重组大鼠GATA4-pEGFP基因在骨髓间充质干细胞的表达及意义

Expression and significance of the recombinant rat GATA4-pEGFP gene in bone marrow mesenchymal stem cells

目的体外获得表达重组大鼠GATA4-pEGFP基因的骨髓间充质干细胞(MSCs),为进一步研究后者在心肌梗死治疗中的作用奠定基础。方法提取胎鼠心肌细胞总RNA,RT-PCR法扩增获得GATA4基因,将目的基因克隆到pEGFP-N3载体并获得重组后GATA4-pEGFP质粒,采用密度梯度离心法联合贴壁法分离骨髓单个核细胞并获得MSCs。将重组质粒GATA4-pEGFP以Lipo2000转染到大鼠MSCs,G418筛选2周,Western blot和荧光检测重组质粒在MSCs中的表达情况。结果成功构建重组大鼠GATA4-pEGFP真核表达载体并转染入MSCs中,Western blot和荧光检测证实GATA4-pEGFP基因在MSCs中呈阳性表达。结论成功建立表达大鼠GATA4-pEGFP基因的MSCs,此为利用组织工程学方法治疗心肌梗死的研究奠定了基础。

Objective To screen bone marrow mesenchymal stem cells（MSCs） expressing GATA4-pEGFP in vitro,so as to provide theoretical foundation for the research of myocardial infarction.Methods Total RNA was extracted from embryonic myocardial cells,GATA4 gene was amplified by RT-PCR,the target gene was cloned to pEGFP-N3 vector to get recombinant GATA4-pEGFP plasmid,the bone marrow mononuclear cells was derived and MSCs were obtained by gradient centrifugation.The recombinant plasmid was transfected into MSCs using lipofectamine 2000,then screened for 2 weeks by G418,Western blot and immunofluroscence staining were used to examine the expression pattern of transfected protein.Results Recombinant rat GATA4-Pegfp eukaryocyte expression vector was constructed and transfected into MSCs successfully,Western blot and immunofluroscence staining showed that GATA4-Pegfp gene expressed positively in the MSCs.Conclusion MSCs expressing GATA4-pEGFP can be constructed successfully,this provides opportunity to study and treat myocardial infarction using tissue engineering technologies.