PDGF-A基因逆转录病毒载体的构建及其在猪骨髓间充质干细胞中表达的研究  

Cloning and expression of platelet-derived growth factor A chain in porcine marrow mesenchymal stem cells by retroviral vector transfection

作　　者：闫国和[1] 杨余亮 侯爱莲[3] 王锋超[1] 许庆娥 熊玮[5] 程天民[1] 王军平[1] 粟永萍[1] 

机构地区：[1]第三军医大学军事预防医学院防原医学教研室,全军复合伤研究所,创伤、烧伤与复合伤国家重点实验室,重庆400038 [2]四川省古蔺县人民医院普外科,四川古蔺646500 [3]重庆医科大学基础医学院生理学教研室,重庆400016 [4]解放军71613部队医院,山东海阳265100 [5]第三军医大学西南医院呼吸内科,重庆400038

出　　处：《第三军医大学学报》2008年第21期2024-2028,共5页Journal of Third Military Medical University

基　　金：国家重点基础研究发展计划(973计划,2005CB522605);国家自然科学基金(30200142,30770929);全军医学科研“十一五”计划科技攻关项目(06G076)~~

摘　　要：目的克隆血小板衍生生长因子A链(platelet-derived growth factor A chain,PDGF-A)基因,以逆转录病毒载体pLXSN为骨架携带PDGF-A基因转染猪骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,bMSCs),为应用PDGF-A基因修饰bMSCs进行创面修复奠定基础。方法采用RT-PCR二步分离法,以人肝癌细胞总RNA为模板,扩增PDGF-A基因的全长cDNA编码序列,构建PDGF-A基因的逆转录病毒载体pLXSN/PDGF-A,并将其导入病毒包装细胞PA317中,经G418筛选并扩增,以病毒液感染NIH3T3细胞,选出抗性克隆,测其病毒滴度,将高滴度病毒液感染bMSCs。采用RT-PCR、Southern blot和ELISA法,分别检测PDGF-A基因转染bMSCs的效率及PDGF-A在bMSCs中的表达水平;光镜、MTT法、成骨与成脂诱导分别检测bMSCs转染PDGF-A基因后的生长状态、增殖情况及干细胞特性。结果经测序验证,克隆到PDGF-A基因的全长cDNA序列与GenBank所报告的该基因序列完全一致。成功构建了PDGF-A链基因的逆转录病毒载体pLXSN/PDGF-A,并实现了PDGF-A基因在bMSCs的稳定表达。bMSCs转染PDGF-A后的生长状态、增殖情况良好,且仍具干细胞特性。结论成功地将PDGF-A基因克隆到pLXSN载体中,并实现了PDGF-A基因在bMSCs中的稳定表达。bMSCs转染PDGF-A后仍具干细胞特性。Objective To clone platelet-derived growth factor A chain （PDGF-A） gene sequence, construct pLXSN-PDGF-A plasmid and express it in porcine bone marrow derived mesenchymal stem cells （bM- SCs）. Methods cDNA fragment encoding human PDGF-A gene were amplified by reverse transcriptase-polymerase chain reaction （RT-PCR） with the total mRNA isolated from human hepatoma cell line SMC-7721 as template. The PCR amplified fragment of PDGF-A gene was first cloned into pMD18-T vector, and then subcloned into the recombinant retroviral vector pLXSN to construct pLXSN/PDGF-A after seqencing. Subsequently, plasmid DNA of pLXSN/PDGF-A was transfected into packaging cell line PA317, and positive cell clones with higher titer were screened, bMSCs were infected with cell-free supernatant of the highest titer clone trans- fected with pLXSN/PDGF-A. The transfection and expression of PDGF-A in bMSCs were identified by RTPCR, Southern blot hybridization and enzyme-linked immunosorbent assay （ELISA）. Light microscopy, methyl thiazolyl tetrazolium （MTF） assay, and induced bone and fat formation were employed to examine the growth and proliferation of bMSCs transfected with PDGF-A and their muhilineage differentiation as stem cells. Results Full-length cDNA sequence encoding human PDGF-A gene was successfully cloned, and recombinant retroviral vector of pLXSN/PDGF-A is constructed. Stable expression of PDGF-A in porcine bMSCs was obtained with transfection of retroviral superuatant from pLXSN/PDGF-A clone, bMSCs after PDGF-A transfection were grown and proliferated well, and still possessed the trait of stem cells. Conclusion cDNA sequence encoding human PDGF-A gene is successfully cloned into pLXSN vector. Stable expression of PDGF-A gene in bMSCs is implemented by retroviral transfection, bMSCs transfected with PDGF-A still possesses the trait of stem cells.

关 键 词：逆转录病毒载体 血小板衍生生长因子A链基因 骨髓间充质干细胞 稳定表达 

分 类 号：R331.2[医药卫生—人体生理学] R394-33[医药卫生—基础医学]