rnyocytes with the help of lipofectamine. Results The endothelial cells and cardiac rnyocytes were successfully transfected with pIRES2-EGFP- hVEGF165 gene, which confirmed by fluoroscopy and immunohistochemistry. Conclusion VEGF eukaryotic can be expressed stably in the endothelial cells and cardiac rnyocytes with the help of lipofectamine, and it is useful for the gene therapy of myocardial ischemia.Part III Ultrasound destruction of microbubbles enhances gene expression after lipofectamine-mediated transfection of cells in vitro Objective To study the effects of ultrasound-mediated microbubbles destruction on endothelial cells transfected with lipofectamine-DNA complexes. Methods One hour after addition of pIRES2-EGFP-hVEGF gene and lipofectamine, endothelial cells on 6-well plates were exposed to ultrasound for 30s, 60s, or 90s in the presence(10L,100L,500L) or absence of ultrasound contrast agent. The transducer(Sonos 5500,83,1.3MHz,MI1.0) was submerged in water and held at a distance of 1 cm. Expression of the marker gene EGFP was observed by fluorescent microscopy 2 days later. Results Ultrasound treatment of endothelial cells on plates for 30S and 60S yielded significant increase in transfection rates without damaging the cells, but 90S treatment killed most of the cells. Ultrasound contrast agent was ableto significantly increase endothelial cells transfection rate by enhancing cavitation effects at a concentration of 100L, and damaged most cells when applied at a concentration of 500L. Conclusion Ultrasound destruction of microbubbles enhances gene expression after lipofectamine-mediated transfection of endothelial cells in vitro and may represent an improved avenue for therapeutic gene delivery in vivo.Part IV Ultrasound destruction of microbubbles directs gene delivery to the ischemic myocardium of rabbitsObjective To investigate the feasibility of gene delivery to the ischemic myocardium of rabbits by ultrasound destruction of microbubbles. Methods We used a ligation model of left circumflex branch coronary artery of rabbits. Three days after ligation, the mixture of gene and microbubbles were infused into the vein of rabbits with or without simultaneous ultrasound. Additional controls included ultrasound of microbubbles that did not contain gene, gene alone, gene plus ultrasound, and blank control. Rabbits were killed after two weeks and examined for VEGF expression. Results The hearts of five rabbitsthat underwent ultrasound-mediated destruction of microbubbles containing gene showed VEGF protein expression in ischemic myocardium.None of the control animals showed VEGF protein expression. Conclusion Ultrasound-mediated destruction of microbubbles is a promi... |
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