摘要
目的构建RNAi表达载体,并检验其对基因的抑制效果,从而在细胞中研究SURVIVIN基因的功能。方法(1)PCR扩增小鼠肝组织中的MU6启动子,并将其连接到TA载体中,构建带有MU6启动子的RNAi载体ps,并构建重组质粒psSURVIVIN和对照载体psN。(2)将psSURVIVIN及psN分别转染入HeLa细胞中,经过稳定筛选细胞,流式细胞术分析各组细胞周期及细胞凋亡率的影响因素,Western-blot和细胞免疫荧光检测两组细胞SUR-VIVIN蛋白质表达情况。(3)Caspase-Glo(tm)3/7 Assay Reagent检测两组HeLa细胞caspase 3/7的活性。结果(1)细胞凋亡率,psSURVIVIN组明显高于psN组与对照组;细胞阻滞在G0/G1期(P<0.01)。(2)在HeLa细胞中,转染带有MU6启动子的抑制Survivin基因表达的RNAi载体质粒(psSURVIVIN),Survivin基因表达明显降低(P<0.01),其抑制率在70%以上。(3)psSURVIVIN组caspase 3/7的活性明显增强(P<0.05)。结论构建的RNAi载体psSURVIVIN能有效抑制SURVIVIN基因的表达,caspase 3/7的活性明显增强。
Objective To investigate the effect of down regulation of SURVIVIN on cell and subsequent apoptosis in cervical cancer cells HeLa. Methods （ 1 ） The U6 promoter was obtained by PCR from liver of mouse and ligated into TA vector. RNAi vector（psSURVIVIN） and psN（ control vector） containing of the U6 promoter was established. （2）Using HeLa cells as a model system, two groups were set stably up transfected with RNAi control plamid and psSURVIVIN（SURVIVIN RNAi plasmid）, respectively. The expression of SURVIVIN in HeLa cells was measured by Western blot and immunofluorescence methods. （3）The activity of caspase 3/7 was detected using caspase-Glo（tm） 3/7 assay reagent. Results （1）Cell apoptotic rate was significantly increased by transfection with RNAi targeting plasmid, and cell was arrested at G0/G1 （P 〈 0. 01 ） ; （2） SURVIVIN expression was significantly decreased by transfection with RNAi targeting plasmid ; the expression proportion was reduced by about 70% （P 〈0. 01 ） ; （3）The activity of caspase 3/7 of psSURVIVIN group obviously increased （P 〈0. 01 ）. Conclusion These results showed that by inducing RNAi-targeting plasmid, SURVIVIN expression could be effectively decreased, moreover, the activity of caspase 3/7 of psSURVIVIN group was obviously increased.
出处
《基础医学与临床》
CSCD
北大核心
2009年第9期965-969,共5页
Basic and Clinical Medicine
