改良鱼精蛋白-脂质体-寡聚核苷酸复合体法转染卵巢癌SKOV3细胞

A Modified Method for Transfection to SKOV3 Cells with Protamine-DOT-AP/Chol Liposome-Oligodeoxynuleotides Complex

目的:优化鱼精蛋白-脂质体-寡聚核苷酸(oligodeoxynuleotides,ODN)复合体(liposome—protamine—ODN,LPD)法以提高SKOV3细胞的转染效率,并探讨该转染法对细胞体外增殖能力的影响。方法:参照文献设计核转录因子-κB(NF-κB)诱捕物ODN和对照物Scrambled ODN,并用FITC标记。LPD表面+/-电荷比例设计为1,2,4,6,并按转染液中是否含有小牛血清分成2组。用荧光光度计、流式细胞仪测定转染效率。用MTT法观察LPD法对SKOV3细胞在体外增殖的影响。结果:SKOV3细胞经LPD法转染,+/-电荷比例为4时转染效率最高,达96.41％,而无鱼精蛋白-脂质体的裸露ODN细胞转染效率为12.87％,两者差异有统计学上的高度显著性意义。培养液中10％小牛血清对转染效率无明显影响。结论:适宜电荷比例的LPD法是一种不受血清影响的高效率转染方法,有望用于体内转染。

Objective: To observe the transfer efficiency of protamine-DOTAP/Chol liposome-oligodeoxynuleotides complex to SKOV3 cells. Methods; Synthesized NF-κB decoy ODNs or scrambled ODNs labeled by FITC on the 5'ends was mixed with rationic DOTAP/cholesteral liposome and protamine, making the charge ratio of positive: negative 4. SK-OV3 cells were transfected by the complex (1 μmol/L) and the efficiency were measured by flow cytometry ( FCM) and fluorescence meter. The direct effect of NF-KB decoy ODNs transfer on the growth of SKOV3 cell line in vitro was measured by MTT method. Results: The efficiency of LPD transfer to SKOV3 cell line was 96. 41% , significantly higher than that of naked DNA transfer which was 12. 86%. The growth of SKOV3 cell line was not affected by 48h incubation after NF-KB decoy ODNs or scrambled ODNs transfection. Conclusion: LPD method is the one with high transfer efficiency not affected by NBS( newborn bovine serum) with possibility of use in vivo.