人端粒酶hTERT可诱导RNAi系统的建立及其对TREx-HeLa细胞生长的影响

Construct tetracycline-inducible H1 RNAi system of hTERT and its function in TREx-HeLa cell line

目的:建立可诱导性沉默hTERT的RNA干扰系统,并初步探讨端粒酶与细胞增殖调控之间的关系。方法:构建沉默端粒酶催化亚基hTERT的Tet-on可诱导性RNAi载体psiRNA-hH1/TO-hTERTshRNA,并稳定转染表达TR的TREx-HeLa细胞,用半定量RT-PCR检测hTERT的转录后水平,TRAP法检测端粒酶活性。MTT法检测细胞的增殖,流式细胞仪和Hoechst33342染色法观察细胞凋亡。结果:构建了可诱导性沉默端粒酶催化亚基hTERT的重组体psiRNA-hH1/TO-hTERTshRNA,并建立了可诱导性沉默hTERT的TREx-HeLa细胞株。短期内的端粒酶活性降低后TREx-HeLa细胞生长增殖有下降趋势,而其凋亡情况则未见明显变化。结论:本研究建立了可诱导沉默hTERT的RNAi系统,为研究端粒酶活性与细胞增殖和衰老的关系提供了实验基础。

AIM ： To construct a tetracycline - inducible H1 RNAi system of hTERT and to study the apoptosis rates and survival rates of tetracycline- inducible hTERT RNAi cell line. METHODS： The tetracycline -inducible H1 RNAi system for hTERT was constructed and transfected into TREx - HeLa cell line. The inhibitory effects were determined by RT - PCR and TRAP. The proliferation of the cell line in two groups was assayed by MTT test. The apoptotic rates were estimated by using Hoechst33342 and FACS. RESULTS： Tetracycline - inducible H1 RNAi system of hTERT was constructed successfully and TREx - HeLa cell induciblely interfered with hTERT was cultured successfully. As to the different cell lines, different influences after telomerase activity were declined. For TREx - HeLa cell, declined telomerase activity reduced proliferation but had no remarkable influence in apoptosis during short time. CONCLUSION： Tetracycline - inducible H1 RNAi system of hTERT and the assay of the proliferation and apoptosis of the TREx - HeLa cell provide the basis for further research.