乙型肝炎表面抗原对单核细胞来源的树突状细胞Toll样受体4信号通路的影响
Effects of hepatitis B surface antigen on toll like receptor 4 signaling pathway of monocyte-derived dendritic cells
目的 探讨HBsAg对脂多糖刺激成熟的单核细胞来源的树突状细胞(MoDC)功能和细胞表面TLR4信号通路的影响.方法 将健康者PBMC诱导分化为未成熟MoDC,加入不同浓度HBsAg体外刺激,再经脂多糖刺激获得成熟MoDC.Western印迹检测各组TLR4、核因子-κB、丝裂原激活蛋白激酶信号通路蛋白表达,ELISA检测细胞因子,细胞增殖与毒性检测试剂盒检测刺激同种异体淋巴细胞增殖的能力.两组间比较采用t检验,多组间比较采用单因素方差分析.结果 未经HBsAg刺激的对照组TLR4、核因子-κB、pp38、磷酸化c-Jun氨基末端激酶1/2 (pJNK1/2)蛋白分别为0.43±0.14、0.69±0.27、0.21±0.09和0.44±0.13,5μg/mL HBsAg刺激后分别增至1.22±0.39、1.37±0.40、0.74±0.17和0.92±0.15(t值分别为-3.14、-3.09、-5.07和-2.93,均P<0.05);但细胞外信号调节激酶1/2表达下降(2.43±0.80比1.13±0.23,t=3.18,P<0.05).5μg/mL HBsAg刺激后的MoDC刺激同种异体T淋巴细胞,其刺激指数高于对照组,且随MoDC/淋巴细胞比值增高而增高(t值分别为-5.72、-4.93和-4.96,均P<0.05).5μg/mL HBsAg刺激后MoDC分泌IL-12、TNF-α分别为(637.8±19.2)和(710.6±47.4)pg/mL,对照组分别为(597.8±15.4)和(568.5±35.0)pg/mL(t值分别为-5.07和-6.70,均P<0.05).结论 HBsAg可激活MoDC表面TLR4信号通路,促进细胞因子分泌,增强刺激T淋巴细胞增殖的能力.
更多Objective To investigate the effects of hepatitis B surface antigen (HBsAg) on the tolllike receptor 4 (TLR4) signaling pathway of lipopolysaccharide (LPS)-stimulated human monocytederived dendritic cells (MoDC).Methods The peripheral blood mononuclear cells (PBMC) were isolated from healthy volunteers,and then induced and proliferated to immature MoDC in vitro.Mature MoDC were obtained by HBsAg and LPS stimulation.The expressions of TLR4,nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) protein were determined by Western blotting assay,and the concentrations of cytokines in the supermatant were detected by enzyme-linked immunosorbent assay (ELISA).In addition,the capacity of MoDC to stimulate allogenic lymphocyte was measured by MTS assay.Statistical analyses were performed by t test and analysis of variance.Results The expressions of TLR4,NF-κB,phosphorylation p38 (pp38),phosphorylation c-Jun N-terminal kinase 1/2 (pJNK1/2) protein were upregulated in the HBsAg-stimulated groups with 5 μg/mL HBsAg compared with those of control group (0.43±0.14 vs 1.22±0.39,0.69±0.27 vs 1.37±0.40,0.21±0.09 vs 0.74±0.17,and 0.44±0.13 vs 0.92±0.15,t=-3.14,-3.09,-5.07,-2.93,respectively,all P<0.05),while the expression of phosphorylation extracellular signal-regulated kinase 1/2 (pERK 1/2) was downregulated in HBsAg stimulated groups,which were compared with that of control group (2.43±0.80 vs 1.13±0.23,t=3.18,P<0.05).The capacity of MoDC treated with 5 μg/mL HBsAg to stimulate allogenic T lymphocytes was enhanced,and the stimulation index increased with DC-to-lymphocyte ratio (t=-5.72,-4.93,-4.96,P<0.05).In addition,the 5 μg/mL HBsAg-pulsed MoDC were found to have a stronger capacity to produce interleukin (IL)-12 and tumor necrosis factor-α (TNF-α) compared with those of control group ([637.8±19.2] pg/mL vs [597.8±15.4] pg/mL and [710.6±47.4] pg/mL vs [568.5± 35.0] pg/mL,respectively; t=-5.07,-6.70,both P<0.05).Conclusion HBsAg can activate TLR4 signaling pathway of MoDC,promote the secretion of cytokines and enhance the ability of MoDC stimulating T cell proliferation.
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