文章摘要
俞〓悦,王学浩.经phoenix细胞包装含人全长HGF基因的逆转录表达载体及体外感染骨髓间充质干细胞的研究[J].南京医科大学学报,2006,(12):1155~1158
经phoenix细胞包装含人全长HGF基因的逆转录表达载体及体外感染骨髓间充质干细胞的研究
Study on construction of hepatocyte growth factor expression vector system and stable expression in rat bone marrow stem cells with phoenix cells
投稿时间:2006-05-24  
DOI:10.7655
中文关键词: 间充质干细胞  肝细胞生长因子  c-met
英文关键词: mesenchymal stem cells  hepatocyte growth factor  c-met
基金项目:国家自然科学基金(30271236)和江苏省卫生厅医学重点人才培养(BJ98025)资助
作者单位
俞〓悦 南京医科大学第一附属医院肝脏外科江苏 南京〓210029 
王学浩  
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中文摘要:
      目的:构建含人全长肝细胞生长因子(HGF)基因与绿色荧光蛋白(GFP)基因融合的逆转录表达载体plEGFP-HGF,经phoenix细胞包装,病毒液感染骨髓间充质干细胞(BM-MSCs),研究phoenix细胞的包装能力与病毒感染效率。方法:将人全长HGF cDNA亚克隆到逆转录病毒载体plEGFP-N1中,获得HGF定向插入重组子plEGFP-HGF,采用酶切法和测序法分别鉴定。用磷酸钙法经phoenix细胞包装,收集病毒上清,感染MSCs,经G418筛选获得阳性克隆,命名为HGF/MSCs细胞。ELISA法检测细胞培养上清中HGF,RT-PCR检测HGF受体c-met在MSCs中的表达。结果:重组逆转录病毒载体体系plEGFP-HGF经限制性内切酶切电泳后显示有2.3 kb目的基因片段和6.9 kb的线性化plEGFP-N1载体片段;荧光显微镜观察到GFP 基因获得了表达。重组子测序结果与Gene bank中HGF-cDNA序列相符。RT-PCR检测转染细胞中c-met基因mRNA表达水平升高,ELISA法检测细胞培养上清中HGF升高。结论:重组真核表达载体构建正确并在MSCs中获得高效、稳定表达,为其在进一步的体内外研究奠定基础。
英文摘要:
      Objective:To construct plEGFP-N1 mammalian expression vector system containing human full-length HGF cDNA, and to study the packaging efficiency of phoenix cells and the expression of HGF in bone marrow stem cells(MSCs). Methods:The plasmid plEGFP-N1 and expression plasmid pCMV-HGF were cut with BamHI enzyme respectively. HGF cDNA was obtained and subcloned into plEGFP-N1 expression vector. The recombinant plEGFP-HGF was identificated with restriction enzyme digestion and sequencing. Subsequently, plEGFP-HGF was transformed into phoenix cells and the supernatant was collected. The expression of HGF in the transfected cells was detected by ELISA. RT-PCR was used to determine the expression the receptor of HGF, c-met mRNA in plEGFP-HGF+MSCs. Results:The mammalian expression vector system plEGFP-HGF was digested with BamHI and HindⅢ. Agarose gel electrophoresis showed that there were fragments of 2.3 kb(HGF) and 6.9 kb(plEGFP-N1). DNA sequencing of HGF was identical to the report in Genebank. The results of RT-PCR showed that c-met transcript strongly increased in pEGFP-HGF/MSCs, suggesting that HGF amplified its own action via stimulation of c-met expression. Conclusion:The plEGFP-HGF mammalian expression vector system containing human full-length HGF cDNA could be constructed successfully, and they could be effectively expressed in MSCs.
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