e抗原阳性慢性乙型肝炎患者外周血树突状细胞Toll样受体3的表达及意义

目的探讨Toll样受体3（TLR3）的表达与HBV感染后宿主免疫清除障碍的关系。方法选取轻度CHB患者50例,健康对照者48名,其外周血用免疫磁珠细胞分选法获得纯化的CD14^＋单核细胞,用RPMI 1640培养基培养,并且用人粒细胞-巨噬细胞集落刺激因子和hIL-4诱导单核细胞成为未成熟的髓样树突状细胞（mDC）,加入聚肌胞刺激后获得成熟的mDC。分别在剌激后0、12、24、48h用流式细胞仪检测TLR3、CD86、HLA-DR和CD1a的表达,实时PCR检测TLR3的表达变化。结果健康对照组中mDC在刺激后24h,TLR3表达较0 h时上调显著（P〈0.05）,48h时TLR3的表达与0h相比差异无统计学意义（P〉0.05）;患者组在刺激后12、24h TLR3的表达与0h时相比,上调不明显,48h时TLR3的表达显著上调（P〈0.05）。实时PCR检测mDC上TLR3 mRNA结果发现,对照组TLR3 mRNA在刺激后12h的表达水平较0h显著上升（P〈0.05）,也显著高于患者组刺激后0、12、24h的表达水平;患者组刺激后48h的TLR3 mRNA表达水平较0h显著上升（P〈0.05）。与0h比较,健康对照组在刺激后12、24h和48h,CD86的表达水平显著高于患者组（P〈0.05）。患者组与对照组间CD1a和HLA-DR的表达差异无统计学意义。结论慢性HBV感染者mDC受聚肌胞刺激后TLR3表达异常,协同刺激因子CD86表达低下,可能造成宿主对HBV感染的免疫清除障碍,导致疾病慢性化。

Expression of Toll-like receptor 3 on peripheral blood dendritic cells in HBeAg positive patients with chronic hepatitis B

OBJECTIVE: To elucidate the roles of Toll-like receptor 3 (TLR3) on dendritic cells (DCs) in HBV infection. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from 48 healthy volunteers (HV) and 50 chronically HBV-infected patients (CH). DCs were induced and proliferated in a culture medium with rhGM-CSF and rhIL-4. We stimulated DCs with poly I:C and then TLR3, HLA-DR, and CD86, and CD1a expressions were examined by flow cytometry at 0 h, 12 h, 24 h and 48 h. The mRNA expressions of TLR3 were quantified by real-time PCR. RESULTS: TLR3 expression on DCs before the poly I:C stimulation and afterwards on the 12 h, 24 h, and 48 h were 69.2%+/-20.4%, 76.0%+/-18.6%, 78.2%+/-19.5% and 85.5%+/-6.9% respectively in the CH group, and 70.8%+/-11.2%, 67.5%+/-20.9%, 86.3%+/-14.7%, 68.6%+/-16.9% in the HV group. The expressions of TLR3 were up-regulated significantly at 24 h and 48 h after stimulation with poly I:C in the HV group, and in the CH group they were not significantly increased at 24 h but obviously increased at 48 h. The mRNA expressions of TLR3 increased significantly at 12 h in the HV groups, and at 48 h in CH group. The rate of CD86 expressions increased after poly I:C stimulation, and the increased rates were 12.6%+/-9.8%, 23.8%+/-20.0%, 20.7%+/-14.3% in the CH group, and 31.0%+/-25.0%, 43.4%+/-24.7%, 44.6%+/-25.5% in the HV group at 12 h, 24 h and 48 h after poly I:C stimulation. There was a marked increase of the expression level of CD86 in the HV group. In contrast, the level was only slightly increased in the CH group (31.0% vs 12.6%). The differences between the two groups were significant at 24 h and 48 h. No significant differences were detected in HLA-DR and CD1a between the two groups. CONCLUSIONS: The increase of expression level of TLR3 is slower in the CH group than that in the HV group. A marked increase of the expression level of CD86 is observed in the HV group. Our results suggest that abnormal expression of TLR3 and CD86 may relate to the persistance of HBV infection.