人甲状腺鳞癌细胞中端粒酶启动子功能分析

Function of hTERTp in SW579 cells

目的观察不同长度的人端粒酶催化亚单位(hTERT)的启动子对启动增强型绿色荧光蛋白(EGFP)能力的差异,评价启动子启动功能与长度的关系。方法克隆4段不同长度的端粒酶基因起始位点(ATG)上游序列长约2 068、1 135、333和142 bp的启动子片段,通过观察EGFP的荧光强度,确定启动效率。结果显微镜观察荧光亮度及荧光分光光度计检测荧光强度随着启动子长度的缩短而减弱,荧光强度由高至低依次为8.56,7.81,6.88,3.62。结论人端粒酶的表达强度及端粒酶的活性变化与其启动子的长度有关,而肿瘤细胞的端粒酶表达相对增强,可通过下调启动子活性,为肿瘤的靶向性基因治疗提供新思路。

Objective To clone DNA sequence of human telomerase reverse transcriptase promoter（hTERTp） and to explore the function of the promoter in promoting enhanced green flurescence protein（EGFP） and the relationship between promoter length and function.Methods The hTERT promoter of 2068bp,1 135 bp,333bp,142bp were amplified.The hTERT promoters were inserted into upstream of EGFP to reconstruct four recombinant plasmids and transfected into the SW579 cells.Then the fluorescence intensity was detected and the promoter rate was identified.Results The promote effiency of four eukaryotic plasmids decreased with the shortening of the promoter length with the fluoreslenic intensity of 8.56,7.81,6.88 and 3.62 from high to low.Conclusion The human telomerase reverse transcriptase expression and activity change are correlated with the length of the promoter.The research gives a way for tumor gene therapy by down regulation of the acitivity of hTERTp.