nm23-H1基因转染前后人高转移大细胞肺癌细胞株双向凝胶电泳图谱分析

为了更全面地了解nm23-H1在肺癌中发挥转移抑制的机理,用双向凝胶电泳技术比较人高转移大细胞肺癌细胞株(L9981)和转染nm23-H1基因的人大细胞肺癌细胞株(L9981-nm23-H1)间蛋白表达的差异.利用固相pH梯度双向凝胶电泳分离人高转移大细胞肺癌细胞株(L9981)和转染nm23-H1基因的人大细胞肺癌细胞株(L9981-nm23-H1)的总蛋白,用图像分析软件比较分析以识别细胞间的差异表达蛋白质.结果成功地获得了两株细胞蛋白组分辨率高、重复性好的双向凝胶电泳图谱.软件分析两种细胞的凝胶电泳图谱后发现,在相同分析条件下识别的蛋白质斑点数L9981为902±169个、L9981-nm23-H1为1160±212个.比较L9981和L9981-nm23-H1人大细胞肺癌细胞株的双向凝胶电泳蛋白质图谱后发现6个蛋白质点仅在L9981中有表达,17个蛋白质点仅在L9981-nm23-H1中有表达.此外,发现13个在两种细胞株中均存在,但表达量差异在2倍以上的蛋白质点(P<0.05).结果提示,nm23-H1基因转染引起人高转移大细胞肺癌细胞株蛋白质表达谱的变化,可能是其逆转肺癌侵袭转移的生物学基础.

Two-Dimensional Gel Electrophoresis of Human High-Metastatic Large Cell Lung Cancer Cell Lines Before and After Transfection of nm23-H1 Gene

nm23-H1 gene is one of the most important tumor metastatic suppressive genes in lung cancer. To understand the mechanism of metastasis suppressive effects of nm23-H1, differential proteomic comparison between L9981 with high metastatic potential and nm23-H1 gene deletion and L9981-nm23-H1 constructed by transfecting nm23 gene into L9981 was conducted using two-dimensional(2-1) gel electrophoresis. The total proteins of two cell lines were separated by immobilized pH gradient (IPG)-based 2-D gel electrophoresis. The differential expression proteins of two cell lines were analyzed using image analysis software. The high resolution and reproducible 2-DE images of two cell lines were successfully obtained. In three different experiments the total number of protein spots was 902 ±169 in L9981 cells and 1160 ±212 in L9981-nm23-H1 cells. The image analysis of silver-stained 2-D gels between L9981 and L9981-nm23-H1 revealed that 33 protein spots showed significant differential expression (student's t-test, p<0.05). 6 protein spots were only detected in L9981 cells, 17 protein spots were only detected in L9981-nm23-H1 cells. Our research results suggested that the changes of protein spectrums after transfection of nm23-H1 gene might be the base of nm23-H1 gene for reversing the invasive and metastatic phenotype in human high-metastatic large cell lung cancer.