转染人CXCR4细胞株的构建及其生物学功能的研究  

Construction of the transfected cell line expressing the human CXCR4 gene and study of its biological function

作　　者：周时勇[1] 杨明峰[1] 王雪峰[1] 于葛华[1] 成中芹[1] 张学光[1] 

机构地区：[1]苏州大学医学生物技术研究所江苏省干细胞研究重点实验室,江苏苏州215007

出　　处：《细胞与分子免疫学杂志》2006年第4期427-429,432,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学重点资助项目(30330540);江苏省干细胞重点实验室基金(2005年度)

摘　　要：目的:构建稳定表达人CXCR4基因的L929细胞株,分析CXCR4分子对转基因细胞迁移能力的影响。方法:TRIzol一步法抽提人外周血单个核细胞(PBMC)总RNA,RT-PCR扩增出CXCR4基因,双酶切装入逆转录病毒载体pEGZ-Term,与辅助病毒载体用脂质体法共转染包装细胞293T,用其培养上清感染L929细胞72h后,经Zeocin筛选出稳定表达CXCR4分子的L929细胞株;利用微孔隔离小室检测转人CXCR4基因的L929细胞在SDF-1α作用下的迁移能力。结果:构建含CXCR4基因的重组逆转录病毒载体,经转染包装细胞293T后,筛选获得能稳定高表达人CXCR4蛋白的L929转基因细胞,转入人CXCR4基因的L929细胞在SDF-1α作用下介导迁移。结论:成功构建转染人CXCR4细胞株,为肿瘤迁移模型的研究和鼠抗人CXCR4mAb的制备打下基础。AIM： To construct the tranfected cell line expressing the human CXCR4 gene and to study the biological function. METHODS： The total RNA was isolated from peripheral blood mononuclear cell （PBMC） with TRIzol, and the CXCR4 gene was amplified by RT-PCR, then digested with restriction endonuclease Pst I and EcoR I, and inserted into retrovirus vector pEGZ-Term. The recombinant vector together with its two helper virus vectors was cotransfected into the package cells 293T with LipofectAMINE 2000. Then the supernatant of the 293T cell culture was used to infect L929 cells, the cell clones stably expressing the CXCR4 molecule were screened in the presence of Zeocin （500 mg/L） after 72 h cultivation. RESULTS： It was found that the full-length of CXCR4 gene was successfully cloned, and the recombinant retrovirus vector carrying the CXCR4 gene was constructed. The CXCR4 cDNA transfected L929 cell could stably express the human CXCR4 on the cell membrane, and the migration ability of transfected cells was well evidenced in the transwell system induced by SDF-1α after the transfection with CXCR4. CONCLUSION： The CXCR4 transfected L929 cell line was successfully established, and it can make the basis for the further research.

关 键 词：CXCR4 逆转录病毒 稳定表达 迁移 

分 类 号：Q785[生物学—分子生物学]