重组真核表达载体pEGFP-N1/PDGF-A的构建及真皮干细胞的转染  

Construction of eukaryotic expressing vector pEGFP-N1/PDGF-A for transducting Dermis-derived mesenchymal stem cells

作　　者：闫国和[1] 粟永萍[1] 王军平[1] 汪代杰[2] 艾国平[1] 王锋超[1] 冉新泽[1] 程天民[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 [2]四川省泸州市人民医院肿瘤科,泸州646000

出　　处：《第三军医大学学报》2005年第20期2005-2008,共4页Journal of Third Military Medical University

基　　金：国家重点基础研究发展规划资助项目("973"项目2005CB522605);国家自然科学基金资助项目(30200142)~~

摘　　要：目的克隆血小板衍生生长因子A链(plateletderivedgrowthfactorAchain,PDGFA)基因,以增强型绿色荧光蛋白(enhancedgreenfluorescentprotein,EGFP)载体pEGFPN1为骨架,携带PDGFA基因进入真皮间充质干细胞(dermisdrivedmesenchymalstemcells,DMSCs),为采用转入PDGFA基因的DMSCs修复创面奠定基础。方法采用RTPCR二步分离法,以人肝癌细胞系(SMC7721)的总RNA为模板,扩增PDGFA基因的全长cDNA编码序列,克隆入载体pMD18T,随后又将PDGFA基因亚克隆入pEGFPN1载体中,构建PDGFA基因的真核表达载体pEGFPN1/PDGFA,并采用Fugene6介导转染技术将PDGFA基因导入DMSCs。结果克隆到PDGFA基因的全长cDNA序列,经测序验证,其序列与GenBank所报告的该基因的序列完全一致。结论成功地将PDGFA基因克隆到pEGFPN1载体中,并实现了PDGFA基因在DMSCs的表达。Objective To clone platelet-derived growth factor A chain （PDGF-A） gene and insert PDGF-A gene into. Enhanced green fluorescent protein （EGFP） vector and then transformed into dermis-drived mesenchymal stem cells （DMSCs）. Methods cDNA clones encoding human PDGF-A gene were isolated from a human hepatoma cell line mRNA by reverse transcription-polymerase chain reaction （RT-PCR）. The PCR amplified fragment of PDGF-A gene was cloned into pMD18-T vector. The eukaryotic expression vector pEGFP- N1/PDGF-A was constructed by subcolone PDGF-A gene into pEGFP-N1 vector. PDGF-A gene was transfected into DMSCs with the help of Fugene 6 transfection reagent. Results Full cDNA sequence encoding human PDGF-A gene had been cloned, which sequence was consistent with the reported sequence in GenBank by sequence assaying. Conclusion cDNA sequence encoding human PDGF-A gene was successfully cloned into pEGFP-N1. The transient expression of PDGF-A gene in DMSCs has been realized.

关 键 词：克隆 真核表达载体 血小板衍生生长因子A链基因 绿色荧光蛋白 真皮间充质干细胞 

分 类 号：Q782[生物学—分子生物学] R322.991[医药卫生—人体解剖和组织胚胎学]