重组腺病毒介导人类白细胞抗原-G基因转染恒河猴树突状细胞对T细胞的增殖作用  

Effects of recombinant adnovirus-mediated HLA-G transfection in macaca mulatta immature dendritic cells on T cell proliferation

作　　者：陈哲[1] 胡明道[1] 田大广[1] 魏晓平[1] 于恒海[1] 陈鹏[1] 

机构地区：[1]昆明医科大学第二附属医院肝胆胰外科,昆明医学硕士研究生650101

出　　处：《医学研究生学报》2017年第1期5-9,共5页Journal of Medical Postgraduates

基　　金：国家自然科学基金(81160069;30960378);云南科技计划项目(2013FZ275)

摘　　要：目的人类白细胞抗原-G(HLA-G)通过与树突状细胞(DC)表面的免疫球蛋白样转录体(ILT2)和ILT4(CD85d)结合,广泛参与机体的免疫耐受的过程。探讨经HLA-G重组腺病毒载体感染后的恒河猴未成熟树突状细胞刺激T细胞的增殖作用。方法麻醉恒河猴获取新鲜骨髓血,采用密度梯度离心法,分离获得的单个核细胞行免疫磁珠法获得CD34+细胞。添加小剂量细胞因子联合诱导分化CD34+细胞,从而获得树突状细胞。介导HLA-G的重组腺病毒经过转染未成熟树突状细胞后,检测病毒感染效率,Western blot检测HLA-G在未成熟树突状细胞中的表达。以恒河猴T细胞作为反应细胞,将介导HLA-G的重组腺病毒转染的不同状态下的DC为刺激细胞,进行混合淋巴细胞实验。设置5组:即成熟树突状细胞组(mDC组);未成熟树突状细胞组(imDC组);imDC(L)组:在第7天成功获得imDC后加入脂多糖100 ng/m L;imDC(V)组:重组腺病毒介导HLA-G感染的imDC,方法同前;imDC(L+V)组:重组腺病毒同样转染imDC,同时在培养过程中添加100ng/m L的脂多糖。根据DC与T细胞不同比例(1∶5、1∶10、1∶20、1∶40)计算各组刺激指数(SI)。结果成功培养获得恒河猴未成熟树突状细胞,HLA-G在该细胞中表达。流式细胞术检测结果发现DC的纯度高达92.3%,imDC组则高达72.39%,CD4+T细胞阳性率纯度>80%。混合淋巴细胞实验分析DC刺激T细胞增殖结果表明,不同DC与T细胞比例(1∶5、1∶10、1∶20、1∶40)下,imDC组SI为1.63±0.03、1.52±0.04、1.39±0.03和1.21±0.01;mDC组SI为2.23±0.05、2.01±0.03、1.65±0.02和1.54±0.05;imDC(L)组SI为2.25±0.04、1.96±0.02、1.62±0.03和1.48±0.01;imDC(V)组SI为1.46±0.04、1.33±0.02、1.20±0.04和1.04±0.02;imDC(L+V)组SI为1.67±0.03、1.59±0.04、1.38±0.03和1.24±0.03。与imDC组比较,mDC组、imDC(L)组SI明显增高(P<0.01);与imDC(V)组比较,imDC组、mDC组、imDC(L)组、imDC(L+V)组SI明显增高(P<0.01);与imDC(L+V)组比较,mDC组、Objective HLA-G widely participates in immune tolerance by its combination with immunoglobulin-like transcripts IL-2 and IL-4 on the surface of dendritic cells（ DCs）. The aim of the article was to explore the effects of recombinant adnovirus-mediated HLA- G transfection in macaca mulatta immature dendritic cells on T cell proliferation. Methods Marrow blood was collected from macaca mulattas by the puncture needle after anesthesia. Density gradient centrifugation method was applied in separating mononuclear from the extracted blood on which CD34＋cells were collected and purified by means of immunomagnetic separation. Small doses of cytokines were added to get the immature dendritic cells after induced differentiation of CD34＋cells. After the recombinant adnovirus-mediated HLA-G transfection in macaca mulatta immature dendritic cells,observation was done on the viral infection efficiency and western blot was used in detecting the expression of HLA-G in immature dendritic cells. Taking T cells in macaca mulatta as responders and DCs transfected by recombinant adnovirus-mediated HLA- G as stimulators,mixed lymphocyte test was conducted. T cells were divided into 5 groups： mDC group（ mature DCs）,imDC group（ immature DCs）,imDC（ L） group（ addition of 100 ng/mL lipopolysaccharide after getting imDC at 7th day）,imDC（ V） group（ imDCs infected by recombinant adnovirus-mediated HLA- G）,imDC（ L＋V） group（ imDCs infected by recombinant adnovirus-mediated HLA- G along with the addition of 100 ng/mL lipopolysaccharide in culture process）. Results We obtained the immature dendritic cells and recombinant adenovirus of HLA-G expressed in these cells. Flow cytometry showed DC purity was up to 92. 3%,imDC purity was up to72.39% and positive percentage of CD4＋T was greater than 80%. In comparison with imDC group,the proliferation of stimulated T cells in mDC and imDC（ L） groups was obviously intensified（ P〈0.01）. In comparison with imDC（ V） group,the proliferation of stimulat

关 键 词：人类白细胞抗原-G基因 T细胞 免疫耐受 恒河猴 

分 类 号：R392.11[医药卫生—免疫学]