论文部分内容阅读
小鼠B7-H3作为协同刺激分子的生物学功能至今尚未明确。本文拟通过构建小鼠B7-H3基因的真核表达载体,获取稳定表达小鼠B7-H3基因的细胞株,并进一步通过体外实验研究其对T淋巴细胞体外活化及功能的调节作用。作者运用RT-PCR技术从小鼠肝脏组织中获得全长小鼠B7-H3基因,并构建真核表达载体pIRES2-B7-H3,转染仓鼠细胞株(CHO)。经过G418抗性筛选和亚克隆,获得稳定表达B7-H3的基因转染细胞,并经RT-PCR、Western-blot及流式细胞术方法鉴定细胞的表达,采用CCK8、酶联免疫吸附试验(ELISA)等方法分析基因转染细胞对T细胞的增殖和IL-2、IFN-γ的分泌。本文成功克隆和构建了稳定表达小鼠B7-H3的基因转染细胞B7-H3/CHO,该细胞株和T细胞共培养能有效促进T细胞的增殖,增强其IL-2、IFN-γ的分泌。小鼠B7-H3在T细胞活化过程中可能发挥正性协同作用。
The biological function of mouse B7-H3 as a costimulatory molecule is not yet known. In this study, we constructed a murine B7-H3 gene eukaryotic expression vector to obtain a stable expression of mouse B7-H3 gene cell lines, and further in vitro experiments on T lymphocytes in vitro activation and function of regulation. The authors obtained the full-length mouse B7-H3 gene from mouse liver tissue by RT-PCR and constructed the eukaryotic expression vector pIRES2-B7-H3, which was transfected into hamster cell line (CHO). After G418 resistance screening and subcloning, the transfected cells stably expressing B7-H3 were obtained, and the expression of B7-H3 was confirmed by RT-PCR, Western-blot and flow cytometry. The expression of B7-H3 was detected by CCK8 and enzyme-linked immunosorbent assay ELISA) and other methods analysis of gene transfected cells on T cell proliferation and IL-2, IFN-γ secretion. This study successfully cloned and constructed B7-H3 / CHO gene transfection cells stably expressing mouse B7-H3. The co-culture of this cell line with T cells can effectively promote the proliferation of T cells and enhance the expression of IL-2 and IFN-γ secretion. Mice B7-H3 may play a positive synergistic effect during T cell activation.