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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Westernblot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1×109TU/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。
Objective To investigate the effect of RNAi on the ability of dendritic cells (DCs) to activate naive T cells by down-regulating DC-SIGN and ManLAM binding signals. Methods The target RNAi of human DC-SIGN was screened out and constructed and packaged to generate lentivirus expression vector. The experiment was divided into: interference group, empty vector group and blank control group. The expression of DC-SIGN on the surface of DCs was down-regulated by lentivirus expression vector. The DC-SIGN level of DCs in each group was detected by flow cytometry, RT-PCR and Western blotting. After cultured with LPS for 18 h, The maturation markers of DCs were detected by cytometry. The ability of DCs to stimulate the differentiation of CD4 + CD45RA + T cells into Th1 cells was detected by ELISA, and the effect of DC-SIGN and ManLAM binding signals on the ability of DCs to activate primary T cells was investigated. Results ① The lentivirus concentration of the lentivirus was 1 × 109TU / ml. ② Flow cytometry showed that DC-SIGN expression on the surface of DCs was significantly reduced (F = 14.19, P <0.05) Compared with the blank control group (0.572 2 ± 0.190 9) and the empty vector group (0.548 7 ± 0.119 3), the gray value decreased significantly (0.252 5 ± 0.139 6) (F = 8.142, P <0.05) The level of DC-SIGN protein in DCs detected by interference group was significantly lower than that in control group and blank vector group (F = 51.376, P <0.05). ③The levels of CD86 (F = 15.59, P = 0.004), CD83 (F = 18.03, P = 0.003) and HLA-DR The group was significantly higher. (4) DCs were co-cultured with naive T cells. The levels of IFN-γ (F = 18.434, P = 0.003) and IL-2 (F = 31.08, P = 0.001) were significantly increased in the DCs compared with the blank control group and the blank control group. Conclusion Down-regulation of DC-SIGN level on the surface of DCs by RNAi lentiviral vector attenuates the binding of DC-SIGN and ManLAM, which can restrain the maturation of DCs and eventually restore the ability of DCs to activate primary T cells.