人4-1BBL转基因细胞株的构建及其生物学功能的研究  

Construction of Human 4-1BBL Transfected Cell Line and the Investigation of Its Bioactivity in Vitro

作　　者：居颂文[1] 居颂光[1] 仇红霞[1] 胡玉敏[1] 王凤鸣[1] 王明元[1] 王勤[1] 张学光[1] 

机构地区：[1]苏州大学生物技术研究所,江苏苏州215007

出　　处：《苏州大学学报（医学版）》2006年第4期539-542,共4页Suzhou University Journal of Medical Science

基　　金：国家自然科学基金青年科学基金资助项目(30400394);江苏省教育厅博士生创新计划资助项目(50320025)

摘　　要：目的为探讨人4-1BBL分子的生物学功能,构建人4-1BBL转基因细胞株。方法利用RT-PCR方法克隆人4-1BBL全长基因,继而重组入逆转录病毒表达载体pEGZ-Term。重组逆转录病毒载体pEGZ-Term/4-1BBL与两个辅助病毒载体用脂质体法共转染包装细胞293T,用含有完整重组逆转录病毒颗粒的293T细胞培养上清感染L929细胞,筛选获得Zeocin抗性的转基因细胞。3H-TdR掺入实验分析4-1BBL转基因细胞对T细胞增殖的影响,ELISA法分析细胞因子的分泌。结果流式细胞仪表型分析结果表明,L929/4-1BBL转基因细胞稳定表达人4-1BBL分子。体外实验表明,L929/4-1BBL细胞能够促进T细胞活化、增殖及IL-2、IL-6和IFN-γ的分泌。结论该研究成功克隆了人4-1BBL基因,构建了能稳定表达4-1BBL分子的转基因细胞株L929/4-1BBL,该株转基因细胞能提供有效的4-1BBL共刺激信号,促进T细胞活化、增殖及细胞因子的分泌。Objective To explore the biological functions of human 4-1BBL, the human 4-1BBL transgenic cell line was constructed. Methods The gene encoding the human 4-1BBL protein was obtained by RT-PCR, then subcloned into the corresponding region of the retrovirous expressing vector pEGZ-Term. The recombinant plasmid pEGZ-Term/4-1BBL together with its two helper virus vectors was co-transfected into the package cell 293T with lipfectamine 2000. Then the supernatant of 293T was used to infect L929 cells. Selected by zeocin, a cell line L929/4-1BBL expressing the human 4-1BBL was established. The expression stability and efficiency of the target molecule were identified by flow cytometry analysis. T cell proliferation induced by L929/4-1BBL was determined by ^3HTdR incorporation assay and cytokine production was analyzed by ELISA assay. Results Flow cytometry analysis showed that cell line L929/4-1BBL stably expressed 4-1BBL molecule. Studies in vitro demonstrated that L929/4-1BBL could promote T cell activation, proliferation and secretion of IL-2, IL-6 and INF-7. Conclusion The gene of the human 4-1BBL is cloned and a stable cell line L929/4-1BBL expressing the human 4-1BBL is established. The human 4-1BBL transgenic cell line can provide costimulatory signal to promote T cell proliferation and cytokine production.

关 键 词：4-1BBL 转基因细胞 T细胞 

分 类 号：R392.11[医药卫生—免疫学]