下调DC-SIGN-ManLAM结合信号恢复树突状细胞活化初始T细胞的能力

目的 探讨利用RNAi下调DC-SIGN与ManLAM结合信号对树突状细胞(dendritic cells,DCs)活化初始T细胞能力的影响.方法 利用筛选出的人DC-SIGN分子RNAi有效靶点,构建并包装产生慢病毒表达载体.实验分为:干扰组、空载体组和空白对照组.利用慢病毒表达载体下调DCs表面DC-SIGN分子水平,流式细胞仪、RT-PCR及Western blot检测各组DCs DC-SIGN分子水平变化,在LPS存在下,与ManLAM共培养18 h后,流式细胞仪检测DCs表面成熟标志物,ELISA方法检测DCs 刺激CD4+CD45RA+初始T细胞向Th1分化能力,以了解下调DC-SIGN与ManLAM结合信号对DCs活化初始T细胞的能力的影响.结果 ①包装慢病毒产生浓缩病毒悬液的滴度为1×109 TU/ml.②流式细胞仪检测干扰组DCs表面DC-SIGN分子表达水平较其余2组显著降低(F=14.19,P<0.05),RT-PCR检测干扰组DC-SIGN目的条带灰度值/内参灰度值(0.252 5±0.139 6)与空白对照组(0.572 2±0.190 9)及空载体组(0.548 7±0.119 3)相比灰度值显著下降 (F=8.142,P<0.05),Western blot检测干扰组DCs内DC-SIGN蛋白水平均显著低于空载体组和空白对照组(F=51.376,P<0.05).③干扰组DCs的成熟标志物CD86(F=15.59,P=0.004)、CD83(F=18.03,P=0.003)、HLA-DR(F=7.684,P=0.022)水平较空载体组和空白对照组明显增高.④DCs与初始T细胞共培养,干扰组分泌IFN-γ(F=18.434,P=0.003)、IL-2(F=31.08,P=0.001)水平较空载体组和空白对照组显著增高.结论 利用RNAi慢病毒载体下调DCs表面DC-SIGN水平,减弱DC-SIGN与ManLAM结合信号,能够恢复DCs的成熟受阻,最终使得DCs活化初始T细胞的能力恢复.

Down-regulation of DC-SIGN-ManLAM binding signal recovers ability of dendritic cells to activate naive T lymphocytes

Objective To study the effect of DC-SIGN-ManLAM binding signal on the ability of dendritic cells(DCs) to activate naive T lymphocytes with RNA interference(RNAi).Methods Effective RNAi targeting human DC-SIGN molecules were used to construct lentivirus vectors.DC were divided into RNAi group,empty carrier group,and blank control group.Changes of DC-SIGN molecules in DC were detected by flow cytometry,RT-PCR and Western blotting,respectively.In the presence of LPS,18 h after DC and naive T lymphocytes were c...