人4-1BBL转基因细胞株的构建及其生物学功能的研究

目的为探讨人4-1BBL分子的生物学功能，构建人4-1BBI。转基因细胞株。方法利用RT-PCR方法克隆人4-1BBI。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／4-1BBI。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。

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

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.