转染人CTLA-4细胞的对树突状细胞B7分子表达的影响  

Construction of human CTLA-4 tansfectant and its effects on the expression of B7 molecule on dendritic cells

作　　者：孙玮丽[1] 陈永井[1] 孙中文[1] 徐耀瑜[1] 胡玲玲[1] 张学光[1] 邱玉华[1] 

机构地区：[1]苏州大学生物技术研究所,苏州215007

出　　处：《现代免疫学》2007年第4期289-293,共5页Current Immunology

基　　金：江苏省自然科学基金(BK2004203);江苏省高校高新技术产业发展基金(JHB05-45)

摘　　要：从PHA活化的人外周血T细胞中抽提总RNA,经RT-PCR扩增出CTLA-4的全长基因,构建逆转录病毒载体pEGZ-Term/CTLA-4,经PCR及电泳鉴定后感染包装细胞293T。收集培养上清感染L929细胞。用Zeocin选择培养。经免疫荧光及流式细胞术进行筛选,获取稳定表达CTLA-4分子的细胞株CTLA-4/L929。将CTLA-4/L929经丝裂霉素处理后与体外细胞因子诱导的树突状细胞共同孵育。分别于24、48及72h,用直接免疫荧光法和流式细胞术分析树突状细胞B7-1和B7-2分子的表达。研究结果显示,构建的重组逆转录病毒载体pEGZ-Term/CTLA-4的PCR产物长约700 bp,与人CTLA-4基因的长度一致。经Zeocin选择及多次亚克隆化培养,CTLA-4基因转染细胞CTLA-4/L929稳定表达膜型CTLA-4分子。以其为刺激细胞与树突状细胞共同培养后,可明显下调树突状细胞B7-1的表达,但对B7-2的表达没有影响。本研究获得的CTLA-4基因转染细胞株,为进一步探讨CTLA-4及其配基分子在免疫应答中的作用与机制提供了手段。The cDNA encoding the human CTLA-4 was obtained from mRNA of the PHA-activated T cells in peripheral blood by RT-PCR and cloned into the retrovirus vector pEGg-Term. This recombinant vector together with its 2 helper virus vectors was co-transfected to the package cells 293T with lipfectamine 2000^TM , and the supernatant of 293T cells was used to transfect L929 cells. The cell line stably expressing CTLA-4 molecule was screened by zeocin after trabsfection and was assayed by immunophenotyping and flow cytometry（FCM）. This cell line CTLA-4/L929 treated with mitomycin was then co-cultivated with dendritic cells induced by cytokine, and the expressions of B7-1 and B7-2 molecules on the dendritic cells were detected by immunofluorescence assay and FCM at 24, 48 and 72 hrs respectively. It was demonstrated that the length of the PCR product constructed by recombinant retrovirus vector pEGZ-Term/CTLA-4 was approximately 700 bp, consistent with that of human CTLA-4 gene. The expression of the B7-1 molecule on dendritic cells was found to be 22.1%, which appeared to be lower than those of control group L929/CTLA-4 and dendritic cells（both of which appeared to be more than 85%）. In addition, this human CTLA-4 tranfectant could effectively down-regulate the expression of BT-1 molecule on dendritic cells, but it showed no any effect on the expression of B7-2 molecule. These results may provide a useful means for the further studies on the role and mechanism of CTLA-4 and its ligands in immune responses.

关 键 词：CTLA-4 B7 基因转染 共刺激分子 树突状细胞 表达 

分 类 号：R392.11[医药卫生—免疫学]