Mmp2基因克隆及其对肝癌细胞的作用

克隆人基质金属蛋白酶-2基因(Mmp2)编码区并构建重组真核表达载体pEYFP-Mmp2,研究其在肝癌细胞中的作用。以肝癌细胞Hep G2的总RNA为模板,通过RT-PCR获得cDNA,并通过PCR获得Mmp2基因编码区。经TA克隆和测序鉴定将无任何突变的Mmp2基因编码区插入到真核表达载体pEYFPN1中,构建p EYFP-Mmp2重组真核表达载体并进行酶切鉴定和测序鉴定。pEYFP-Mmp2稳定性转染肝癌细胞,经G418筛选获得Mmp2稳转单克隆细胞株,并用Western blotting分析鉴定。用实时无标记细胞增殖分析技术(RTCA)分析Mmp2基因对肝癌细胞生长增殖的作用,细胞划痕愈合实验分析Mmp2基因对肝癌细胞迁移的作用。结果证明成功构建重组真核表达载体pEYFP-Mmp2,Western Blotting证实稳转株中高表达外源融合蛋白MMP2-YFP。细胞增殖和迁移结果表明,Mmp2基因可以抑制肝癌细胞增殖但能够促进肝癌细胞迁移。以上研究表明,Mmp2基因能够促进肝癌细胞迁移。

Mmp2 Gene Cloning and Its Effect on Liver Cancer Cells

To clone human matrix metalloproteinase 2 gene(Mmp2)coding region and construct recombinant eukaryotic expression vector pEYFP-Mmp2 to explore its function on hepatocellular carcinoma cell(HCC).Taking the total RNA of Hep G2 as a template,c DNA was obtained by RT-PCR,and the Mmp2 gene coding region was amplified by PCR.After TA cloning and DNA sequencing,Mmp2 gene coding region was inserted into the eukaryotic expression vector pEYFP-N1 to construct a pEYFP-Mmp2 recombinant eukaryotic expression vector which was identified by enzyme digestion and DNA sequencing.pEYFP-Mmp2 was stably transfected into Hep G2 cell and screened by drug G418 to get Mmp2 stable transfected monoclonal cell(St.cell).Western blotting was employed to analysis the expression of MMP2 or fusion protein MMP2-YFP in St.cell.Real time cellular analysis(RTCA)was explored to uncover the function of MMP2 on cell proliferation of HCC and cell scratch healing on cell migration.Results showed that coding region of Mmp2 was cloned and the recombinant eukaryotic expression vector pEYFP-Mmp2 was successfully constructed.Mmp2 gene stable transfected monoclonal HCC was built and the fusion protein YFP-Mmp2 expressed in it.Moreover,MMP2 could inhibit HCC cell proliferation but promote cell migration.Altogether,Mmp2 gene was cloned and could induce HCC migration.