重组人pEGFP-heNOS质粒的构建及其在人内皮祖细胞中的表达

Construction of recombinant pEGFP-heNOS plasmid and its expression in EPC

目的构建含有增强绿色荧光蛋白报道基因(EGFP)的人内皮一氧化氮合酶(heNOS)重组质粒,观察其在人骨髓来源的内皮祖细胞(EPC)中的表达。方法构建重组人pEGFP-heNOS质粒,脂质体转染人骨髓来源的内皮祖细胞,荧光显微镜下观测绿色荧光蛋白的表达；逆转录-聚合酶链反应(RT-PCR)和Western blot方法检测heNOS在内皮祖细胞中的表达。结果重组人pEGFP-heNOS质粒构建成功,体外转染人人内皮祖细胞中,在荧光显微镜下可见强绿色荧光蛋白的表达,RT-PCR和Western blot分别从mRNA和蛋白水平检测到heNOS的表达。结论重组人pEGFP-heNOS质粒体外转染人内皮祖细胞后,目的基因能够在细胞中有效表达,为下一步基因治疗提供了理论支持。

Objective To construct a recombinant plasmid containing an enhanced green fluorescent protein （EGFP） reporter gene for the role of human endothelial nitric oxide synthase （heNOS）, and observe its expression in endothelial progenitor cells （EPCs） derived from human bone marrow. Methods Recombinant plasmid pEGFP-heNOS was constructed by techniques of gene recombination, and transfected into EPCs by liposome. The GFP expression was observed by fluorescence microscopy and the heNOS expression in EPCs was detected by reverse transcriptase polymerase chain reaction （RT-PCR） and Western blot. Results Recombinant plasmid pEGFP-heNOS was constructed and transfected into EPCs successfully. The strong expression of GFP was observed by fluorecensce microscopy. The heNOS mRNA and protein could be detected within the transfected cells by RT-PCR and Western blot respec- tively. Conclusion Recombinant plasmid pEGFP-heNOS is effectively expressed after being transfected into EPCs in vitro,which provides theoretical support for further gene therapy.