ShRNA沉默基因DNA甲基转移酶3b的表达对膀胱癌T24细胞增殖的影响  

Effect of silencing gene DNA methyltransferase 3 b expression by shRNA on proliferation of bladder cancer T24 cells

作　　者：张士龙[1] 曾甫清[1] 董继华[2] 朱朝辉[1] 廖贵益[1] 彭世波[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院泌尿外科,湖北武汉430022 [2]华中科技大学同济医学院附属协和医院中心实验室

出　　处：《中国医师杂志》2008年第9期1156-1159,共4页Journal of Chinese Physician

基　　金：湖北省科技公关计划项臼（2006AA301B56-1）

摘　　要：目的体外研究重组质粒pshRNA-DNMT3b对膀胱癌T24细胞中DNMT3bmRNA和蛋白的沉默效应以及细胞增殖抑制的影响，初步探讨DNMT3b在膀胱肿瘤发生过程中的作用。方法实验分为空白对照组、HK组及pshRNA-DNMT3b组（24、48、72h）；常规培养T24细胞，用脂质体lipofectamine2000将重组质粒pshRNA-DNMT3b转染进入T24细胞，后进行RT．PCR、WesternBlot、MTY检测，观察pshRNA-DNMT3b对T24细胞中DNMT3bmRNA、蛋白质水平变化以及细胞增殖的影响。结果重组质粒pshRNA-DNMT3b成功的转染进入T24细胞；RT-PCR电泳结果用Gel-proanalyzer图像分析系统进行灰度分析，空白对照组、HK组、pshRNA-DNMT3b组（24、48、72h）条带的IOD相对比值（DNMT3／β-actin）分别是（99．56±1．24）％、（99．12±1．35）％、（75．77±1．42）％、（44．69±1．05）％和（20．52±0．89）％；Westernblot图像分析结果各组的相对比值分别是（99．43±1．28）％、（98．90±1．31）％、（67．83±1．02）％、（43．43±1．05）％和（21．92±0．89）％；在MTF检测中空白对照组和HK组之间差异无统计学意义（P〉0．05），pshRNA-DNMT3b组仅在72h后和前2组之间差异有统计学意义（P〈0．01），但抑制率仅增加0．45％左右。结论重组质粒pshRNA-DNMT3b能有效的降低膀胱癌1、24细胞中基因DNMT3bmRNA的转录及蛋白的表达，但在抑制细胞增殖上作用甚微。Objective To investigate the effect of recombinant plasmid pshRNA-DNMT3b on expression of DNMT3b mRNA and protein and on the proliferation of bladder cancer T24 cells, and research the function of DNMT3b in the process of bladder tumor formation. Methods There were three groups in this study, which are blank controller, HK and pshRNA-DNMT3b （24h,48h,72h） , respectively. T24 cells were cultured routinely and transfected by the recombinant plasmids with lipfectamine 2000. The cells were detected by methods of RT-PCR, western blot and MTF. The varying level of DNMT3b mRNA and expression protein, and the conditions of cellular survival rate were observed. Results The recombinant plasmids were successfully transfected into T24 cell lines. The grey value of RT-PCR elctrophoretogram was analyzed by the software of Gel-pro analyzer, the rate of blank controller, HK and pshRNA-DNMT3b （24h, 48h, 72h）, was （99. 56 ± 1.24） %, （99. 12±1.35 ） %, （75.77 ± 1.42） %, （44.69±1.05 ） % and （ 20. 52 ±0. 89） %, respectively. The analytical result of western blot image was （99.43 ±1.28） %, （98.90 ± 1.31 ） %, （67.83 ± 1.02） %, （43.43 ± 1.05 ） % and （21.92± 0. 89） %. There was no statistically difference in survival between blank control and HK （ P 〉 0.05 ）. The group of pshRNA-DNMT3b and other two groups had statistical difference only at the 72th hour and the cell inhibitory growth rate only increase 0. 45%. Conclusions The recombinant plasmid pshRNA-DNMT3b can inhibit the expression of mRNA and protein of DNMT3b effectively. However, it has slight function on inhibiting cell proliferation.

关 键 词：DNA(胞嘧啶-5-)-甲基转移酶 RNA干扰 膀胱肿瘤 

分 类 号：R737[医药卫生—肿瘤]