中文摘要：

目的构建靶向肿瘤坏死因子转换酶（TACE）的小发夹结构RNA（shRNA）的真核表达载体,观察干扰TA-CE表达对HeLa细胞增殖和凋亡的影响。方法设计针对TACE基因的4个shRNA序列,构建真核表达载体shR-NA1、shRNA2、shRNA3、shRNA4。通过酶切和测序等方法进行鉴定。转染HeLa细胞后,用Realtime PCR的方法检测其对HeLa细胞TACE基因表达的影响;用ELISA检测细胞分泌的sTNF-α,间接反映干扰TACE的效果;用MTT法检测细胞增殖活性;用DNA ladder和流式细胞术检测细胞凋亡。结果成功构建和筛选出了针对TACE基因的3个有效的特异性真核表达载体,且均在不同程度上抑制了HeLa细胞TACE基因的表达。ELISA检测结果与Realtime PCR实验结果相符。同时发现干扰TACE基因后,HeLa细胞的生长受到抑制,凋亡率升高,与对照组相比差异有显著性意义（P〈0.05）。实验组转染shRNA1~3后72h可见特征性的基因组DNA ladder条带。结论所构建的TACEshR-NA真核表达载体能显著抑制TACE的表达。干扰TACE基因的表达可有效抑制HeLa细胞的增殖并诱导其凋亡。

英文摘要：

Objective To construct the shRNA eukaryotic expression vectors of TACE gene and to investigate the effects of TACE expression interference on proliferation and apoptosis of transfected HeLa cells in vitro. Methods Four specific shRNAs of TACE gene were designed, and cloned into the pGenesil-1, which was proved by the restriction map and the sequence analysis. Transient transfection into HeLa cells via Lipofectamine 2000 was conducted. Real time quantitative PCR was used to detect the TACE mRNA expression. The sTNF-α was tested by ELISA kit to reflect indirectly the inhibitory effect of the vectors. The cell growth was assessed by MTT assay and the apoptosis was evaluated by flow cytometry and DNA ladder. Results Three specific shRNA eukaryotic expression vectors （shRNA1, shRNA2, shRNA3） targeting TACE were successfully produced. All of these could remarkably inhibit the expression of TACE to varying degrees. The results of ELISA also supported the data of real-time quantitative PCR. Upon shRNA vector transfection, HeLa cell growth was also inhibited significantly （P 〈0.05）, and apoptotic rate increased remarkably. Genomic DNA agarose gel electrophoresis showed ＂Ladder＂ bands 72 h after transfection. Conclusion TACE targeting shRNA silenced the TACE gene remarkably, and induced HeLa cell growth inhibition and apoptosis.