利用RNAi阻抑hTERT和Bi-1双基因表达的RNAi作用效果的研究

The effect of RNA interference induced by inhibition of hTERT and Bi-1 gene expression

目的利用LipofectamineTM2000将针对靶向人类端粒酶末端逆转录酶(hTERT)和Bax inhibitor-1(Bi-1)基因设计并构建的质粒载体转染至CNE-2Z鼻咽癌细胞内,诱导序列特异性的基因沉默,研究其产生的shRNA阻抑hTERT和Bi-1基因表达的效果。方法收集CNE-2Z细胞,设未处理组、pEGFP-N1组、pEGFP-N1/Lip组,采用流式细胞术检测Lip对CNE-2Z细胞的转染能力,RT-PCR和Western blot法分析表达shRNA重组质粒载体对hTERT和Bi-1基因mRNA表达的抑制效应。结果转染CNE-2Z细胞的质粒和Lip最佳组合:质粒为2.5μg,Lip为6.25μL。结论成功构建的针对人hTERT和Bi-1基因的shRNA真核表达质粒能特异、有效地阻抑hTERT和Bi-1基因的表达。

Objective In this study ,we constructed a series of recombinant plasmids carriers expressing shRNA targeting hTERT and Bi‐1 gene .These recombinant plasmids carriers were transfected into CNE‐2Z cell lines using Lip and continuously in‐duced the expression of shRNAs .Furthermore ,the shRNAs caused the degradation of mRNAs homologous in sequence with the target genes ,which lead to a sequence‐specific gene silencing .Methods The CNE‐2Z cells was divided into untreated group ,pEG‐FP‐N1 group and pEGFP‐N1/Lip group .Flow cytometry（FCM ） was applied to determine the transfection efficiency .The changes of hTERT and Bi‐1 gene expression were detected by Real‐time RT‐PCR and Western blotting .Results The best transfection effi‐ciency between plasmid and Lip was 2 .5 μg plasmid and 6 .25μL Lip .Conclusion We constructed several shRNA recombinant eu‐karyotic expression plasmids successfully .The recombinant plasmid can inhibit the expression of hTERT and Bi‐1 gene specifically and effectively .