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建立人协同刺激分子HVEM的基因转染细胞株,探讨膜型HVEM体外对T细胞活化与增殖的协同刺激作用。从此前已插入人HVEM编码区全长基因的pMD18-T/HVEM中用PCR法扩增出目的片段,经EcoRⅠ和BamHⅠ双酶切后插入pIRES2-EGFP真核表达载体构建成pIRES2-EGFP/HVEM重组载体,以脂质体法转染鼠L929细胞。经G418长期加压筛选,以免疫荧光标记和流式细胞术分析HVEM分子在转染细胞膜上的表达情况,以MTT法和ELISA法测定获得HVEM转染细胞L929体外对T淋巴细胞增殖及细胞因子分泌的影响。结果:基因转染细胞株L929/HVEM的细胞膜上能稳定高表达人HVEM分子。体外细胞共培养试验表明,与未转染HVEM基因的L929/mock细胞相比,L929/HVEM能显著地促进抗人CD3单抗(mAb)刺激的T细胞增殖,以及促进T细胞IL-2、IFN-γ、IL-10和TNF-α的分泌。建立了稳定高表达人HVEM分子的基因转染细胞株,并发现膜型HVEM体外对T细胞增殖和相关细胞因子的分泌具有显著促进作用。
Human costimulatory molecule HVEM was constructed and transfected into cell lines to investigate the synergistic stimulatory effect of membrane type HVEM on T cell activation and proliferation in vitro. The target fragment was amplified by PCR from pMD18-T / HVEM which had been inserted into the full-length gene of human HVEM coding region and inserted into the eukaryotic expression vector pIRES2-EGFP after digested with EcoRⅠ and BamHⅠ to construct pIRES2-EGFP / HVEM The recombinant vector was transfected into murine L929 cells by lipofectamine. After long-term pressurized screening by G418, the expression of HVEM in transfected cell membrane was analyzed by immunofluorescence staining and flow cytometry. The proliferation of T lymphocytes and the expression of cytokines were detected by MTT assay and ELISA assay Secretion effects. Results: Human HVEM was stably expressed on the cell membrane of gene transfection cell line L929 / HVEM. In vitro cell coculture experiments showed that L929 / HVEM could significantly promote the proliferation of T cells stimulated by anti-human CD3 mAb and promote the expression of IL-2 in T cells compared with L929 / mock cells without HVEM gene transfection. IFN-γ, IL-10 and TNF-α secretion. A stable cell line transfected with human HVEM was established. It was found that HVEMEM could significantly promote the proliferation of T cells and the secretion of related cytokines in vitro.