超声联合微泡介导基因转染对内皮细胞骨架的影响

目的 探讨白蛋白微泡在治疗性超声条件下对介导基因转染以及对细胞骨架的影响,同时探讨超声联合微泡介导基因转染的最佳声学参数.方法 六孔板培养大鼠微血管内皮细胞,一组每孔加入eGFP质粒10μg,加或不加微泡,超声条件为连续波,频率2 MHz,机械指数是0.50～1.80.照射时间是30～180 S,24～48 h后观察细胞转染情况.一组超声照射后免疫荧光染色细胞骨架,荧光显微镜观察并测定荧光强度.结果 机械指数为0.50～1.00范围内,基因转染率与机械指数呈正相关(P<0.001),再增加机械指数基因转染率差别无统计学意义.超声照射时间30～90 S范围内,基因转染率以及微管荧光强度改变与超声照射时间呈正相关(P<0.001).再延长照射时间基因转染率差别无统计学意义.同种条件下,微丝蛋白荧光强度改变无统计学意义(P>0.05).结论 超声联合微泡能够显著增加报告基因的转染率并且对细胞骨架无明显的损伤.微泡可以作为临床冠心病基因治疗的安全有效的载体.

Ultrasound enhances microbubble-mediated gene transfection without affecting the cytoskeletons of endothelial cells

OBJECTIVE: To investigate the effect of therapeutic ultrasound on enhancing albumin microbubble-mediated gene delivery and evaluate the cytoskeletal damages in endothelial cells (ECs). METHODS: The ECs cultured in 6-well plates were transfected with the plasmid eGFP in the presence or absence of the microbubbles at different concentrations, followed by ultrasound exposure for 30 to 180 s at 2 MHz, with the mechanical index (MI) of 0.5-1.8. The gene transfection efficiency was evaluated by observing the green fluorescence of the cells, and the cytoskeletal damage was assessed using immunofluorescence staining 24 to 48 h after ultrasound exposure. RESULTS: Within the MI range of ultrasound exposure between 0.50 and 1.00, the gene transfection efficiency was positively correlated to MI (P<0.001), and further increment of the MI failed to further increase the transfection efficiency. The duration of ultrasound exposure, within the range of 30-90 s, was also positively correlated to the gene transfection efficiency and microtubule fluorescence intensity(P<0.001), and prolonged exposure did not further enhance the effects. Variation of the MI and ultrasound exposure time within the above ranges did not cause significant changes in the florescence intensity of the microfilaments (P>0.05). CONCLUSION: Albumin microbubbles in the presence of ultrasound exposure can substantially enhance the gene transfection efficiency due to therapeutic ultrasound-mediated microbubble destruction without causing obvious damage of the cytoskeletons, and allows safe and efficient nonviral gene delivery in gene therapy.