Ultrasound enhanced gene therapy: Development of ultrasonic methods of gene transfection for cardiovascular system.

超声触发的微泡破坏已被认为是一种针对特定器官的基因治疗手段。已有报道使用这种方法成功地表达了报告基因。然而，在心血管细胞或组织中，用这种方法对治疗基因产物进行量化还没有被阐明。我们利用新生大鼠心肌细胞研究了基因数量、微泡浓度和孵育时间之间的剂量-反应关系。采用大肠杆菌β-半乳糖苷酶基因表达载体、大鼠肝细胞生长因子基因巨细胞病毒-β肌动蛋白杂合启动子表达载体和培养的新生大鼠心肌细胞进行体外实验。由于临床上的相关性，我们使用频率为2.5 MHz，最大强度为0.5W/cm2的连续波多普勒超声。1.重复暴露于超声触发的半乳糖/棕榈酸微泡破坏中，可以有效地将基因转移到心肌细胞。2.使用导管在大鼠左室腔内结合胸腔内超声照射，研究荧光素酶基因导入心肌的可行性。在体外实验中，荧光素酶的表达水平与体外培养的人脐静脉内皮细胞相似，很难超出内皮屏障。3.在静脉注射基因和微泡的情况下，我们研究了经胸前超声照射将荧光素酶基因导入心肌的可行性。我们在心肌中没有检测到荧光素酶的表达，在肝脏中检测到了一些荧光素酶的表达。4.综上所述，我们不妨(1)通过修饰增强子/启动子来开发更特异的心肌基因打靶方法；(2)改进微泡外壳，使其对质粒-基因和心肌细胞膜都具有更高的亲和力。

Ultrasound-triggered microbubble destruction has been proposed as a means of targeting gene therapy to specific organs. Successful reporter gene expression using this approach has been reported. However, quantification of therapeutic gene products with this method has not been elucidated yet in cardiovascular cells or tissues. We examined the dose-response relations among the amount of gene, microbubble concentration and the duration of incubation using neonatal rat cardiomyocytes. We employed expression plasmid of Escheririchia Coli β-Galactosidase gene, expression plasmid of rat hepatocyte growth factor (HGF) gene containing CMV-β actin hybrid promoter, and cultured rat neonatal cardiomyocytes for in vitro experimental setups. We used continuous-wave Doppler ultrasound with frequency of 2.5MHz and maximal intensity of 0.5W/cm^2 because of clinical relevance.1.Repetitive exposure to ultrasound-triggered galactose/palmitic acid microbubble destruction may yield efficient transfection of genes to cardiomyocytes.2.Using catheter in the rat left ventricular cavity with precordial ultrasound irradiation, we examined the feasibility of luciferase gene transfection into the myocardium. Luciferase activity was detected only in the anterior myocardium, and the level of expression was similar to that in HUVEC cells for in vitro experiments for transfection, suggesting difficulty to transfect beyond the endothelial barrier.3.We examined the feasibility of luciferase gene transfection into the myocardium with precordial ultrasound irradiation in the setting of intravenous administration of gene and microbubble. We detected no expression of luciferase in the myocardium, and some expression in the liver.4.Taken together, we might as well (1)develop more specific gene targeting method for myocardial transfection with modifying enhancer/promoter ; and (2)improve microbubble shells with higher affinity for both plasmid-gene and myocardial cell membrane.