慢病毒载体介导apelin基因转染人脐带间充质干细胞的实验研究  

Transfection of Lentivirus Vector Containing Apelin Gene into Umbilical Cord Mesenchymal Stem Cells in Vitro

作　　者：王力[1,2] 张宁坤[2] 徐小红[2] 郑楠[2] 高连如[2] 朱智明[2] 

机构地区：[1]第二军医大学海军临床医学院,北京100048 [2]中国人民解放军海军总医院心脏中心

出　　处：《天津医药》2013年第12期1137-1141,I0001,共6页Tianjin Medical Journal

基　　金：国家自然科学基金面上项目(项目编号:81170094)

摘　　要：目的构建带有荧光标记基因增强型绿色荧光蛋白(EGFP)和apelin基因的重组质粒pUbi-apelinFLAG-pSV40-EGFP并进行慢病毒包装,探讨其转染人脐带间充质干细胞的最佳感染复数(MOI)值及目的基因表达情况。方法化学合成目的基因片段并扩增。采用In-Fusion技术将酶切后的目的基因片段与线性质粒载体连接,转化入感受态DH5α细胞中后筛选阳性克隆并进行测序。重组质粒慢病毒载体转染293T细胞,包装慢病毒并测定滴度。将重组质粒慢病毒按不同MOI值转染人脐带间充质干细胞,根据转染效率得到最佳MOI值,并采用Real-timePCR及Western blot方法检测目的基因表达情况。结果通过PCR扩增获得酶切位点碱基修饰后的大小约284 bp的目的基因片段,与慢病毒质粒载体连接后形成pUbi-apelin-FLAG-pSV40-EGFP重组质粒,测序结果与预期完全符合,并成功包装慢病毒颗粒。最佳MOI值为20,转染效率为(90.32±3.61)%。慢病毒载体能高效转染人脐带间充质干细胞且2周内持续稳定上调目的基因mRNA及蛋白的表达。结论重组质粒慢病毒载体pUbi-apelin-FLAGpSV40-EGFP可有效转染人脐带间充质干细胞,并可持续高表达apelin基因。Objective To construct a recombinant plasmid pUbi-apelin-FLAG-pSV40-EGFP and package with lentivirus to co-express enhanced green fluorescent protein （EGFP） and apelin, and to investigate optimal multiple of infec-tion （MOI） to transfect human umbilical cord mesenchymal stem cells （hUCMSCs） and expression of target gene. Methods The apelin gene was chemically synthesized and amplified by polymerase chain reaction （PCR）, and which was inserted into linear plasmid vector. The gene fragment and linear plasmid vector were connected by In-Fusion technology after enzyme di-gestion and transformed into competent DH5αcells. The positive clones of lentiviral expression vector were obtained after screening and followed by sequencing. The lentiviral vector was used to transfect 293T cells and package virus, and then the virus titers were determined. HUCMSCs were transfected with lentivirus vector in vitro via different values of MOI. The trans-fection efficiency was obtained according to the optimal MOI. The expression of target gene was detected by RT-PCR and Western blot assay. Results A 284 bp target gene fragment with the restriction sites was obtained by PCR and connected to the lentiviral vector. The positive clones of lentiviral expression vector were corresponded to the expected result. The lentivi-ral particles were successfully packaged. HUCMSCs could be transfected by the lentivirus vector with high efficiency. The mRNA and protein levels of target gene were stably up-regulated within 2 weeks. Conclusion The lentivirus vector pUbi-apelin-FLAG-pSV40-EGFP can transfect apelin gene into hUCMSCs with high efficiency. The infected cells can express high levels of apelin gene in two weeks.

关 键 词：间质干细胞 慢病毒属 遗传载体 转染 基因表达 APELIN 

分 类 号：R722.12[医药卫生—儿科]