超声微泡联合脂质体介导双自杀基因杀伤MCF-7细胞的实验研究

Study of ultrasound microbubbles combined with liposome transecting double suicide gene into MCF-7 cells and its killing effects

目的探讨超声辐照微泡联合脂质体介导双自杀基因(CD/TK)对MCF-7细胞的体外杀伤作用。方法将培养的MCF-7细胞分为5组:裸质粒组、脂质体组、超声辐照微泡组、超声辐照脂质体组、超声辐照微泡联合脂质体组。转染pEGFP-KDRp-CD/TK质粒于MCF-7细胞,用荧光显微镜及流式细胞仪检测转染效率。转染后,再分为空白对照组、未转染组、转染组;未转染组和转染组又各分为3个亚组,给予前药丙氧鸟苷(GCV)、5-氟胞嘧啶(5-Fc)、GCV+5-Fc。四甲基偶氮唑盐(MTT)法检测双自杀基因对MCF-7细胞的体外杀伤作用。结果超声辐照微泡联合脂质体组绿色荧光蛋白(GFP)表达最多、最强。超声辐照微泡联合脂质体组转染率(39.59%±1.19%)最高(P<0.05)。MTT检测细胞抑制率结果显示,转染组细胞抑制率明显高于未转染组(P<0.01),转染组中联合用药组细胞抑制率明显高于单一用药组(90.77%±2.68%vs 64.75%±2.27%、67.81%±2.43%;P<0.05)。转染组各前药组细胞抑制率均显著高于超声辐照微泡联合脂质体对MCF-7细胞的转染率(P<0.05)。结论超声辐照微泡联合脂质体能明显提高基因转染效率,是较理想的乳腺癌基因治疗策略。

Objective To transfect the pEGFP-KDRp-CD/TK into MCF-7 cells in vitro by ultrasound mediated microbubble destruction combined with liposome, and investigate the transfection efficiency and the killing effect after treatment with the prodrugs. Methods The MCF-7 ceils were divided into 5 groups, ①naked plasmid group, ②liposome group, ③ultrasound irradiated microbubble group, ④ultrasound irradiated liposome group, ⑤ultrasound irradiated microbubble ＋ liposome group. The transferction efficiency of pEGFP-KDRp-CD/TK in MCF-7 cells was detected by fluorescence microscope and flow cytometry. After transfection, then divided into control, untransfected and transfected groups, each untransfected or transfected group divided into 3 subgroups given prodrugs of gancyclovir（GCV）, 5-flucylosine（5-Fc） or combination of GCV and 5-Fc. The killing effect of double suicide gene on MCF-7 cells treated with gancyclovir（GCV）, 5-flucylosine（5-Fc） or combination of GCV and 5-Fc were determined by MTI＂ method. Results The expression of green fluorescent protein（GFP） in the fifth group was the strongest when observed with fluorescence microscope. The results of flow cytometry showed that the transferction efficiency of the fifth group was the highest （39.59 % ± 1.19 %） compared with the other groups（P 〈 0.05）, and that of transfected MCF-7 cells treated with GCV and 5-Fc was obviously higher than the single prodrug group GCV or 5-Fc（90.77 % ± 2.68 % vs 64.75 % ± 2.27 %, 67.81% ± 2.43 %; P 〈 0.05）. Every inhibition ratio of transected groups was obviously higher than the transfection efficiency of MCF -7 cells （P 〈 0.05）. Conclusion It is demonstrated that ultrasound mediated microbubble destruction combined with liposome can enhance the efficiency of gene transfection. Ultrasound mediated microbubble destruction combined with liposome transfecting double suicide gene is an ideal strategy for breast cancer gene therapy.