超声联合微泡增强脂质体介导基因转染RPE细胞的实验研究

摘要

目的 比较声诺维联合超声与脂质体法介导基因转染RPE细胞的转染率和安全性,探讨超声联合微泡增强脂质体转染率的可行性.方法 RPE细胞培养在24孔板,质粒用量2 μg/孔,分为对照组、超声辐照组、超声联合微泡组(包括2 W/cm2和3 W/cm2两个亚组)、脂质体组、脂质体+超声+微泡组5组,每组10孔细胞.荧光显微镜观察基因转染情况,流式细胞仪检测基因转染率,Annexin Ⅴ-FITC和PI双染流式细胞法检测凋亡率.结果 单纯超声辐照组基因转染率为(1.06±0.13)%,超声联合微泡组分别为(15.81±1.70)%和(20.86±2.63)%,脂质体组为(30.06±3.49)%,明显高于超声联合微泡组(P<0.05).脂质体+超声+微泡组为(44.51±1.28)%,明显高于脂质体组(P<0.05).结论 脂质体介导EGFP转染RPE细胞的效率高于超声联合微泡的转染率.超声联合微泡能明显提高脂质体的转染率.%Objective To compare the transfection rate and safety between SonoVue + ultrasound and liposome mediated gene transfection to retinal pigment epithelium (RPE) in vitro, and to approach the feasibility of SonoVue and ultrasound enhancing liposome mediated gene transfection to RPE in vitro. Methods RPE were cultured in 24 well dishes with green fluorescent protein plasmid of 2 μg/well. All the cells were divided into 5 groups: control group, ultrasound irradiance group, SonoVue + ultrasound irradiance group(2 W/cm2 and 3 W/cm2),liposome group and liposome + SonoVue + ultrasound irradiance group. There were 10 wells in every group. Gene transfection results were observed under inversion fluorescent microscope. Seventy-two hours later, gene transfer was guantified under flow cytometry. The apoptosis rates of RPE were determined with Annexin Ⅴ-FITC and PI staining by flow cytometry. Results The transfection rate of ultrasound exposure group was (1.06 ± 0.13)%. The transfection rate of SonoVue + ultrasound group was (15.81 ± 1.70)% and (20.86 ± 2.63)%. The transfection rate of liposome group was (30.06 ± 3.49)% ,and the differences between above mentioned sets were significant(P<0.05). The transfection rate of liposome + SonoVue + ultrasound was (44.51 ± 1.28) %,and the differences between above mentioned two sets were significant (P<0.05). Conclusions The transfection rate of liposome is higher than that of SonoVue + ultrasound group. SonoVue and ultrasound can obviously enhance liposome mediated EGFP gene transfection to RPE in vitro.