超声微泡介导体外转染hAng-1基因最佳条件的初步探讨

目的 探讨SonoVue微泡携带人血管生成素-1(hAng-1)基因在体外转染时超声照射强度、微泡浓度、DNA浓度等对转染效率和细胞活性的影响,以确定最佳转染条件.方法 将连接有eGFP-C_3-hAng-1质粒的SonoVue微泡以不同方式与293 T细胞混合,在超声照射下进行基因转染.分别检测不同超声照射强度、照射时间、不同微泡浓度和DNA浓度下的基因转染效率和细胞存活率.结果 随着超声照射强度、照射时间、微泡浓度和DNA浓度的增加,基因转染效率显著增加,细胞存活率在90%以上;当照射强度达到1.5 W/cm~2以上,照射时间超过30 s,微泡浓度和DNA浓度分别达到20%和15 mg/L后hAng-1基因的转染效率不再显著升高,而细胞存活率显著下降(P<0.01).结论 SonoVue微泡体外转染hAng-1基因的最佳条件为以细胞贴壁方式混合载基因微泡.照射强度1.5 W/cm~2,照射时间30 s,微泡浓度和DNA浓度分别为20%和15 mg/L.

The primary study of optimization parameters of ultrasonic microbubbles delivery hAng -1 gene into 2 9 3 T cells in vitro

Objective To certificate the effects on transfection ratio and cells viability of ultrasound (US) acoustic intensity, radiation duration, microbubbles concentration and DNA concentration in delivery human angiopioetin-1 gene (hAng-1) into 293T cells by SonoVue microbubbles and decide the optimal transfection parameters. Methods Mix 293T cells and SonoVue microbubbles linked with eGFP-C_3-hAng-1 in a different way, detect the gene transfection ratio and cells viability under the various US intensity, radiation duration, microbubbles and DNA concentrations. Results The gene expression would be increased if enhanced the intenstiy of US,radiation time,microbubbles and DNA concentrations,and the cells viability would be kept more than 90% ( P <0. 01). Whereas,if the US intensity increased over 1. 5 W/cm~2 ,the duration over 30 s and microbubbles and DNA concentrations over 20% and 15 mg/L respectively,the gene expression would not increase significantly ( P > 0. 05),whereas coupled with obviously decreased cells viability( P <0. 01). Conclusions The optimal conditions of deliver hAng-1 gene into 293T cells by SonoVue microbubbles was mixing cells and microbubbles in a cell wall-sticky way,US intensity was 1. 5 W/cm~2, duration 30 s,20% microbubbles and 15 mg/L DNA concentration.