慢病毒载体系统介导hUC-MSC绿色荧光蛋白和荧光素酶共表达技术体系的建立

Establishment of technology of co-expression of green fluorescent protein and luciferase in human umbilical cord mesenchymal stem cell via lentiviral expression system

目的建立慢病毒载体系统介导的人脐带间充质干细胞(hUC-MSC)绿色荧光蛋白(GFP)和荧光素酶共表达技术体系。方法 GFP和荧光素酶共表达慢病毒载体与相应包装质粒psPAX2和pMD2.G经聚乙烯亚胺介导共转染HEK293T细胞以包装病毒;病毒感染P4代hUC-MSC 12 h后,再行嘌呤霉素筛选24 h,普通光学显微镜观察细胞形态,荧光显微镜下观察GFP的表达情况,IVIS Kinetic成像系统拍照以观察和记录慢病毒感染后hUC-MSC荧光素酶的表达情况;MTS法行细胞生长曲线作图,同时,普通和实时定量RTPCR法检测细胞周期调控相关蛋白Cyclin D1、Cyclin E1和p21WAF1/CIP1的表达。采用方差分析和t检验进行统计学分析。结果慢病毒感染并不会造成体外培养hUC-MSC形态的明显改变,而荧光显微镜和IVIS Kinetic成像系统的观察结果则分别证实,GFP和荧光素酶经慢病毒载体系统的介导可在hUC-MSC中成功地共表达。此外,细胞生长曲线作图结果表明,对照和GFP及荧光素酶共表达慢病毒感染后hUC-MSC的生长增殖速率相仿(P>0.01);实时定量RT-PCR法检测结果则显示,与对照慢病毒感染相比,GFP和荧光素酶共表达慢病毒感染后其细胞周期调控相关蛋白Cyclin D1、Cyclin E1和p21WAF1/CIP1mRNA表达水平分别是对照组的1.11倍(P=0.130)、0.54倍(P=0.000)和0.78倍(P=0.005),表明外源GFP和荧光素酶共表达对体外培养的hUC-MSC生长增殖等表型无显著影响。结论慢病毒载体系统可有效介导外源基因在hUC-MSC中的表达;同时,GFP和荧光素酶在hUC-MSC中的共表达也将极大地方便其体内转归的示踪。

Objective To establish technology of co-expression of green fluorescent protein（GFP） and luciferase in human umbilical cord mesenchymal stem cell（hUC-MSC） via lentiviral system. Methods The HEK293T cells were co-transfected with lentiviral coexpressing vector of GFP and luciferase and two package plasmids（psPAX2 and pMD2.G） to yield lentivirus. Human umbilical cord mesenchymal stem cells（P4） were infected with harvested lentivirus for 12 hrs and then subjected for selection with Puromycin for additional 24 hrs.Cell morphology was examined under microscope. The expression of GFP and luciferase was detected by fluorescent microscopy and IVIS Kinetic image system, respectively. MTS assay was applied to survey the proliferation of infected cells. The regular and real-time RT-PCR was carried out to analyze the mRNA expression of cell cycle regulators Cyclin D1, Cyclin E1 and p21WAF1/CIP1. Analysis of variance and t test was used for statistical analysis. Results Lentiviral infection resulted in no significant morphological change of hUC-MSC cultured in vitro. The coexpression of GFP and luciferase in hUC-MSC via lentiviral expression system was visualized by microscopy and IVIS Kinetic image system. The proliferation rate of hUCMSC infected with GFP and luciferase co-expressing lentiviruses was the same as that of control lentiviruses infected hUC-MSC as indicated by MTS assay results（P ＆gt; 0.01）. In addition, the realtime RT-PCR results showed that the lentiviral system mediated co-expression of exogenous GFP and luciferase in hUC-MSC wouldn’t cause any significant change of expression of cell cycle regulators Cyclin D1（1.11 fold, P = 0.130）, Cyclin E1（0.54 fold, P = 0.000） and p21WAF1/CIP1（0.78 fold, P = 0.005） as compared with that of control viral infection. Conclusions The lentiviral expression system can be used to mediate expression of exogenous genes in hUCMSC.The introduced co-expression of GFP and luciferase in hUC-MSC will facilitate the in vivo tracing after infusion.