含miR-193b前体基因的重组腺病毒感染K562细胞可抑制其增殖  

Recombinant adenovirus of microRNA-193b inhibits proliferation of K562 cells

作　　者：舒燕[1] 齐杰玉[1] 王灿蔚[1] 于淑静[1] 陶崑[1] 

机构地区：[1]重庆医科大学基础医学院免疫学教研室分子医学与肿瘤研究中心,重庆400016

出　　处：《细胞与分子免疫学杂志》2015年第4期452-455,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：重庆市科委课题(CSTC:2010BB5369S)

摘　　要：目的构建小RNA193前体(pre-miR-193b)重组腺病毒,观察miR-193b对慢性髓系白血病K562细胞增殖的影响。方法化学合成miR-193b cDNA序列,克隆进入pAdTrack-CMV穿梭质粒;重组穿梭质粒经PmeⅠ酶切线性化后,转入含有腺病毒骨架质粒pAdEasy-1的大肠杆菌BJ5183感受态细胞中进行同源重组,获得pAd-miR-193b重组腺病毒质粒,经PacI线性化后转入HEK293细胞包装、扩增。倍比稀释法测定病毒滴度。实时荧光定量PCR检测K562细胞中miR-193b表达水平,MTT法检测K562细胞增殖能力。结果pAd-miR-193b重组腺病毒质粒,经限制性内切酶鉴定和测序分析显示构建成功,并能够高效转染K562细胞;与对照组相比,荧光定量PCR检测显示重组腺病毒载体感染K562细胞后,miR-193b基因表达明显增加;同时高表达miR-193b基因的K562细胞增殖水平低于对照组。结论K562细胞高效表达的miR-193b可抑制K562细胞的增殖。Objective To construct a recombinant adenovirus vector containing human pre-miR-193b and investigate its effect on the proliferation of chronic myelocytic leukemia K562 cells.Methods The c DNA of pre-miR-193b was obtainedby chemical synthesis and inserted into the adenoviral shuttle vector p Ad Track-CMV.The recombinant shuttle plasmid was linearizedby Pme I and transformed into Ad Easier cells for homologous recombination in E.coli BJ5183 with the adenoviralbackbone plasmid p Ad Easy-1.The recombinant adenoviral plasmid was linearizedby Pac I and then used for transfecting HEK293 cells.After package and amplification in HEK293 cells,the virus titer was determinedby serial dilution assay.The expression level of miR-193b in K562 cells was detectedby real-time quantitative PCR.The cel proliferation was observedby MTT assay.Results The recombinant plasmid named p Ad-miR-193b was confirmedby restriction enzyme analysis and sequencing.The recombinant adenovirus could infect K562 cells efficiently.Compared with control group,real-time quantitative PCR revealed that the expression of miR-193b was significantly up-regulated in K562 cells infected with p Ad-pre-miR-193b,and the cell proliferation was suppressed significantly in K562 cells with up-regulated miR-193b expression.Conclusion The over-expression of miR-193b may suppress the proliferation of K562 cells.

关 键 词：腺病毒 微小RNA193前体 K562细胞 

分 类 号：Q279[生物学—细胞生物学] Q253