【摘要】 目的对超声促进基因转染的方法进行系统的优化研究,初步确定超声介导微泡破裂(UMMD)增强体外基因转染的最优参数。方法选用Ishikawa、Hela和MCF-73种细胞系为研究对象,用1MHz超声仪,超声强度为1.0W/cm2,系统研究不同参数下的细胞活力及两种DNA质粒[红色荧光蛋白质粒(DsRed)和荧光素酶质粒(pCMV-LUC)]的基因转染情况,优化UMMD的转染条件(质粒浓度、占空比及辐照时间),分析SonoVue微泡对基因转染的增强作用。结果基因转染率随着质粒浓度的增加而增高,当质粒浓度达到30μg/孔时转染率最高,两种DNA质粒的最佳转染浓度相同。与10%占空比的超声辐照相比,20%占空比的转染率显著提高(P<0.01)。辐照3min时基因表达率最高,但存活率无明显下降(89.03±2.01)%,为最佳辐照时间。无超声辐照时,单独应用质粒或微泡+质粒的样本几乎不表达红色荧光蛋白。与单纯超声辐照相比,超声辐照联合SonoVue微泡可显著提高基因转染效率(P<0.01)。结论UMMD的转染参数影响转染效率和细胞活力,优化的参数有利于促进基因转染。更多还原

【Abstract】 Objective To perform the systematic optimization study on ultrasound facilitating gene transfection to cells,and to establish the optimal parameters of ultrasound-mediated microbubble destruction (UMMD) enhanced gene transfection in vitro. Methods The influence of parameters on transfection efficiency and cell viability of three cell lines (Ishikawa, Hela and MCF-7) by two different DNA plasmids, DsRed and luciferase recombinant plasmid (pCMV-LUC) was studied. Ultrasound was generated by a 1 MHz ultrasound machine. The intensity of ultrasound was 1.0 W/cm2. Varied parameters, such as DNA concentration, duty cycle and the duration of exposure were systematically observed in order to optimize gene transfection, and the enhancement of SonoVue microbubble was analyzed as well. Results The increase of transfection efficiency related to plasmid concentration and saturated when DNA concentration reached 30 μg/well. The optimal concentration was the same for the two different DNA plasmids. Transfection efficiency of 20% duty cycle increased significantly compared with that of 10% duty cycle (P<0.01). Exposure 3 min was used to optimize experiments to obtain the highest expression efficiency without obvious decreasing of survival rate (89.03±2.01)%. Plasmid alone or plasmid plus microbubbles barely expressed red fluorescent protein. Compared with the ultrasound irradiation alone, ultrasound irradiation associated with SonoVue microbubble significantly increased transfection efficiency of optimal UMMD (P<0.01). Conclusion The varying parameters of UMMD have effectiveness on both transfection efficiency and cell viability. Optimal parameters may facilitate gene transfection.更多还原