高表达CypA肝癌细胞系的建立及其功能初步研究

目的：构建含人全长CypA cDNA的真核表达质粒,建立高表达CypA蛋白的肝癌细胞系,为进一步研究CypA在肝癌中的功能和作用机制提供细胞模型。方法：将人全长CypA cDNA序列克隆后,连接至真核表达载体pcDNA3.1中,再将构建的真核表达载体转染至FHCC98肝癌细胞中,经G418加压筛选后获得稳定表达CypA的肝癌细胞株;免疫印迹法检测目的蛋白的表达情况;MTT法测定细胞的增殖速率。结果：真核表达载体pcDNA3.1-CypA经酶切、测序鉴定,结果正确;免疫印迹结果显示,稳定转染后细胞的CypA表达水平显著高于空载体转染和未转染的细胞;与对照相比,稳定高表达CypA的细胞增殖速度加快。结论：成功构建了含有人全长CypA cDNA序列的真核表达载体pcDNA3.1-CypA;建立了稳定高表达CypA的FHCC-98肝癌细胞系;CypA的高表达可以促进FHCC98细胞的增殖。

Establishment and functional analysis of hepatocellular carcinoma cell line which overexpressed CypA

Objective： To construct a recombinant CypA eukaryocyte expression vector,and to establish a high expression CypA hepatocellular carcinoma（HCC） cell model for further studying the function and mechanism of CypA in HCC.Methods： The full gene fragment of human CypA was cloned,and subcloned into the eukaryotic gene expression vector pcDNA3.1（＋）.Then the recombinant vector was transfected into the FHCC98 cells,a hepatocellular carcinoma cell line,which were cultured selectively with 800 μg/ml G4l8 to get a stable CypA expression cell line.The expression of CypA was examined by immunobloting and the proliferation rate was determined by MTT.Results： The eukaryotic expression vector pcDNA3.1-CypA was examined by two restrictive endonuclease digesting and sequence measuring,and both results were correct.The immunobloting results showed that the stable transfected pcDNA3.1-CypA-FHCC98 cell expressed higher level of CypA than the stable transfected pcDNA3.1-FHCC98 cell or untransfected FHCC98 cell.Comparing to control,pcDNA3.1-CypA-FHCC98 cell had a higher proliferation rate.Conclusion： The recombinant eukaryocyte expression vector and its stable expression HCC cell line were established.Over expression of CypA in FHCC98 cell could stimulate its proliferation.