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目的观察慢性乙型肝炎(CHB)患者单核细胞来源树突状细胞(moDC)Toll样受体(TLR)的表达,并分析抗病毒相关TLR(TLR3、TLR4、TLR7、TLR8、TLR9)在CHB患者moDC的表达特征及意义。方法用羟乙基淀粉(HES)分离10例CHB患者及15例健康对照外周血单个核细胞(PBMC),经重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)、重组人白细胞介素4(rhlL-4)诱导培养树突状细胞(DC),经流式细胞仪用特异性单克隆抗体检测不同成熟阶段DC的TLR表达,并以HBcAg负载未成熟DC,应用FACS分析HBcAg的负载效率及细胞内IFN-γ、IL-4表达水平。结果moDC不同成熟阶段TLR的表达水平不同;CHB患者的未成熟moDC(imDC)表达的TLR7、TLR8低于健康对照(75.9%、1.0%比98.4%、15.4%,P<0.05),CHB患者成熟moDC(mDC)表达TLR3、TLR7低于健康对照(9.5%、79.7%比11.5%、90.7%,P<0.05);应用HBcAg分别于细胞培养第3天和第5天冲击DC,第1次冲击后48 h HBcAg的膜内、膜外负载率分别为53.0%和7.5%,第2次冲击后分别为80.2%和19.0%;HBcAg负载或未负载的moDC,均刺激淋巴细胞高表达IFN-γ,较少表达或不表达IL-4,呈现显著的Th1免疫应答。结论CHB患者moDe的抗病毒相关TLR表达低下,且可能是其DC功能低下的重要原因之一;应用HBcAg冲击imDC,可使其有效负载HBcAg,负载后DC的成熟过程及功能不受影响,仍以诱导Th1免疫应答反应为主。
Objective To investigate the expression of Toll-like receptor (TLR) in monocyte-derived dendritic cells (DCs) from patients with chronic hepatitis B (CHB) and to analyze the role of TLR3, TLR4, TLR7, TLR8 and TLR9 in CHB The expression and significance of moDC in patients. Methods 10 cases of CHB patients and 15 healthy control peripheral blood mononuclear cells (PBMCs) were isolated by using hydroxyethyl starch (HES). Recombinant human leukocyte-antigen (rhGM-CSF) Dendritic cells (DCs) were induced by rhlL-4, TLR expression was detected by flow cytometry with specific monoclonal antibodies at different stages of maturation, and immature DCs were loaded with HBcAg. FACS analysis of HBcAg Load efficiency and intracellular IFN-γ, IL-4 expression levels. Results The expression levels of TLR in different stages of moDC were different. The expression of TLR7 and TLR8 in imDC in immature CHB patients was lower than that in healthy controls (75.9%, 1.0% vs 98.4%, 15.4%, P <0.05) The expression of TLR3 in moDC (mDC) was lower than that in healthy controls (9.5%, 79.7% vs. 11.5%, 90.7%, P <0.05). DCs were challenged with HBcAg on day 3 and day 5, After 48 h HBcAg intra-membrane and extracellular loading rates were 53.0% and 7.5%, respectively, after the second shock were 80.2% and 19.0%; HBcAg loaded or not loaded moDC, were stimulated lymphocyte highly expressed IFN-γ , Less or no IL-4 expression, showing a significant Th1 immune response. Conclusions The expression of antiviral TLR of moDe in CHB patients is low, and may be one of the important reasons for its low DC function. Impacting imDC with HBcAg can effectively load HBcAg and the maturation process and function of DC after loading are unaffected To induce Th1 immune response.