转染人BTLA基因细胞株的建立及其生物学功能的初步研究_医学论文.docVIP

转染人BTLA基因细胞株的建立及其生物学功能的初步研究_医学论文 转染人BTLA基因细胞株的建立及其生物学功能的初步研究_医学论文 作者：王雪峰, 陈永井, 王勤, 杨科芳, 代群, 王凤鸣, 张学光 【关键词】 B、,T淋巴细胞衰减子,基因转染,T细胞,增殖 　　Establishment of cell line transfected with human BTLA gene and preliminary study on its biological function 　　　[Abstract] AIM: To establish 293T cells expressing human BTLA gene, a novel attenuator for B and T lymphocyte. METHODS: The gene encoding human BTLA was amplified by RTPCR from PHA activated T cells, and then the target gene fragment was inserted into retrovirus vector pEGZTerm after being digested with EcoR I and BamH I. The recombinant vector was transfected into 293T cells with LipfectAMINETM 2000, and the cells was further selected with Zeocin. Effect of 293T/BTLA cells on T cells proliferation and activation in vitro was been studied by methods of MTT and cytometry. RESULTS: The stable expression of human BTLA on the transfected cell line was identified by flow cytometry analysis. In vitro, 293T/BTLA cells had an inhibitory effect on the proliferation of T cells stimulated by antiCD3 mAb and downregulated the expression of activation marker CD25 on the surface of T cells and decreased the secretion of IFNγ and IL10. CONCLUSION: A cell line 293T/BTLA stably expressing the human BTLA protein has been obtained and it can partially inhibit the proliferation and activation of T cells in vitro. 　　[Keywords]B and T lymphocyte gene transfection T cell proliferation 　　[摘 要] 目的: 构建B、 T淋巴细胞衰减子（BTLA）基因转染的细胞作为免疫原, 并探讨该基因转染细胞在体外的生物学功能。方法: 通过RTPCR法从经PHA活化的人外周血T细胞中克隆出人BTLA编码区全长基因, 经EcoR I和BamH I双酶切后插入逆转录病毒载体pEGZTerm中构建成重组载体pEGZTerm/BTLA。用脂质体法以重组逆转录病毒载体转染293T细胞, 并用Zeocin抗生素进行长期筛选 流式细胞术分析BTLA在基因转染细胞膜上的表达 通过MTT法和流式细胞术探讨基因转染细胞在体外对T淋巴细胞增殖与活化的影响。结果: 流式细胞术检测表明BTLA基因转染的293T细胞膜上能稳定地高表达人BTLA蛋白。BTLA基因转染的293T细胞和T细胞体外共培养显示, 与未转染的293T细胞相比, 该基因转染的细胞能部分地抑制抗人CD3单克隆抗体（mAb）刺激的T细胞增殖 流式细胞术和ELISA法分析揭示, 该基因转染的细胞能够下调T细胞表面活化标志CD25的表达并降低IFNγ和IL10 的分泌。结论: 获得稳定高表达人BTLA基因转染的细胞株。该细胞株在体外对抗人CD3 mAb刺激的T细胞的增殖与活化具有部分地抑制作用。 　　[关键词]B、 T淋巴细胞衰减子 基因转染 T细胞 增殖 　　B、 T淋巴细胞衰减子（B a