摘要
目的:构建针对h TERT基因的人工mi RNA表达框架,并验证其对Hep G2细胞端粒酶活性的抑制作用。方法:应用融合PCR技术针对不同位点设计并构建3个靶向h TERT的人工mi RNA表达框架,对各表达框架鉴定后,将其单独或两种共转染Hep G2细胞,采用TRAP-银染法和TRAP-二聚体蝎形探针荧光定量PCR法检测细胞端粒酶活性。结果:3个针对h TERT基因的人工mi RNA表达框架均成功构建,单独或共转染Hep G2细胞后,Hep G2细胞的端粒酶活性均被不同程度的抑制,且两种表达框架共转染的抑制作用明显大于单独转染,差异均有统计学意义(均P<0.05)。结论:靶向h TERT基因的人工mi RNA表达框架能有效而特异抑制Hep G2细胞端粒酶活性,多位点联合抑制是一种有效的实验方案。
Objective： To construct the artiicial miRNA expression cassettes targeting hTERT gene and validate their inhibitory effect on telomerase activity in HepG2cells.Methods： Three artificial mi RNA expression cassettes targeting different sites of h TERT gene were designed and constructed by overlap extension PCR method. After identification, the cassettes were transfected alone or cotransfected into HepG2cells, and then the telomerase activities in HepG2cells were detected by TRAP-silver staining and TRAP-duplex scorpion probe fluorescence quantitative PCR.Results： The three artificial mi RNA expression cassettes targeting h TERT gene were all successfully constructed, and after they were transfected alone or co-transfected into the HepG2 cells, the telomerase activities in HepG2cells were inhibited to different degrees, and the inhibitory effect of co-transfection with two mi RNA expression cassettes was significantly stronger than those of any lone transfection. All the differences reached statistical significance（all P〈0.05）.Conclusion： The artificial mi RNA expression cassettes targeting h TERT gene can effectively inhibit telomerase activity in HepG2cells, and combined inhibition is an effective experimental program.
出处
《中国普通外科杂志》
CAS
CSCD
北大核心
2014年第12期1647-1651,共5页
China Journal of General Surgery
基金
中南大学本科生自由探索计划项目资助(2282014bks193)
