新型人工miRNA表达框架的构建及其有效性验证  

Construction of novel expression cassettes of artificial miRNA and validation of its inhibition effect

作　　者：徐慧[1] 范张玲 张旺[1] 杨伦宇 彭剑雄[1] 

机构地区：[1]中南大学湘雅医学院医学检验系,长沙410013

出　　处：《临床检验杂志》2014年第12期939-943,共5页Chinese Journal of Clinical Laboratory Science

基　　金：中南大学本科生自由探索计划项目(2282014bks193)

摘　　要：目的构建针对EGFP基因的人工miRNA表达框架并验证其有效性。方法用融合PCR技术在体外构建针对EGFP基因的人工miRNA表达框架。经测序比对后,以该表达框架与报告基因质粒p EGFP-N2分别共转染人肝癌细胞系Hep G2、人宫颈癌细胞系He La和人肺腺癌细胞系A549,48 h后用倒置荧光显微镜观察增强型绿色荧光蛋白(EGFP)的表达情况并用流式细胞仪定量检测其抑制效率。结果构建的人工miRNA表达框架经测序比对正确;共转染该表达框架和报告基因质粒p EGFP-N2后,倒置荧光显微镜观察结果显示,3种细胞中发出荧光的细胞数量减少,荧光强度明显降低。流式细胞术检测结果显示,Hep G2细胞、He La细胞和A549细胞中共转染组和单独转染组的细胞荧光表达率差异均有统计学意义(t分别为20.80、30.35和20.34,P均<0.01)。结论成功构建靶向EGFP的人工miRNA表达框架,且此表达框架能有效而特异地抑制EGFP蛋白在Hep G2细胞、He La细胞和肺癌A549细胞中的表达。Objective To construct the expression cassettes of artificial miRNA targeting EGFP and validate its inhibition effect.Methods The expression cassettes of artificial miRNA targeting EGFP were designed by overlap extension PCR and co-transfected into Hep G2 cells,He La cells and lung carcinoma A549 cells with reporter plasmid p EGFP-N2 after DNA sequencing. The expression of EGFP was observed by inverted fluorescence microscope and the flow cytometry was used to detect the inhibition efficiency after 48 hours. Results DNA sequencing demonstrated the expression cassettes of artificial miRNA were constructed correctly. After the cotransfection with expression cassettes and reporter plasmid p EGFP-N2,inverted fluorescence microscope showed that both the number and intensity in the 3 different cell lines were significantly reduced. Flow cytometry showed that the fluorescent expression rate of cotransfection and individual transfection in Hep G2 cells,He La cells and lung carcinoma A549 cells group were significantly different（ t = 20. 80,30. 35,20. 34,all P〈0. 01）. Conclusion The expression cassettes of artificial miRNA targeting EGFP were constructed successfully which could effectively and specifically inhibit the expression of EGFP protein in Hep G2 cells,He La cells and lung carcinoma A549 cells.

关 键 词：人工miRNA表达框架 EGFP基因 HEP G2细胞 HE La细胞 A549细胞 

分 类 号：R34[医药卫生—基础医学]