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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 human endothelial nitric oxide synthase (heNOS) with enhanced green fluorescent protein reporter gene (EGFP) and observe its expression in human bone marrow-derived endothelial progenitor cells (EPCs). Methods Recombinant human pEGFP-heNOS plasmid was constructed and transfected into human bone marrow-derived endothelial progenitor cells by lipofectamine 2000. The expression of green fluorescent protein (GFP) was observed by fluorescence microscopy. The expression of heNOS was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot In endothelial progenitor cells. Results The recombinant plasmid pEGFP-heNOS was successfully constructed and transfected into human endothelial progenitor cells in vitro. The expression of strong green fluorescent protein (GFP) was observed under fluorescence microscope. The expression of heNOS mRNA and protein was detected by RT-PCR and Western blot respectively. Conclusion Recombinant human pEGFP-heNOS plasmid transfected human endothelial progenitor cells in vitro, the target gene can be effectively expressed in cells, which provides theoretical support for the next gene therapy.