shRNA沉默VEGF基因表达对大肠癌细胞生物学特性的影响要点解析.doc

shRNA沉默VEGF基因表达对大肠癌细胞生物学特性的影响 　　　 提　要:目的　观察Pavu6+27-VEGF siRNA重组体在细胞体内表达的VEGF短发夹状RNA(short hairpin RNA, shRNA)对人大肠癌HCT116细胞株生物学特性的影响。方法　将自行构建的表达短发夹状RNA的重组质粒转染到大肠 癌HCT116细胞株中,以空质粒Pavu6+27转染为对照,经G418筛选出稳定表达shRNA细胞,行RT-PCR检测VEGF-mR- NA表达的改变,Western blot检测VEGF蛋白表达,流式细胞技术分析细胞周期分布,MTT比色法检测细胞的生长抑制率。结果　成功转染重组体的HCT116细胞中VEGF-mRNA表达明显下降,约降低为对照组的61. 2% (P0. 05)。VEGF蛋白 表达明显下降,光密度分析比较差异有显著性(P0. 05)。G0/G1期细胞比例增高,另一方面S期和G2/M期细胞比例降 低,细胞的增殖指数明显降低,实验组细胞增殖指数为(25. 63±4. 54)%,对照组细胞的增殖指数为(31. 90±2·19)% (P0·05)。结论　Pavu6+27-VEGF siRNA重组体在细胞内表达的短发夹状RNA能有效抑制人大肠癌细胞VEGF-mRNA和VEGF蛋白表达,细胞增殖能力减弱,为质粒介导的RNAi技术运用于大肠癌的基因治疗提供一定的实验依据。 　　关键词: RNA干扰;短发夹状RNA;VEGF;增殖 　　中图法分类号:R394-33;R394. 2;R735. 34　　　文献标识码:A Effect of knockdown ofVEGF gene expression by shRNA on characteristics of human colorectal cancer cell lineLUWei,ZHANG Chao,HAO Jia,LIUWei,HAO Ying-xue,YU Pei-wu(DepartmentofGeneralSurgery,SouthwestHospital,ThirdMilitaryMedicalUniversity,Chongqing 400038,China) 　　Abstract:Objective　To study the effect of knockdown ofVEGF expression by recombinant plasmidPavu6+27-VEGF siRNA on the characteristics ofhuman colorectal carcinoma cell lineHCT116.Methods　The subcloned recombinantplasmid shRNA was transfected into human colorectal carcinoma cell lineHCT116with DOTAPmethod. The positive transfected cell cloneswere screenedwithG418. The stable transfection andexpression ofVEGFmRNA inHCT116 were determined by semi-quantitative RT-PCR. The protein expressionofVEGFwas examined byWestern blotting. Distribution ofcell cyclewas assessed by flow cytometry. The cellgrowth suppressionwas analysed byMTT assay.Results　Comparedwith the negative control cells,multiRTPCR andWestern blotting showed the expression ofVEGFmRNA and proteinwas down-regulated in the transfected cells(P0. 05). The subcloned recombinant plasmid expressing shRNA effectively inhibitedHCT116cell growth and proliferation while empty plasmid had no such specific effec.t The proliferation index of thecellls of recombinantplasmid transfectionwas(25. 63±4.