摘要
目的 建立2IgB7-H3基因转染细胞株和4IgB7-H3基因转染细胞株,探讨B7-H3两种异构体对T细胞的协同刺激作用.方法 RT-PCR 法从诱导成熟的DC细胞中克隆人B7-H3两种异构体基因2IgB7-H3和4IgB7-H3的编码区,经EcoR Ⅰ和BamH Ⅰ双酶切后插入pIRES2-EGFP真核表达载体构建pIRES2-EGFP/2IgB7-H3和pIRES2-EGFP/4IgB7-H3重组子,采用脂质体法转染脑胶质瘤细胞株SHG44.经G418抗性筛选,采用免疫荧光标记和流式细胞术分析2IgB7-H3和4IgB7-H3在SHG44细胞上的表达.继而,利用MTT法和酶联免疫吸附测定法（ELISA）分析两种异构体基因转染细胞株对T细胞体外增殖和细胞因子的分泌.结果 成功构建了可以稳定表达2IgB7-H3和4IgB7-H3的基因转染细胞株.体外生物学功能分析表明,与转染空载体的SHG44/mock细胞相比,SHG44/2IgB7-H3和SHG44/4IgB7-H3均能有效抑制T细胞增殖以及对IFN-γ的分泌,且两者的协同抑制作用不存在显著差异.结论 B7-H3两种异构体分子均能负性调控T细胞介导的免疫应答,但两者的生物学功能并不存在显著的差异.
Objictive To establish two cell lines carrying human 2IgB7-H3 gene and 4IgB7-H3 gene,which can be stably expressed,and to study their costimulatory effects on T lymphocytes.Mcthods The gene encoding human 2IgB7-H3 gene and 4IgB7-H3 gene were amplified by RT-PCR from mature DC cells by induction,then the two target gene fragments were inserted into eukaryotic vector plRES2-EGFP after being digested with EcoR I and BamH I .The two recombinant vectors were transfected into SHG44 cells with LipofectamineTM 2000,and the two cells were further screened with G418.Then the expression of human 2IgB7-H3 gene and 4IgB7-H3 gene were analysed by Immunofluorescence and flow cytometry.Effect of cell lines transfected with two human B7- H3 isoforms on T cells proliferation and cytokine production in vitro was studied by MTT and ELISA. Results The stable expressions of human 2IgB7-H3 and 4IgBT-H3 on the transfected cell lines were identified by flow cytometry analysis.In vitro,SHG44/2IgB7-H3 and SHG44/4IgB7-H3 cells could both significantly down-regulate the proliferation of T cells stimulated by anti-CD3 mAb and inhabit the production of IFN-γ ,which is shown by ELISA results.And there is no significant difference of their negative costimulatory effect on T cells.Conclusion Two isoforms of human B7-H3 have negative costimulatory effect on T cells,but there is no significant different effect.
出处
《中国血液流变学杂志》
CAS
2011年第3期379-383,共5页
Chinese Journal of Hemorheology
基金
国家自然科学基金资助项目（30771958）
