反义寡核苷酸转染A375细胞实验条件的优化  

Experimental studies of optimized transfection condition on human malignant melanoma cell by survivin antisense oligonucleotide

作　　者：王梅[1] 王冰[2] 谭升顺[1] 

机构地区：[1]西安交通大学医学院第二附属医院皮肤科,陕西西安710004 [2]西安交通大学医学院病理系,陕西西安710061

出　　处：《西安交通大学学报（医学版）》2007年第5期502-505,510,共5页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：陕西省科技攻关基金资助项目(No.2003K10-G10)

摘　　要：目的研究不同的转染条件下,Survivin反义寡核苷酸(ASODN)对体外培养的人恶性黑色素瘤细胞A375生长的影响。方法通过对ASODN进行硫代修饰、脂质体运转及培养条件的改变,用倒置显微镜、台盼蓝计数观察Survivin ASODN对A375细胞增殖的抑制作用。结果硫代修饰ASODN经脂质体包裹对细胞生长抑制作用明显强于未经脂质体包裹组(P<0.01);②细胞培养6 h贴壁后进行转染对细胞的生长抑制率明显高于24 h后进行转染(P<0.05);③转染期间(4 h)无血清存在组与相应对照组比较差异有显著性意义(P<0.01);④ASODN转染后12-72 h,对细胞的抑制效应逐渐升高,72 h达顶峰,之后开始回落;⑤与混杂组(SODN)及错配组(MODN)相比,反义组在各个浓度对细胞生长抑制作用最明显,各浓度组比较差异有统计学意义(P<0.05)。结论许多因素影响ASODN的转运,不同的实验应选择不同的实验优化条件。Objective To observe effects of survivin antisense oligonucleotide on proliferation of human malignant melanoma cell in vitro in different transfection condition. Methods By using trypan blue exclusion method, this study screened optimized transfection condition that inhibited A375 cell viability. Results ① By the phosphorothiote-modification and the liposome-encapsulation, there were significant differences in ASODN with ASODNL. ASODNL were stronger in inhibiting A375 cell proliferation （P〈0.01）. ② 6 hours after plating, antisense oligonucleotide transfected A375 cell, growth suppression rate of cells was higher than 24h. ③ During 4h transfection, compared to the serum-present group, the serum-free group had greater effect on the cell growth （P〈 0.01）. ④ The inhibitory effect of ASODN gradually rose at 12-72 h after transfection, 72 h reached the peak, and after that the inhibitory rate began to fall. ⑤ Compared with MODN and SODN, ASODN at distinct concentration had the highest inhibition on the cell growth, and there were significant differences in different groups （P〈0.01）. Conclusion Many factors influenced antisense oligonucleotide uptake in vitro. Different experiment should select different optimal transfection condition.

关 键 词：SURVIVIN 恶性黑色素瘤 反义寡核苷酸 脂质体 细胞培养 

分 类 号：R739.5[医药卫生—肿瘤]