E2F“诱骗”策略抑制膀胱出口梗阻后逼尿肌细胞的表型转化  

Inhibition on phenotypic transformation of detrusor smooth muscle cells after bladder outlet obstruction by E2F decoy strategy

作　　者：龚宇[1] 杜传军[1] 丁砺蠡[2] 宋波[2] 金锡御[2] 

机构地区：[1]浙江大学医学院第二附属医院泌尿外科,杭州310009 [2]第三军医大学第一附属医院全军泌尿外科研究所

出　　处：《中华医学杂志》2007年第12期829-832,共4页National Medical Journal of China

基　　金：国家自然科学基金(30300350);浙江省医学会临床科研基金(2006ZYC06)

摘　　要：目的利用自建的膀胱出口梗阻(BOO)细胞水平的模型,探讨转录因子 E2F"诱骗"(decoy)策略抑制逼尿肌细胞(DSMC)的表型转化作用。方法对培养的 DSMC 施加周期性张力负荷以建立细胞水平的 BOO 模型;以 Lipofectamine 2000介导 E2F-decoy 脱氧寡核苷酸(ODN)转染DSMC,设立 E2F-decoy ODN 转染组(Decoy 组),错配 E2F-decoy ODN 转染组(Mis-decoy 组)及空白对照组(非转染组);四甲基偶氮唑盐比色法(MTT)测定细胞的增殖活性,逆转录-聚合酶链反应(RT-PCR)分析增殖细胞核抗原(PCNA)mRNA 表达水平,Western 印迹检测 PCNA 和细胞周期依赖性蛋白激酶cdk2表达。结果 Lipofectamine 介导转染 E2F-decoy ODN 后24 h 可获得表达;Decoy 组增殖活性显著低于非转染组和 Mis-decoy 组(均 P<0.01);Decoy 组 PCNA mRNA 表达显著低于非转染组(P<0.01);Decoy 组 PCNA 和 cdk2蛋白表达显著低于非转染组(126±14 vs 180±10;155±6 vs210±22,均 P<0.01);而 Mis-decoy 组与非转染组之间以上指标差异均无统计学意义(均 P>0.05)。结论 E2F-decoy ODN 能够转染 DSMC 并获得表达;转录因子 E2F"诱骗"策略有效抑制了 BOO 后DSMC 的表型转化,显示了从逼尿肌结构的角度改善梗阻后膀胱功能的前景。Objective To investigate the inhibitory effect of E2F decoy strategy on the phenotypic transformation of detrusor smooth muscle cells （DSMCs） so as to verify the effect of the E2F decoy strategy in improvrmrnt of the bladder function after bladder outlet obstruction （BOO）. Methods Rat DSMCs were cultured, underwent cyclic mechanical stretch so as to establish BOO model, and then were divided into 3 groups： E2F-ODN decoy group [ transfected with E2F-decoy ODN tagged with carboxy-fluorescein （FAM）, a at its 3＇ end], Mis-decoy group （trnsfected with mismatch E2F-decoy ODN）, and control group （without transfection）. Inverted fluorescence microscopy was used to detect the green fluorescence of FAM in the successfully transfected cells. The proliferation of the cells was observed by MTT method. RT-PCR was used to examine the mRNA exprerssion of proliferating cell nuclear antigen （PCNA）. Western blotting was used to detect the protein expression of PCNA and cdk2 kinase. Results FAM-labeled E2F-ODN was detected stably in the DSMCs of the E2F-ODN decoy group 24 hours after transfection. The proliferation of the DSMCs of the E2F-ODN decoy group was decreased significantly compared with the mismatch E2F-ODN decoy and control groups （ both P 〈 0.01 ）. The mRNA expression of PCNA, protein expression of PCNA and cdk2 kinase of the E2F-ODN decoy group were all significantly lower than those of the other 2 groups （ all P 〈 0. 01 ）. Conclusion E2F-decoy ODN can be transfected and stably expressed in DSMCs. The phenotypic transformation of DSMCs can be successfully inhibited by E2F decoy strategy, which clarifies the potential role of structural stability-based method on improvement of bladder function recovery after BOO.

关 键 词：膀胱颈梗阻 表型 转录因子 逼尿肌 

分 类 号：R694[医药卫生—泌尿科学]