【作者】 舒雄；

【导师】 周立军；

【作者基本信息】 天津大学 ， 微生物与生化药学， 2010， 硕士

【摘要】 目的:构建含人的肝细胞生长因子(Human hepatocyte growth factor)基因真核表达载体pEGFP-N1-HGF,探讨其在体外培养人的成纤维细胞的转染与表达,为其在体内应用提供实验依据。方法: RT-PCR扩增人的间充质干细胞中hHGF全长cDNA片段,克隆入pEGFP-N1真核表达载体中,获得pEGFP-N1-HGF重组质粒。应用脂质体技术以绿色荧光蛋白(GFP)作为报告基因,采用脂质体Lipofectamine 2000包裹pEGFP-N1-HGF重组质粒转染到人的皮肤成纤维细胞中,研究了细胞接种密度、DNA用量、脂质体与DNA的比例等因素对脂质体转染效率的影响,获得表达克隆。采用荧光显微镜观察、逆转录聚合酶链反应(RT-PCR)、蛋白质印记方法(Western Blotting)鉴定和ELISA方法定性和定量的测定hHGF的表达。结果: 1.质粒酶切鉴定结果:提取的质粒经双酶切、琼脂糖凝胶电泳鉴定后可得到4660bp和2200bp两个片段,与原质粒图谱符合,表明所提取的质粒为重组pEGFP- N1- HGF。2.人的皮肤成纤维细胞经2-5次传代后转染重组质粒pEGFP- N1- HGF后,荧光显微镜下观察到细胞内发出绿色荧光,在细胞接种密度为2×10~5、0.8ugDNA用量、2.5:1的脂质体与DNA比例时,转染效率最高分别为22.73±0.44%、21.88±0.34%、22.29±0.42%。3.转染后细胞提取总RNA后经过RT-PCR能扩增HGF cDNA预期的756 bp片段,证实转染质粒在成纤维细胞内有转录,Western blotting证实转染HGF基因在成纤维细胞内有HGF蛋白的表达,同时通过ELISA法检测转染细胞的上清液,表明转染成纤维细胞中有分泌,其分泌量为(5.9～7.55 ng/ 8×10~5 cells)。结论:成功构建重组质粒pEGFP-N1-HGF,并证实其能够在成纤维细胞中转录与表达分泌,提示我们构建的成纤维细胞模型能够替代毛乳头细胞分泌肝细胞生长因子的功能,为脱发疾病的基因治疗提供依据。更多还原

【Abstract】 Objective: To construct a plasmid carrying human hepatocyte growth factor (hHGF) gene and determine t he effects of the hepatocyte growth factor (HGF) gene o n transfection and expression of human fibroblast cells in vitro. Methods: The full length c DNA o f hHGF, which was amplified from human mesenchymal stem cells mRNA by RT-PCR, was cloned into pEGFP-N1 vector to construct pEGFP-N1-HGF recombinant p lasmid. With Lipofectamine 2000 liposome, the recombinant plasmid pEGFP-N1-HGF was used to transfect t he c ulture human fibroblast c ells.After pEGFP-N1-HGF transfection, the following transfection conditions were optimized: cell density, DNA a mount, ratio of liposome and DNA. The t ransfection a nd expression of H GF i n hum an f ibroblast cells were tested by RT-PCR and Western Blotting, the level o f s ecreted HGF p rotein in s upernatant was d etermined w ith enzyme-linked immunosorbent assay (ELISA). Result: 1. Two DNA segments, about 4.7kb and 2.2kb in length, were obtained after digestion with BamHⅠand SacⅠ.The recombinant plasmid pEGFP-N1-HGF was identified by restriction endonuclease digestion and nucleotide sequencing. 2. The transfection efficiency was achieved with the following optimized conditions:At passage 2-5 later, 2×10~5 cells, 4ug DNA, 2.5:1 ratio for liposome and DNA, the results showed the highest transfection efficiency of human fibroblast cells ,which were 22.73±0.44%, 21.88±0.34%, 22.29±0.42%. 3. RT-PCR and Western-blotting proved that there was transcription of HGF gene in transfection c ells a nd t here w as e xpression of HGF in these c ells. When hHGF detected with ELISA in cell culture, there was a high level of hHGF expressed by transfected human fibroblast in 48 hours (5.9～7.55 ng/ 8×10~5 cells). Conclusion: The recombinant of pEGFP-N1-HGF is an effective expression vector. The recombinant plasmid pEGFP-N1-HGF can transcript and express in the transfected cells. HGF is thus a potent agent for strategies designed to promote therapeutic angiogenesis.更多还原