人TNF-α重组慢病毒载体构建及其脐血间质干细胞表达  

CONSTRUCTION OF LENTIVIRAL VECTOR CARRYING TNF-α GENE AND ITS EXPRESSION IN HUMAN UMBILICAL CORD BLOOD MESENCHYMAL STEM CELLS

作　　者：马贵亮[1] 毛伟征[1] 杨堃[1] 安岗 岱震波 

机构地区：[1]青岛大学医学院附属医院普外科,山东青岛266003 [2]潍坊市寒亭区医院 [3]天津肿瘤医院

出　　处：《青岛大学医学院学报》2010年第3期189-192,195,共5页Acta Academiae Medicinae Qingdao Universitatis

基　　金：国家自然科学基金资助项目(30772542)

摘　　要：目的构建带有人TNF-α基因的慢病毒载体,并且观察该基因在体外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(RT-PCR)从PCD DNA-TNF-α质粒中获得人TNF-α基因,利用Infusion技术重组构建慢病毒载体穿梭质粒pGC-FU-TNF-α,在脂质体Lipofectamine 2000介导下与结构质粒pHelper 1.0和包膜质粒pHelper 2.0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-TNF-α)、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方法检测TNF-α表达。结果所获TNF-α基因测序证明与GenBank中序列一致;重组慢病毒载体质粒pGC-FU-TNF-α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2×107TU/L,感染脐血间质干细胞后RT-PCR、ELISA检测3组细胞均有TNF-α表达,其中实验组大量表达TNF-α,与其余两组比较差异有显著性(F=11.677、21.321,P<0.01)。结论成功构建带有TNF-α基因的慢病毒载体并实现在脐血间质干细胞中的表达,为脐血间质干细胞转基因治疗胃癌的应用奠定基础。Objective To construct a lentiviral vector carrying human TNF-α gene,and observe its expression in human umbilical cord blood mesenchymal stem cells（UCBMSCS）. Methods Human TNF-α gene was obtained with reverse transcription polymerase chain reaction（RT-PCR） from PCD DNA-TNF-α plasmid.The TNF-α gene was recombined to construct the transfer plasmid pGC-FU-TNF-α by infusion technique.The 293T cells were cotransfected with the transfer plasmid pGC-FU-TNF-α,the construction plasmid Helper 1.0 and the envelope plasmid Helper 2.0 with the help of lipofectamine 2000 to produce lentiviral particles.Human UCBMSCS were divided into experimental group（pGC-FU-TNF-α）,mock group（pGC-FU-EGFP） and blank group（UCBMSCS）,which were infected by recombinant lentiviral particles,empty lentiviral particles and PBS,respectively.The expression of TNF-α was observed by RT-PCR and ELISA. Results The results of gene sequencing showed that the cloned TNF-α gene was consistent with the sequence reported in GenBank.The pGC-FU-TNF-α plasmid was identified to have correct sequence.After the three plasmids of lentiviral vectors were cotransfected to the 293T cells,the supernatant was collected and concentrated.The titer of the lentiviral vector particles was 2×107 TU/L.After the constructed lentiviral vectors infected the UCBMSCS,TNF-α expression detected with RT-PCR and ELISA was found in all three groups,but the expression of TNF-α in the pGC-FU-TNF-α group was significantly higher than that in the pGC-FU-EGFP group and the UCBMSCS group at both mRNA and protein levels（F=11.677,21.321;P0.01）. Conclusion Lentiviral vector carrying TNF-α gene has been successfully constructed.The infected human UCBMSCS can express TNF-α protein.The results of present study provide basis for application of UCBMSCS gene transfer therapy in the treatment of gastric carcinoma.

关 键 词：慢病毒感染 遗传载体 肿瘤坏死因子α 基因 脐血 间质干细胞 

分 类 号：R730.2[医药卫生—肿瘤]