人LIGHT基因重组慢病毒的构建以及在脐血间质干细胞中的表达

Construction of Lentiviral Vector Carrying the LIGHT Gene and its Expression in the UCBMSCs

目的构建带有人LIGHT基因的慢病毒载体,观察其在人脐血间质干细胞中的表达。方法通过逆转录聚合酶链反应从PCD DNA-LIGHI、质粒中获得人LIGHT基因,利用infusion技术重组构建慢病毒载体质粒pGC-FU-LIGHT,在脂质体lipofectamine 2000介导下与结构质粒pHelper1.0及包膜蛋白质粒pHelper2.0共转染293T细胞包装生产慢病毒。将人脐血间质干细胞分为实验组(pGC-FU-LIGHT)、空载体对照组(pGC-FU-EGFP)及空白组(脐血间质干细胞),分别用重组慢病毒、空载慢病毒、PBS感染后,采用RT-PCR以及Elisa检测IIGHT表达情况。结果所获LIGHT基因经测序后与Gene Bank报道序列完全一致;重组慢病毒载体质粒pGC-FU-LIGHT经鉴定正确;三质粒共转染293T细胞成功,收集、浓缩病毒后测定其滴度为2×107TUJ/L,感染UJCBMSCs后RT-PCR、Elisa检测各组细胞均有LIGHT的表达,其中试验组pGC-FU-LIGHT组更大最表达LIGHT,与其余2组(pGC-FU-EGFP、UJCBMSCs)比较差异具有统计学意义。结论成功构建带有LIGHT基因的慢病毒载体并实现在脐血间质干细胞中的表达,为间充质干细胞移植治疗胃癌的应用奠定了基础。

Objective To construct a lentiviral vector carrying human LIGHT gene and to observe its expression in the human umbilical cord blood mesenchymal stem cells.Methods The human LIGHT gene was obtained with reverse transcription polymerase chain reaction(RT-PCR) and the LIGHT gene was recombined to construct the transfer plasmid pGC-FU-LIGHT by infusion technique.The 293T cells were cotransfected with the transfer plasmid pGC-FU-LIGHT the construction plasmid Helper 1.0 and the envelope plasmid Helper 2.0 with the help of lipofectamine 2000 to produce lentiviral particles.The human UCBMSCs were divided into the experimental group(pGC-FU-LIGHT),the mock group(pGC-FU-EGFP)and the blank group(UCBMSCs),which were respectively infected by recombinant lentiviral particles,empty lentiviral particles and PBS.The expression of LIGHT was detected by RT-PCR and Elisa method.Results The result of sequencing showed that the cloned LIGHT gene was consistent with the sequence reported in Gene Bank.The insertion of LIGHT gene in viral genome was conformed by PCR.The pGC-FU-LIGHT plasmid was identified to have correct sequence.After the three plasmids of lentiviral vectors were contransfected to the 293T cells.The supernatant was collected and concentrated.The titer of the lentiviral vector particles was found to be 1×10~7 TU / L.After the constructed lentiviral vectors infected the UCBMSCs,the results obtained using RT-PCR and Elisa method showed that the expression of LIGHT in the pGC-FU-LIGHT group(the experimental group,EG) was significantly higher than those of in the pGC-FU-EGFP group(the mock group,MG) and those of in the UCBMSCs group(the control group,CG)at both mRNA and protein levels.Conclusions Lentiviral vector carrying LIGHT gene has been successfully constructed.The infected human umbilical cord blood mesenchymal stem cells are able to express the LIGHT protein.This results showed that transplantation of mesenchymal stem cells could help to treat the patients with gastric carcinoma.