反义调控DNA甲基转移酶1基因对人胆管癌细胞生长的抑制效应  

Inhibitory effects of antisense regulating DNMT1 on growth of human cholangiocarcinoma cell line

作　　者：左石[1] 刘民锋[1] 罗剑[1] 徐立宁[1] 陈勇军[1] 唐启彬[2] 邹声泉[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院普外Ⅱ科,武汉市430030 [2]中山大学附属第二医院普通外科,广州市510120

出　　处：《中华肝胆外科杂志》2007年第3期196-199,共4页Chinese Journal of Hepatobiliary Surgery

基　　金：本课题受国家高技术研究发展计划（863计划）资助（项目编号：2002AA214061）

摘　　要：目的观察转染反义DNMT1基因真核表达载体对人胆管癌细胞QBC-939生长的影响，初步探讨DNMT1基因在胆管癌发生中的表遗传学机制。方法将构建好的反义DNMT1基因真核表达载体用脂质体介导法转入人胆管癌细胞QBC-939，Western blot检测转染前后DNMT1蛋白的表达变化，MTT法和软琼脂克隆形成试验观察各组细胞的生长增殖能力，流式细胞术观察细胞生长周期及凋亡率的变化。结果（1）Western blot检测证实转染反义基因能使DNMT1蛋白表达水平降低；（2）转染反义DNMT1基因能抑制QBC-939的生长曲线，并使其软琼脂克隆形成率从（38．020±4．120）％减少至（14．860±2．129）％，差异有显著性意义（P=0．000）；（3）转染反义DNMT1基因能影响QBC-939的细胞周期，使之阻滞于G1期，细胞凋亡率从（1．63±0．27）％增加到（6．19±0．78）％，差异亦有显著性意义（P=0．000）。结论通过转染反义DNMT1基因真核表达载体，可下调DNMT1在QBC-939细胞中的表达水平，并能抑制胆管癌细胞的生长和增殖，促进凋亡的发生，提示DNMT1可能通过甲基化途径与胆管癌的发生有关。Objective To study the effect of transfection with antisense DNMT1 gene eukaryotic expression plasmid on the growth of human cholangiocarcinoma cell line QBC-939 to explore the epigenetic mechanism of DNMT1 in pathogenesis of cholangiocarcinoma. Methods The constructed antisense DNMT1 gene eukaryotic expression plasmid was transfected into QBC-939 cells using liposome. The expression level of DNMT1 protein was detected by Western blotting after stable transfection. The growth curves of the transfected cells and un-transfected cells were observed by MTT. The cell proliferation ability was also monitored by the experiment of colony formation in soft agar. The alterations of the cell cycle and apoptotic rate were determined by FCM. Results 1） The level of DNMT1 significantly decreased after the transfection. 2） Transfection with antisense DNMT1 gene eukaryotic expression plasmid delayed the cell growth curve of QBC-939 and caused the cell colony formation rate to decrease from （38. 020 ±4.120） % to （ 14.860 ± 2. 129 ） %. There was significant difference betwean the transfection group and non-transfection group （P=0. 000）. 3） Transfection with antisense DNMT1 gene blocked the cell cycle at G1 phase and increased the apoptotic rate from （1.63±0. 27）% to （6. 19±0. 78）%. There was also marked difference between the transfection group and non-transfection group （P=0.000）. Conclusions Transfection with antisense DNMT1 gene eukaryotic expression plasmid can down-regulate the expression level of DNMT1. It can inhibit the growth and proliferation of human cholangiocarcinoma cell line QBC-939 and induce the cell apoptosis. DNMT1 gene might be related to the tumorigenesis in cholangiocarcinoma via DNA methylation pathway.

关 键 词：胆管肿瘤 DNA甲基转移酶1 反义RNA 细胞凋亡 DNA甲基化 

分 类 号：R686[医药卫生—骨科学]