【摘要】 目的探讨不同治疗性超声(TUS)参数对基因转染的作用,优化TUS参数以实现高效率的转染,减少对细胞活力和质粒完整性的影响。方法将质粒和Sono Vue微泡加入培养的Hela细胞后行超声辐照,改变超声强度、占空比以及辐照时间等参数,比较不同的TUS辐照策略对细胞活力及红色荧光蛋白(DsRed)表达效率的影响,以确定最佳辐照参数,并对质粒完整性进行分析。结果低超声强度(0.4 W/cm~2、1.0 W/cm~2)、低占空比(10%和20%)时,细胞存活率较高(>80%),辐照1 min和3 min之间的细胞活力差异不明显(P>0.05)。当增加超声强度(1.6 W/cm~2、2.2 W/cm~2)和占空比(50%)时,细胞活力显著下降(P<0.05)。当20%占空比,1.0 W/cm~2的TUS辐照3 min时,可实现最高的转染率。质粒DNA结构的完整性不受优化的TUS参数影响。结论TUS是一种有效的基因输送方法,优化的参数在无显著细胞死亡和DNA损伤的前提下增加转染,这种非侵袭性的基因转染方法可能是临床基因疗法的一种有用工具。更多还原

【Abstract】 Objective To study the contribution of different parameters of therapeutic ultrasound(TUS)for gene transfec- tion,and to optimize TUS parameters in order to obtain high transfection efficiency with minimal loss in cell viability and structural integrity of plasmid DNA.Methods Plasmids were added to cultured Hela cells followed by TUS exposure with Sono Vue microbubbles.Different TUS parameters(intensity,duty cycle,exposure time)that would affect transfection effi- ciency of red fluorescent protein(DsRed)and cell viability of HeLa cells were used,and the optimal exposure protocols were determined.The effects of sonication on plasmid DNA were investigated.Results A series of cytotoxicity experiments re- vealed that cell viability maintained at the normal level(>80%)when the intensity was 0.4 W/cm~2 or 1.0 W/cm~2 and the duty cycle was 10% or 20%.The cell viability of exposure l rain was not significantly different from that of 3 min(P>0.05),but higher intensity(1.6 W/cm~2,2.2 W/cm~2)and duty cycle(50%)of TUS caused obviously decreasing of cells survival rate(P<0.05).Optimal setting of TUS was achieved with 1.0 W/cm~2,20% duty cycle for 3 min.The result of agarose gel electrophoresis indicated that the structural integrity of plasmid DNA was not affected by the optimal TUS pa- rameters.Conclusion TUS is an effective method of gene delivery.The optimal conditions can enhance transfection efficien- cy without significant cell death and DNA damage.This noninvasive gene transfection method may be a useful tool for clini- cal gene therapy.更多还原