表达大鼠融合蛋白NKX2.5-pEGFP的骨髓间充质干细胞的建立

Establishment of marrow mesenchymal stem cells expressing rat NKX2.5-pEGFP fusion protein

目的:构建大鼠NKX2.5融合绿色荧光蛋白真核表达质粒NKX2.5-pEGFP,并筛选表达大鼠融合蛋白NKX2.5-pEGFP的骨髓间充质干细胞(MSCs),为后续性细胞和基因治疗心肌梗死提供理论基础。方法:提取胎鼠心肌细胞总RNA,RT-PCR扩增获得大鼠NKX2.5基因,将目的基因克隆到pEGFP-N3载体并获得重组后NKX2.5-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NKX2.5-pEGFP以Lipo2000转染到大鼠MSCs,G418筛选2周。蛋白免疫印记和荧光检测重组后质粒在骨髓间充质干细胞中的表达情况。结果:电泳检测证实经RT-PCR获得NKX2.5基因大小为981bp,与理论值相符。NKX2.5-pEGFP重组质粒经BamHⅠ、SalⅠ双酶切,PCR鉴定得到2条大小约为981和4 700bp的酶切片段,鉴定结果与预期结果完全一致。蛋白免疫印记检测到转染重组质粒的MSCs中有相对分子质量为65 000的蛋白表达,与理论值相符。荧光检测NKX2.5-pEGFP重组质粒转染的MSCs中可见绿色荧光,证实表达阳性。结论:成功建立表达大鼠NKX2.5-pEGFP基因的MSCs。

Objective To construct the rat NKX2.5-pEGFP fusion eukaryotic expression plasmid and screen bone marrow mesenchymal stem cells（MSCs） expressing rat fusion protein NKX2.5-pEGFP and provide theoretical foundation for the research of cell and gene therapy of myocardial infarction.Methods The total RNAs were extracted from myocardial cells of embryo rats,RT-PCR amplification was performed to produce rat gene of NKX2.5.The recombinant plasmid NKX2.5-pEGFP was obtained by inserting the aim gene into pEGFP-N3.The MSCs derived from bone marrow mononuclear cells were obtained by gradient centrifugation and adherent culture.The recombinant plasmids NKX2.5-pEGFP were transfected into rat MSCs by Lipo2000 and screened with G418 for 2 weeks.The expression of recombinant plasmid in MSCs was detected by Western blotting and fluoroscope analysis.Results The length of NKX2.5 gene obtained by RT-PCR was 981 bp which was consistent with theoretical numerus.The recombinant plasmid NKX2.5-pEGFP was identified by endonuclease digestion and PCR,which could be digested into two fragments with BamHⅠ and SalⅠ,about 981 and 4 700 bp,the results were coincident with the anticipated result.The protein with molecular weight of 65 000 was found in MSCs transfected with the recombinant plasmid NKX2.5-pEGFP by Western blotting which was coincident with theoretical numerus.Green fluorescence was positive expression in MSCs transfected with the recombinant plasmid NKX2.5-pEGFP by fluorescence detection.Conclusion The rat MSCs expressing NKX2.5-pEGFP fusion protein is successfully constructed.