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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。
Objective: To construct human stem cell marker molecule CD133-2 transgenic cells and to explore the biological function of CD133-2 molecule. METHODS: Human CD133-2 full-length cDNA was cloned from human fetal liver cDNA by segmented and overlap PCR. The full-length cDNA of human CD133-2 was cloned into the eukaryotic expression vector pIRES2-EGFP by liposome method L929 cells were stained with G418 selective medium for screening. Transgenic cells were identified by RT-PCR, Western blot and flow cytometry (FCM). The proliferation of T cells was analyzed by MTT assay. The expression of T-cell surface markers CD4CD25 and CD8CD25 were analyzed by flow cytometry . RESULTS: CD133-2 / L929 was successfully cloned and constructed. The co-culture of human CD133-2 cells with T cells inhibited the proliferation and down-regulated the expression of CD4 CD25 and CD8 CD25 on T cells. Conclusion: The establishment of transgenic cell line stably expressing CD133-2 protein laid the material foundation for the subsequent study on the function of this gene.