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重组人血管抑素表达载体的构建及在人肝癌细胞系中的表达
引用本文:陶开山,窦科峰.重组人血管抑素表达载体的构建及在人肝癌细胞系中的表达[J].第四军医大学学报,2005,26(18):1668-1672.
作者姓名:陶开山  窦科峰
作者单位:第四军医大学西京医院肝胆外科,陕西,西安,710033
摘    要:目的:构建重组人血管抑素(hANG)的表达载体并检测其在肝癌细胞系中的稳定表达. 方法:利用RT-PCR从人胚胎肝组织中扩增出hANG cDNA片段,将其克隆到pCR-Blunt Ⅱ-TOPO载体和pcDNA3.1(+)表达载体,利用克隆PCR、限制性内切酶消化以及序列测定对获得的hANG基因片段及重组载体进行验证. 将重组pcDNA3.1-hANG真核表达载体转染到人肝癌细胞SMMC-7721对其表达状况进行检测. 结果:所获得的hANG片段(1.1 kb)序列与报道的序列完全一致. 酶切鉴定的结果表明含血管抑素基因的重组pcDNA3.1-hANG表达载体构建成功. 转染重组pcDNA3.1-hANG的人肝癌细胞SMMC-7721表达了血管抑素. 另外,从生长曲线结果来看,转染pcDNA3.1-hANG真核表达载体和空载体的人肝癌细胞SSMC-7721和未转染的SSMC-7721细胞的生长速度无明显差别. 结论:成功地构建了重组人血管抑素真核表达载体并获得能稳定表达人血管抑素的SSMC-7721肝癌细胞,为开展下一步的实验奠定了基础.

关 键 词:血管抑制素类    肝细胞  血管生成  表达载体
文章编号:1000-2790(2005)18-1668-05
收稿时间:2005-04-29
修稿时间:2005-06-20

Construction of recombinant human angiostatin expression vector and stable expression on human hepatocelluar cancer cells
TAO Kai-Shan,DOU Ke-Feng.Construction of recombinant human angiostatin expression vector and stable expression on human hepatocelluar cancer cells[J].Journal of the Fourth Military Medical University,2005,26(18):1668-1672.
Authors:TAO Kai-Shan  DOU Ke-Feng
Abstract:AIM: To construct the recombinant human angiostatin (hANG) expression vector and to detect its expression in hepatocellular carcinoma cells. METHODS: hANG cDNA fragment was obtained by RT-PCR from human embryonic tissue and the fragment was cloned into pCR-Blunt II-TOPO vector and into pcDNA3.1 (+) expression vector. Colony PCR, restriction enzyme digestion and sequencing were used to verify the obtained hANG cDNA fragment. The recombinant pcDNA3.1-hANG plasmid was then transfected into hepatocellular carcinoma cells to detect the expression of hANG in these cells. RESULTS: The obtained hANG fragment (1.1 kb) was identical to the sequence of reported human angiostatin. Restriction enzyme digestion demonstrated that the recombinant pcDNA3.1-hANG expression vector was successfully constructed. The transfected SMMC-7221 cells successfully expressed hANG. In addition, no difference in the growth speed was observed between the untransfected SMMC-7221 cells, the SMMC-7221 cells transfected with pcDNA3.1(+) vector and the SMMC-7211 cells transfected with recombinant pcDNA3.1-hANG. CONCLUSION: We have successfully constructed the recombinant pcDNA3.1-hANG expression vector and obtained SMMC-7221 cells that can stable express hANG.
Keywords:angiostatins  carcinoma  hepatocellular  angiogenesis  expression vector
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