HBsAg对健康成人外周血单核细胞来源树突状细胞Tim-3信号通路的影响

Effects of hepatitis B virus surface antigen on Tim-3 signaling pathway of dendritic cells

目的探讨HBsAg对健康成人外周血单核细胞来源树突状细胞(MD—DCs)的免疫活性及对T细胞免疫球蛋白黏蛋白分子3(Tim-3)和下游信号分子核因子-κB(NF—κB)蛋白表达的影响。方法分离健康成人外周血单核细胞,经GM—CSF、IL-4联合诱导为树突状细胞(DCs),流式细胞术检测DCs表面标志物表达水平。MD—DCs分4组添加不同浓度HBsAg(0、1、2、5μg/ml),分别设为对照组、+1μg/ml组、+2μg/ml组、+5μg/nl组,Western印迹法检测Tim-3、NF—κB信号蛋白表达,细胞增殖与毒性检测试剂检测MD—DCs刺激同种异体淋巴细胞增殖的能力,ELISA法检测细胞因子分泌水平。结果外周血来源单核细胞成功诱导分化为DCs。与对照组相比,+2μg/ml及+5μg/ml组MD—DCs Tim-3及NF—κB表达水平均上调,刺激同种异体T淋巴细胞增殖能力均增强,炎症因子IL-6、IL-10、IFN—γ分泌水平均增高(均P<0.05),而+1μg/ml组较对照组无统计学差异(均P>0.05)。与其他3组相比,+5μg/ml组MD—DCs Tim-3、NF—κB、炎症因子分泌水平增高,刺激同种异体淋巴细胞增殖能力增强(均P<0.05)。结论 HBsAg上调MD—DCs Tim-3及NF—κB表达水平,增强MD—DCs免疫活性,且刺激作用随HBsAg浓度的增高而增强。

Objective TO investigate the effect of hepatitis B virus surface antigen （HBsAg） on the expression of T cell im- munoglobulin and mucin domain-containing molecules-3 （Tim-3） and the downstream signal molecule Nuclear Factor-kB （NF-kB） of human menocyte-derived dendritic cells （MD-DCs）. Methods Monocytes obtained from normal adult peripheral blood were co-cultured with GM-CSF and IL-4 to induce to dendritic cells, the cell phenotypes expressions on MD-DCs were detected by flow cytornetry. Different concentrations of HBsAg （0,1μg/ml, 2μg/ml, 5μg/ml） were added in the culture medium. The expressions of signaling proteins were determined by Western blotting assay, the lymphocytes-stimulatory capacity of MD-DCs was determined with a proliferation and cytotoxicity detection kit （MTS）, and the concentrations of cytokines in the supernatant were determined by enzyme linked immunosorbent assay （ELISA）. Results Monocytes were successfully induced to dendritic cells. Compared with the control group, the expressions of signaling proteins Tim-3 and NF- K B of the HBsAg （2#g/ml or 51Jg/ml） stimulation group were both up-regulated, the lymphocytes-stimulation capacity was enhanced, and the secretion levels of IL-6, IL-10 and IFN-.,/ were increased （P〈0.05）, while there was no significant difference between llJg/rnl HBsAg stimulation group and control group. Compared with the other three groups, the treatment with 5#g/ml HBsAg led to increased expressions of Tim-3, NF-K B and cytokines, and enhanced lymphocytes-stimulation capacity of MD-DCs （P〈0.05）. Conclusion HBsAg stimulation can increase the expressions of Tim-3 and downstream signaling molecule NF-kB of MD-DCs, and enhance the immune function of MD-DCs in a dose-dependent manner.