论文部分内容阅读
建立人协同刺激分子LIGHT的基因转染细胞株,探讨其体外对T细胞活化与增殖的协同刺激作用。采用RT-PCR法从活化的人外周血T细胞中克隆人LIGHT编码区全长基因,经EcoR I和BamH I双酶切后插入pIRES2-EGFP真核表达载体构建成pIRES2-EGFP-LIGHT重组子,脂质体法以重组质粒pIRES2-EGFP-LIGHT转染鼠L929细胞。经G418长期加压筛选,免疫荧光标记和流式细胞术分析LIGHT分子在基因转染的L929细胞膜上的表达,MTT法和酶联免疫吸附测定法(ELISA)探讨获得的基因转染细胞L929/LIGHT体外对T淋巴细胞增殖与活化的影响。结果:流式细胞术检测转基因L929/LIGHT细胞膜上能稳定高表达人LIGHT分子。体外细胞共培养试验表明,与未转染LIGHT基因的L929/mock细胞相比,L929/LIGHT能显著地促进抗人CD3单抗(mAb)刺激的T细胞增殖;同时L929/LIGHT亦能明显促进T细胞对IL-2、IFN-γ和IL-10的分泌。建立了稳定高表达人LIGHT分子的基因转染细胞株,LIGHT介导的信号在体外对T细胞增殖和相关细胞因子的分泌具有显著地促进作用。
Human costimulatory molecules LIGHT gene transfection cell lines established to explore its synergistic stimulation of T cell activation and proliferation. The full length of human LIGHT coding region was cloned by RT-PCR from activated human peripheral blood T cells. After digested with EcoR I and BamH I, the recombinant plasmid was inserted into pIRES2-EGFP eukaryotic expression vector to construct pIRES2-EGFP-LIGHT recombinant L929 cells were transfected with recombinant plasmid pIRES2-EGFP-LIGHT by liposome method. The expression of LIGHT in L929 cells transfected by gene was analyzed by G418 long-term pressure screening, immunofluorescence labeling and flow cytometry. MTT assay and enzyme linked immunosorbent assay (ELISA) Effect of LIGHT on Proliferation and Activation of T Lymphocytes in. Results: The results of flow cytometry showed that human LIGHT was stably overexpressed in the L929 / LIGHT cell membrane. In vitro co-culture experiments showed that L929 / LIGHT significantly promoted the proliferation of T cells stimulated with anti-human CD3 mAb compared with L929 / mock cells without LIGHT transfected cells. L929 / LIGHT also significantly promoted T cells on the secretion of IL-2, IFN-γ and IL-10. A gene-transfected cell line stably expressing human LIGHT molecule was established. The LIGHT-mediated signal significantly promoted the proliferation of T cells and the secretion of related cytokines in vitro.