MGr1-Ag表达上调及下调的胃癌细胞株的建立及鉴定

为研究胃癌细胞耐药相关新基因MGr1 Ag对胃癌耐药细胞多药耐药性的调节作用及其机制。克隆MGr1 Ag的全长cDNA并构建其正反义真核表达载体 ,以脂质体介导法将正、反义真核表达载体分别转染入人胃癌细胞系MGC80 3与人胃癌多药耐药细胞系SGC790 1/VCR中。结果显示 ,经RT PCR扩增出大小约为 1.0kb的基因片段 ,成功克隆入pUCm T载体 ,经DNA测序证实为MGr1 Ag基因。将其克隆入pCDNA3 .1/V5 His后 ,经限制性核酸内切酶酶切鉴定 ,获得携有MGr1 Ag基因真核表达载体pCD NA3 1/V5 His MGr1 Ag及其反义表达载体pCDNA3 .1/V5 His anMGr1 Ag。以脂质体介导法将MGr1 Ag的正、反义真核表达载体分别转染入MGC80 3与SGC790 1/VCR中 ,经Westernblot证实 ,成功建立了MGr1 Ag表达上调及下调的胃癌细胞株 ,为研究MGr1 Ag参与耐药的分子机制奠定了基础

CONSTRUCTION AND CHARACTERIZATION OF THE MULTIDRUG-RESISTANT GASTRIC CANCER CELL STRAINS WITH UP AND DOWN-REGULATED MGr1-Ag GENE EXPRESSION

To study the role of a novel MDR-related gene MGr1-Ag in the multidrug resistant (MDR) gastric cancer. The whole length MGr1-Ag gene was cloned, and eukaryotic vectors carrying the full length of MGr1-Ag cDNA and its antisense expression vector were constructed. The sense vector and anti-sense were then transfected into MGC803 cells and SGC7901/VCR cells respectively by lipofectamine. The result showed that a 1.0kb fragment was successfully amplified by RT-PCR and cloned into pUCm-T vector. DNA sequencing suggested that the fragment was the properly encoded MGr1-Ag gene. Recombinant eukaryotic plasmids harboring MGr1-Ag and its antisense expression vector were also obtained by subcloning the gene into pCDNA3.1/V5-His, which was confirmed by endonuclease digestion. As confirmed by Western blot, stable cell strains with up and down-regulated MGr1-Ag expression were constructed. Those cell strains provided the basis for further study on the MGr1-Ag in MDR of gastric cancer.