小鼠趋化因子受体7重组慢病毒感染对DC2.4细胞免疫原性和迁移功能的影响  

Influence of infection of murine chemokine receptor-7 recombinant lentivirus on the immunogenicity and migration of DC 2.4 cells

作　　者：董志伟[1] 彭毅志[1] 张帅[1] 陈渝[1] 董凤娟[2] 

机构地区：[1]创伤、烧伤与复合伤国家重点实验室第三军医大学西南医院全军烧伤研究所,400038 [2]河北北方学院医学检验学院

出　　处：《中华烧伤杂志》2013年第1期41-45,共5页Chinese Journal of Burns

基　　金：国家自然科学基金（81071548）

摘　　要：目的 观察含有趋化因子受体7（CCR7）基因的重组慢病毒感染树突细胞（DC）株DC2.4细胞,对其免疫和迁移功能的影响. 方法 常规培养DC 2.4细胞,构建带有绿色荧光蛋白（GFP）的空载慢病毒和带有上调CCR7基因的慢病毒.按照随机数字表法将细胞分为3组：DC 2.4组（DC2.4细胞未经任何处理）、GFP-DC 2.4组（用GFP空载慢病毒感染DC 2.4细胞）和CCR7-DC2.4组（用GFP标记CCR7上升基因的慢病毒感染DC 2.4细胞）.流式细胞术、蛋白质印迹法、激光扫描共聚焦显微镜分别观测各组细胞表面分子组织相容性复合物Ⅱ（MHCⅡ）、CD80、CD86、CCR7的表达,体外趋化实验检测细胞迁移功能的变化；混合淋巴细胞反应检测各组细胞免疫功能的差别,另设LPS-DC 2.4组作为阳性对照.对数据进行单因素方差分析和t检验. 结果 成功构建表达稳定的慢病毒,感染DC 2.4细胞的效率为87.4％.流式细胞检测结果显示,3组之间MHCⅡ、CD80及CD86的表达差异无统计学意义（F值为0.17～1.19,P值均大于0.05）.CCR7-DC 2.4组CCR7蛋白表达量为45.1 ±2.1,明显高于DC 2.4组的25.3±1.4和GFP-DC 2.4组的28.6±0.9（F=162.90,P ＜0.01）；后2组之间比较,差异无统计学意义（t=2.20,P＞0.05）.激光扫描共聚焦显微镜显示,CCR7-DC 2.4组较DC 2.4组CCR7荧光强度明显升高.CCR7-DC 2.4组细胞体外趋化迁移率为（41.0±2.0）％,明显高于DC 2.4组的（6.0±0.5）％和GFP-DC 2.4组的（6.8±0.3）％（F=84.21,P ＜0.01）；后2组之间比较,差异无统计学意义（t=0.45,P＞0.05）.混合淋巴细胞反应显示,DC2.4、GFP-DC 2.4、CCR7-DC 2.4及LPS-DC 2.4组吸光度值分别为1.6±0.4、1.9±0.4、1.7±0.4、3.8±0.4,前3组之间差无统计学意义（F =1.56,P＞0.05）,LPS-DC 2.4组对T淋巴细胞刺激能力明显高于其他3组（t值为1.53～1.82,P值均小于0.01）. 结论 成功构建能高效表达CCR7的DC 2.4细胞对CCL19有较高的趋化性且对免疫功能无明显Objective To observe the influence of infection of murine chemokine receptor-7 recombinant lentivirus on the immunogenicity and migration of dendritic cell strain DC 2.4 cells.Methods DC 2.4 cells were routinely cultured.Lentiviruses carrying GFP and those with up-regulated CCR7 were constructed.DC 2.4 cells were divided into DC 2.4 group （without any treatment）,GFP-DC 2.4 group （infected with GFP-carrying lentivirus）,and CCR7-DC 2.4 group （infected with CCR7-carrying lentivirus labeled by GFP） according to the random number table.The expressions of surface molecules MHC Ⅱ,CD80,CD86,and CCR7 were detected by flow cytometry,Western blotting,and confocal laser scanning microscope.The migration of cells was detected by chemotaxis assay in vitro.The immunogenicity of cells was detected with mixed lymphocyte reaction.LPS-DC 2.4 group was set up as positive control.Data were processed with one-way analysis of variance and t test.Results Lentiviruses carrying stably-expressing CCR7 were constructed,and the transfection rate of which into DC 2.4 cells was 87.4％.There was no statistically significant difference among DC 2.4,GFP-DC 2.4,and CCR7-DC 2.4 groups in the expressions of MHC Ⅱ,CD80,and CD86 as showed by flow cytometry （with F values from 0.17 to 1.19,P values all above 0.05）.The protein expression of CCR7 of cells in CCR7-DC 2.4 group （45.1 ± 2.1） was obviously higher than that in DC 2.4 and GFP-DC 2.4 groups （25.3 ± 1.4,28.6 ± 0.9,F =162.90,P ＜ 0.01）,while the difference of which between DC 2.4 group and GFP-DC 2.4 group was not statistically significant （t =2.20,P ＞ 0.05）.The fluorescence intensity of CCR7 in CCR7-DC 2.4 group was obviously increased compared with that of DC 2.4 group.The chemotaxic migration rate of cells in CCR7-DC 2.4 group with the influence of CCL19 was （41.0 ± 2.0） ％,which was significantly higher than that of DC 2.4 and GFP-DC 2.4 groups [（6.0 ± 0.5） ％,（6.8 ± 0.3） ％,F =84.21,P ＜ 0.01].There was no statistically significant di

关 键 词：慢病毒感染 受体 趋化因子 树突细胞 免疫原性 细胞运动 

分 类 号：R[医药卫生]