反义人端粒酶RNA亚基对胆囊癌端粒酶活性及细胞生长的抑制作用

Effect of antisense RNA for hTR on telomerase activity and cell apoptosis in human gallbladder cancer

目的 探讨反义RNA技术对胆囊癌细胞端粒酶活性的影响及对胆囊癌细胞生长增殖的作用。方法 根据测定的胆囊癌人端粒酶RNA亚基 (hTR)基因的序列结果 ,并根据真核表达载体多克隆酶切位点的物理图谱 ,体外合成反义RNA及正义RNA的基因序列 ,并构建入 pTriEx 4真核表达载体 ,酶切鉴定正确后 ,采用脂质转染法导入胆囊癌细胞。TRAP法检测端粒酶活性 ,流式细胞仪检测细胞周期及凋亡情况 ,电镜观察细胞微观形态变化。结果 实验组胆囊癌细胞的端粒酶活性较对照组明显减低 ,正义组、转染空载体及单纯脂质体转染的细胞端粒酶活性与未转染细胞比较则变化不明显。反义RNA基因作用后 ,G0 /G1期细胞比例明显增高 ,S期细胞比例明显降低。细胞的分裂、增殖受到明显抑制。反义RNA基因转化后 ,10d凋亡率为 11.10 %。 13d后凋亡率为 2 9.0 2 %。电镜下可见明显凋亡细胞。结论 反义RNA技术对胆囊癌细胞端粒酶活性有明显的抑制作用 ;并抑制胆囊癌细胞的生长 ,促进细胞的凋亡 ;且克服了反义寡核苷酸作用时间短的缺点。

Objective To explore the effect of antisense telomerase RNA on telomerase activity and cell apoptosis in human gallbladder cancer.Methods According to the hTR cDNA sequence and multiclone site's physical map of eukaryon express vector,the antisense RNA and sense RNA sequence were synthesized in vitro and transduced into the eukaryon expression vector pTriEx 4 separately.By using the method of Lipofectamine transfection,the carcinoma of gallbladder was transfected.Telomerase activity of GBC SD cells was examined by telomeric repeat amplification protocol (TRAP).Apoptosis was detected by morphology and flow cytometry.Results Antisense RNA could inhibit telomerase activity and cell proliferation of GBC SD cell.After antisense RNA transfection,the cell ratio of G 0/G 1 phase was increased obviously,and the cell ratio of S phase decreased obviously.The apoptosis rate was 11.10% and 29.02% on the day 10 and 13 after antisense RNA transfection.Conclusion Antisense RNA can inhibit telomerase activity of GBC SD cell effectively and induce apoptosis.