hBSP与GFP非融合表达载体的构建及乳腺癌细胞转染条件优化

Construction of hBSP Eukaryon Expression Vector Carrying GFP and Optimization of Transfection Conditions for Breast Cancer Cells

为进一步研究骨唾液蛋白(BSP)在乳腺癌细胞骨转移的整个过程中的作用,首先要建立稳定高效表达BSP的乳腺癌细胞系.文中首先从构建好的pB-hBSP载体中亚克隆hbsp基因,构建重组质粒pIRES2-hBSP-EGFP,然后利用脂质体介导转染特异性骨转移的乳腺癌细胞MDA-MB-231BO,优化一系列转染条件以提高转染效率,再利用G418和流式细胞仪筛选获得稳定转染细胞.结果发现:在96孔板中,细胞融合度达到90%;用1μg脂质体介导280 ng重组质粒转染10 h,转染效率最高可达(19.70±1.10)%.

In order to study the role of bone sialoprotein （BSP） in the metastasis of breast cancer cells to bones, a breast cancer cell line stably expressing BSP should be established. In this paper, hbsp gene was subcloned from constructed pB-hBSP vector and was then inserted into pIRES2-EGFP, thus constructing the recombinant plasmid pIRES2-hBSP-EGFP. Moreover, by the transfection of breast cancer cell MDA-MB-231BO mediated by Lipofectamine^TM 2000, the corresponding transfection conditions were optimized to improve the transfection efficiency. Stably transfected breast cancer cells were finally obtained by the selection via G418 and flow cytometry. It is found that, in a 96-well cell culture plate, the cell growth confluence is up to 90%, and that when 1μg of Lipofectamine^TM 2000 is used to mediate 280ng of recombinant plasmid transfecting breast cancer cells for 10h, the tranfection efficiency is up to （19.70 ±1.10）%.