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目的 构建针对人端粒酶催化亚单位 (humantelomerasecatalyticsubunit,hTERT)基因的小分子干扰RNA(smallinterferingRNA ,siRNA)表达载体pU6 hTERT siRNAs,观察其对胃癌SGC790 1细胞hTERT基因的特异性抑制作用。方法 采用报告基因质粒pCX GFP( 5 5 10bp)和含有鼠U6启动子pU6 ( 330 0bp)质粒 ,设计合成针对绿色荧光蛋白 (GFP)基因的发夹RNA(shRNA)序列 ,构建SHi pU6 GFP质粒。应用LipofectamineTM2 0 0 0将pCX GFP质粒和SHi pU6 GFP质粒转染K5 6 2细胞、SGC790 1细胞 ,并用荧光显微镜观察转染效果。设计合成针对hTERT的siRNAs ,构建重组pU6 hTERT siRNAs质粒。LipofectamineTM2 0 0 0介导pU6 hTERT siRNAs质粒转染SGC790 1细胞。分别应用逆转录聚合酶链反应 (RT PCR)和荧光定量聚合酶链式反应 (FQ PCR)定性和定量检测hTERT基因表达情况。结果 质粒pU6和SHi pU6 GFP经EcoRⅤ和XbaⅠ酶切电泳后 ,前者出现 330 0bp和约 30 0bp条带 ,而后者出现 330 0bp和约 35 0bp条带 ,与实验设计的针对GFP的shRNA长度相符。说明本实验已成功构建了针对绿色荧光蛋白GFP基因的SHi pU6 GFP质粒。K5 6 2细胞和SGC790 1细胞中分别观察到转染pCX GFP质粒和SHi pU6 GFP质粒前后发绿色荧光的细胞数目明显减少。定性、定
OBJECTIVE: To construct a siRNA targeting small interfering RNA (siRNA) targeting human telomerase catalytic subunit (hTERT) gene, pU6 hTERT siRNAs, and observe its specific inhibitory effect on hTERT gene of gastric cancer SGC7901 cells. Methods SHi pU6 GFP plasmids were designed and synthesized by using the reporter gene plasmid pCX GFP (5 5 10bp) and the mouse U6 promoter pU6 (330 0 bp) plasmid. The pCX GFP plasmid and the SHi pU6 GFP plasmid were transfected into K562 cells and SGC7901 cells using LipofectamineTM 2000, and the transfection efficiency was observed under a fluorescence microscope. Design and synthesis of siRNA targeting hTERT and construction of recombinant pU6 hTERT siRNAs plasmid. LipofectamineTM2 0 0 0 mediated pU6 hTERT siRNAs plasmid transfected SGC790 1 cells. Reverse transcriptase polymerase chain reaction (RT PCR) and fluorescent quantitative polymerase chain reaction (FQ PCR) were used to detect the expression of hTERT gene. Results After digestion with EcoRV and XbaI, the plasmids pU6 and SHi pU6 GFP showed 330 0 bp and 30 0 bp bands in the former and 330 bp and 35 bp bands in the latter, which was consistent with the length of GFP-designed shRNA. This experiment has successfully constructed SHi pU6 GFP plasmid targeting the green fluorescent protein GFP gene. K562 cells and SGC7901 cells were observed in the transfected pCX GFP plasmid and SHi pU6 GFP plasmid before and after the number of green fluorescent cells decreased significantly. Qualitative, fixed