【摘要】 目的:构建人干细胞标记分子CD133-2转基因细胞并探讨CD133-2分子的生物学功能。方法:利用分段克隆和重叠PCR方法从人胎肝cDNA文库中克隆出人CD133-2全长基因,经双酶切后装入真核表达载体p IRES2-EGFP中,用脂质体法转染L929细胞,加入G418选择性培养基进行筛选。经RT-PCR、Western blot、流式细胞术(FCM)等方法鉴定转基因细胞;MTT法分析转基因细胞对T细胞的体外增殖作用;流式细胞仪分析T细胞表面活化分子标记CD4CD25、CD8CD25的表达。结果:成功克隆和构建了能稳定表达人CD133-2分子的转基因细胞CD133-2/L929,该转基因细胞与T细胞共培养可抑制其体外增殖,下调T细胞表面分子CD4CD25和CD8CD25的表达。结论:稳定表达CD133-2蛋白的转基因细胞株的建立为该基因功能的后续研究奠定了物质基础。更多还原

【Abstract】 AIM:To generate an engineered L929 cell line harboring human CD133-2 and perform the functional study of the gene-modified cell line.METHODS:The human CD133-2 gene was obtained by PCR from a cDNA library of foetus liver.After digested with Hind III and BamH I,the PCR product was cloned into pIRES2-EGFP vector.The recombinant plasmid CD133-2/pIRES2-EGFP was transfected into L929 cell line using lipofectamine,followed by G418 selection.RT-PCR,Western blot and flow cytometry(FCM)were used to detect the expression of CD133-2.MTT was used to analyze the effect of the CD133-2/L929 cells on proliferation of T cells.FCM was performed to monitor T cell activation by detecting T cell surface markers of CD4CD25 and CD8CD25 after T cells were cocultured with the CD133-2/L929 cells in the presence of an anti-CD3 mAb.RESULTS:A stable cell line constitutively expressing the human CD133-2 was established successfully.In the presence of the anti-CD3 mAb,CD133-2/L929 cells caused inhibition of T cell proliferation and down-regulation of the activation markers of CD4CD25 and CD8CD25 on T cells.CONCLUSION:The engineered CD133-2/L929 cell line provides a gain-of-function cell model for further understanding the biological role of CD133-2.更多还原