Zhou Zhong-jiang, Ye Hai-yan, Cui Kai, Wang Yu-yan
Department of Cardiology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2010 Nov;30(11):2505-8.
To evaluate the effect of therapeutic ultrasound-induced microbubble's cavitation on plasmid gene transduction in rat pulmonary endothelial cells in relation to the changes of membrane fluidity and cytoskeleton structure.
Rat endothelial cells cultured in vitro were transfected with EGFP plasmid in the presence of protein microbubbles. During the transfection process, the cells were exposed to continuous 2 MHz ultrasonic irradiation for 30, 60, 90, 120 and 180 s (groups A, B, C, D and E, respectively) with the constant mechanical index (MI) of 1.0, or for 60 s with different mechanical index (MI) of 0.5, 0.75, 1.0, 1.5, and 1.8 (groups B1, B2, B3, B4 and B5, respectively). The changes of endothelial cytoskeletal structure and membrane fluidity were evaluated by immunofluorescence staining after the exposure.
EGFP gene transduction increase obviously with prolonged echo irradiation and increased MI. The intensity of immunofluorescence staining, which represented endothelial membrane fluidity, was 0.173±0.013, 0.250±0.037, 0.364±0.022, 0.381±0.019, and 0.395±0.009 in groups A-E, as compared with 0.171±0.017, 0.255±0.026, 0.378±0.007, 0.382±0.009 and 0.397±0.008 in groups B1-B5, respectively. The recovery intensity of the immunofluorescence staining representing the changes in microtubulin of the cytoskeleton structure was 159.15±4.79, 188.23±6.20, 205.80±4.48, 208.99±8.34, and 213.70±5.09 in groups A-E, and was 176.84±3.10, 187.57±14.52, 206.41±11.66, 220.12±13.39 and 221.16±12.78 in groups B1-B5, respectively. The endothelial membrane fluidity and microtubule fluorescence recovery intensity increased remarkably compared with the baseline (P<0.01) within the MI range of 0.50-1.0 and the exposure time of 30-90 s, but underwent no further changes in response to prolonged exposure time (180 s) at the MI of 1.5 (P>0.05). No changes in microfilament fluorescence intensity were observed after exposure to different MI or irradiation time.
Therapeutic ultrasound-mediated albumin microbubble cavitation allows enhances plasmid gene transduction without causing cytoskeleton damages. Increased endothelial membrane fluidity and changes in cytoskeleton structure, especially microtubulin, partially contribute to this enhancement.
评估治疗性超声诱导微泡空化对大鼠肺内皮细胞中质粒基因转导的影响，并探讨其与细胞膜流动性和细胞骨架结构变化的关系。
体外培养的大鼠内皮细胞在存在蛋白微泡的情况下用EGFP质粒转染。在转染过程中，细胞分别接受持续2 MHz超声照射30、60、90、120和180秒（分别为A、B、C、D和E组），机械指数（MI）恒定为1.0，或接受不同机械指数（MI）为0.5、0.75、1.0、1.5和1.8的60秒照射（分别为B1、B2、B3、B4和B5组）。照射后通过免疫荧光染色评估内皮细胞骨架结构和膜流动性的变化。
随着超声照射时间延长和MI增加，EGFP基因转导明显增加。代表内皮细胞膜流动性的免疫荧光染色强度在A - E组分别为0.173±0.013、0.250±0.037、0.364±0.022、0.381±0.019和0.395±0.009，而在B1 - B5组分别为0.171±0.017、0.255±0.026、0.378±0.007、0.382±0.009和0.397±0.008。代表细胞骨架结构中微管蛋白变化的免疫荧光染色恢复强度在A - E组分别为159.15±4.79、188.23±6.20、205.80±4.48、208.99±8.34和213.70±5.09，在B1 - B5组分别为176.84±3.10、187.57±14.52、206.41±11.66、220.12±13.39和221.16±12.78。在MI范围为0.50 - 1.0和照射时间为30 - 90秒内，内皮细胞膜流动性和微管荧光恢复强度与基线相比显著增加（P<0.01），但在MI为1.5时，延长照射时间（180秒）后无进一步变化（P>0.05）。暴露于不同MI或照射时间后，微丝荧光强度未观察到变化。
治疗性超声介导的白蛋白微泡空化可增强质粒基因转导，且不引起细胞骨架损伤。内皮细胞膜流动性增加和细胞骨架结构变化，尤其是微管蛋白的变化，部分促成了这种增强作用。
