Department of Cardiology, Beijing Anzhen Hospital, Capital Medical University, Beijing Institute of Heart Lung and Vessel Diseases, Beijing 100029, China.
Chin Med J (Engl). 2011 May;124(9):1395-400.
It has been proven that ultrasonic destruction of microbubbles can enhance gene transfection efficiency into the noncardiac cells, but there are few reports about cardiac myocytes. Moreover, the exact mechanisms are not yet clear; whether the characteristic of microbubbles can affect the gene transfection efficiency or not is still controversial. This study was designed to investigate whether the ultrasound destruction of gene-loaded microbubbles could enhance the plasmids carried reporter gene transfection in primary cultured myocardial cell, and evaluate the effects of microbubbles characteristics on the transgene expression in cardiac myocytes.
The β-galactosidase plasmids attached to the two types of microbubbles, air-contained sonicated dextrose albumin (ASDA) and perfluoropropane-exposed sonicated dextrose albumin (PESDA) were prepared. The gene transfection into cardiac myocytes was performed in vitro by naked plasmids, ultrasound exposure, ultrasonic destruction of gene-loaded microbubbles and calcium phosphate precipitation, and then the gene expression and cell viability were analyzed.
The ultrasonic destruction of gene-loaded microbubbles enhanced gene expression in cardiac myocytes compared with naked plasmid transfection ((51.95 ± 2.41) U/g or (29.28 ± 3.65) U/g vs. (0.84 ± 0.21) U/g, P < 0.01), and ultrasonic destruction PESDA resulted in more significant gene expression than ASDA ((51.95 ± 2.41) U/g vs. (29.28 ± 3.65) U/g, P < 0.05). Ultrasonic destruction of microbubbles during calcium phosphate precipitation gene transfection enhanced β-galactosidase activity nearly 8-fold compared with calcium phosphate precipitation gene transfection alone ((111.35 ± 11.21) U/g protein vs. (14.13 ± 2.58) U/g protein, P < 0.01). Even 6 hours after calcium phosphate precipitation gene transfection, ultrasound-mediated microbubbles destruction resulted in more intense gene expression ((35.63 ± 7.65) U/g vs. (14.13 ± 2.58) U/g, P < 0.05).
Ultrasonic destruction of microbubbles might be a promising method for the delivery of non-viral DNA into cardiac myocytes, and the gene tranfection is related to the characteristics of microbubbles.
已有研究证明，超声破坏微泡可增强非心肌细胞的基因转染效率，但关于心肌细胞的研究较少。而且，确切机制尚不清楚；微泡的特性是否会影响基因转染效率仍存在争议。本研究旨在探讨超声破坏载基因微泡能否增强原代心肌细胞中质粒携带报告基因的转染，并评价微泡特性对心肌细胞中转基因表达的影响。
制备载有两种类型微泡（含气超声处理的葡聚糖白蛋白 ASDA 和全氟丙烷暴露超声处理的葡聚糖白蛋白 PESDA）的β-半乳糖苷酶质粒。通过裸质粒、超声照射、超声破坏载基因微泡和磷酸钙沉淀体外进行基因转染，然后分析基因表达和细胞活力。
与裸质粒转染相比，超声破坏载基因微泡可增强心肌细胞中的基因表达[（51.95±2.41）U/g 或（29.28±3.65）U/g 比（0.84±0.21）U/g，P<0.01]，超声破坏 PESDA 导致的基因表达更为显著[（51.95±2.41）U/g 比（29.28±3.65）U/g，P<0.05]。在磷酸钙沉淀基因转染过程中进行超声破坏微泡可使β-半乳糖苷酶活性比单独进行磷酸钙沉淀基因转染增强近 8 倍[（111.35±11.21）U/g 蛋白比（14.13±2.58）U/g 蛋白，P<0.01]。即使在磷酸钙沉淀基因转染 6 小时后，超声介导的微泡破坏仍可导致更强的基因表达[（35.63±7.65）U/g 比（14.13±2.58）U/g 蛋白，P<0.05]。
超声破坏微泡可能是一种有前途的将非病毒 DNA 递送至心肌细胞的方法，基因转染与微泡的特性有关。
