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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 phenotypic transformation of detrusor cells (DSMC) induced by the transcription factor E2F decoy strategy using a model of self-built bladder outlet obstruction (BOO) cells. Methods The cultured DSMCs were subjected to cyclic tension loading to establish the BOO model at the cellular level. The E2F-decoy ODN transfection group (Decoy group) was established by Lipofectamine 2000-mediated E2F-decoy deoxynucleotide (ODN) , Mismatched E2F-decoy ODN transfected group (Mis-decoy group) and blank control group (untransfected group). MTT assay was used to determine the cell proliferation, reverse transcription-polymerase chain reaction The expression of proliferating cell nuclear antigen (PCNA) mRNA was analyzed by enzyme linked immunosorbent assay (RT-PCR), and the expression of PCNA and cdk2 protein was detected by Western blotting. Results After transfected with E2F-decoy ODN, Lipofectamine could be expressed at 24 h. The proliferation of Decoy group was significantly lower than that of non-transfected group and Mis-decoy group (all P <0.01). The expression of PCNA mRNA in Decoy group was significantly lower than that in non- (P <0.01). The expressions of PCNA and cdk2 protein in Decoy group were significantly lower than those in non-transfected group (126 ± 14 vs 180 ± 10; 155 ± 6 vs 210 ± 22, all P <0.01) There was no significant difference in the above indexes between non-transfected groups (all P> 0.05). Conclusion E2F-decoy ODN can be transfected into DSMC and obtain its expression. The transcription factor E2F “decoy” strategy effectively inhibits the phenotype of DSMC after BOO, which shows the prospect of improving bladder function after obstruction from detrusor structure.