人VSIG4基因转染细胞的构建及其对T细胞的共刺激效应的初步研究  

Establishment of human VSIG4 gene transfectants and study of its costimulatory effects on T lymphocyte

作　　者：张世杰[1] 王蕾[2] 王月颖[1] 蒋靓[1] 蒋玉平[1] 高超[1] 张学光[1] 顾宗江[1] 

机构地区：[1]苏州大学医学生物技术研究所,苏州215007 [2]苏州大学附属第一医院心内科,苏州215006

出　　处：《中国免疫学杂志》2009年第5期397-401,共5页Chinese Journal of Immunology

摘　　要：目的:分别构建含有人VSIG4基因两种不同变异体(VSIG4L和VSIG4S)的重组逆转录病毒载体,获得稳定表达VSIG4L和VSIG4S基因的L929细胞并初步研究其对T细胞的共刺激作用及可能机制。方法:从人肺cDNA文库中扩增出VSIG4L和VSIG4S的全长基因,并分别克隆入T载体中,再双酶切装入逆转录病毒载体pEGZ-Term中,与辅助病毒载体用脂质体法共转染包装细胞293T,以其培养上清感染L929细胞72小时后,经Zeocin筛选出稳定表达VSIG4L或VSIG4S分子的L929细胞株;采用RT-PCR和流式细胞仪分析VSIG4分子的表达,MTT和ELISA分别检测T细胞增殖及IL-2的分泌。结果:构建了含人VSIG4L基因和VSIG4S基因的重组逆转录病毒载体,并获得含有VSIG4L基因和VSIG4S基因的重组病毒,筛选获得能稳定高表达人VSIG4L和VSIG4S分子的L929转基因细胞;该转基因细胞具有体外抑制T细胞增殖、活化和IL-2分泌的作用。结论:构建了稳定表达人VSIG4L和VSIG4S膜型分子的细胞株,该分子两种不同变异体在体外均可抑制T细胞增殖和IL-2分泌。Objective：To construct recombinant retrovirus vectors carrying human VSIG4 gene （VSIG4L and VSIG4S）,which can be stably expressed in cell line L929, and to study the costimulatory effects to T lymphocytes and the mechanism in volved. Methods： The full length human VSIG4 genes （VSIG4L and VSIG4S） were amplified by RT-PCR from the human lung cDNA library. Digested with restriction en- donuclease Pst I and EcoR 1, each of the VSIG4 genes was inserted into retrovirus vector pEGZ-Term. The recombinant vector together with its two helper virus vectors was co-transfected into the package cell 293T with LipofectAMINE 2000^TM. The supernatant of 293T was used to infect L929 cells. L929 cells, and stably expressing VSIG4L and VSIG4S protein was selected in the presence of zeocin, which was then analyzed by PCR and FCM. The functions of VSICAL and VSIG4S for proliferation and IL-2 secretion of T cells were studied by MTF and ELISA respectively. Results： Both of the genes VSIG4L and VSIG4S were successfully cloned, and their recombinant retrovirus vectors were constructed, The selected L929 cells stably expressed VSIG4L and VSICAS protein on the membrane of cells. The transfected cells have inhibitory effects on the activation, proliferation and IL-2 secretion of T ceils. Conclusion： L929 cells lines stably expressing VSIG4L or VSICAS protein were established and VSIG4 may play a negative role in regulation of T cell proliferation and IL-2 production.

关 键 词：VSIG4 逆转录病毒 基因转染 共刺激分子 

分 类 号：R392.11[医药卫生—免疫学]