Department of Medical Imaging Center, First Affiliated Hospital, Jinan University, 613 Huangpu Dadao Xi, 510632 Guangzhou, Guangdong, China.
J Ultrasound Med. 2011 Sep;30(9):1247-58. doi: 10.7863/jum.2011.30.9.1247.
Gene transfer to cardiomyocytes in vivo has received much research attention in the last decade but remains a substantial hurdle. Gene transfer using ultrasound-targeted microbubble destruction is a promising tool for gene therapy. Little data have shown the feasibility and optimization of this method for primary myocardial disease. In this study, we sought to determine the feasibility and efficiency of in vivo gene transfer to the myocardium mediated by ultrasound-targeted microbubble destruction accompanied by polyethylenimine.
Three plasmids (luciferase reporter, red fluorescent protein reporter, and enhanced green fluorescent protein reporter) were used in this study. The ultrasound parameters were also optimized. A solution containing phosphate-buffered saline, a plasmid, plasmid complex, or polyethylenimine/plasmid, and liposome microbubbles was injected via a tail vein with (study) or without (control) transthoracic ultrasound irradiation. The efficiency of reporter gene transfer was determined by detection of luciferase activity or microscopy, and histologic investigations of the tissue specimens were performed.
Ultrasound-targeted microbubble destruction significantly increased luciferase activity in vivo compared to plasmids and microbubbles alone (P < .001). More importantly, the increase in transgene expression was significantly related to ultrasound-targeted microbubble destruction in the presence of polyethylenimine (P < .001). In addition, fluorescein expression was present in all sections that received ultrasound-targeted microbubble destruction. The fluorescent reporter genes and luciferase plasmid all had similar results. Regardless of ultrasound exposure, expression in other organs was close to a background level except for the liver and lung. Hematoxylin-eosin staining showed no notable myocardial injury or death in control and treated mice.
An atraumatic targeted gene delivery technique based on ultrasound-targeted microbubble destruction and polyethylenimine has been developed to transfect cardiomyocytes in vivo. If a suitable target gene is added, the novel technique could be highly effective in many kinds of heart disease.
在过去的十年中，体内基因转移已受到广泛关注，但仍然是一个巨大的障碍。超声靶向微泡破坏的基因转移是基因治疗的一种有前途的工具。很少有数据表明该方法对原发性心肌疾病的可行性和优化。在这项研究中，我们试图确定超声靶向微泡破坏联合聚乙烯亚胺介导的体内基因转移到心肌的可行性和效率。
本研究使用了三种质粒（荧光素酶报告基因、红色荧光蛋白报告基因和增强型绿色荧光蛋白报告基因）。还优化了超声参数。将含有磷酸盐缓冲盐水、质粒、质粒复合物或聚乙烯亚胺/质粒和脂质体微泡的溶液通过尾静脉注入，同时（研究组）或不（对照组）进行经胸超声辐射。通过检测荧光素酶活性或显微镜观察，确定报告基因转移的效率，并对组织标本进行组织学研究。
与单独使用质粒和微泡相比，超声靶向微泡破坏显著增加了体内的荧光素酶活性（P <.001）。更重要的是，在聚乙烯亚胺存在的情况下，转基因表达的增加与超声靶向微泡破坏有显著相关性（P <.001）。此外，所有接受超声靶向微泡破坏的切片中均有荧光素表达。荧光报告基因和荧光素酶质粒都有类似的结果。无论是否进行超声照射，除肝脏和肺部外，其他器官的表达水平均接近背景水平。苏木精-伊红染色显示对照组和处理组的小鼠心肌均无明显损伤或死亡。
已经开发出一种基于超声靶向微泡破坏和聚乙烯亚胺的非创伤性靶向基因传递技术，用于体内转染心肌细胞。如果添加合适的靶基因，该新技术在许多种心脏病中可能非常有效。
