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

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。

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

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.