慢性乙型肝炎患者单核细胞来源树突状细胞Toll样受体的表达特征分析

The expression profile of Toll-like receptor on monocyte-derived dendritic cells in patients with chronic hepatitis B virus infection

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 evaluate the expression of Toll-like receptor（TLR） on the monocytederived dendritic cells（DC） from chronic hepatitis B（CHB） patients and to analyze the expression profile and significance of the TLR such as TLR3, TLR4, TLR7, TLR8 and TLR9, which are associated with immune response to viral infection. Methods Peripheral blood mononuclear cell（PBMC） centrifugated by the hydroxyethyl starch（HES） centrifugation were cultured and induced into DC by granulocyte-macrophage colony stimulating factor（GM-CSF） and interleukin-4 （ IL-4）, and their morphology and phenotype were detected by the inverted microscope and flow cytometry respectively. Monocyte-derived DC were obtained from 10 chronically hepatitis B virus（HBV）-infected patients and 15 healthy volunteers. TLR3, TLR4, TLR7, TLRS, TLR9 expression on immature and mature DC were analyzed by FACS Calibur. DC was pulsed with HBcAg on day 3 and 5, then DC maturation and ability to process HBcAg and to stimulate autogeneic T cells were evaluated. Results Monocyte-derived DC developed different TLR expression patterns as they went through different maturation stages. TLRT, TLR8 expressions on immature DC and TLR3, TLR7 expressions on mature DC were lower in CHB than in control（for TLR7, TLR8 expression on immature DC： 75.9%, 1. 0% vs 98.4% ,15.4%, P〈 0.05, for TLR3, TLR7 expression on mature DC： 9.5%, 79.7% vs 11.5%, 90.7%, P〈 0. 05）. The HBcAg loading ratio on surface or intracellular of DC was 7. 5% and 53.0% respectively 48 hours after the first antigen pulse, while the ratio was 19.0% and 80. 2% respectively after the second pulse. The mixed culture of pulsed DC and autogeneic lymphocytes showed high level expression of interferon （IFN）-γ and less or even no expression of IL-4 （P〈 0.05）. The ability of De to induce T ceils producing IFN-7 was not different between the groups. Conclusions In chronically HBV infection, the expressions of antiviral immune associated TLR on monocyte-derived DC are reduced and may be involved in a mechanism of persistent HBV infection. HBcAg can be detected on DC＇s surface and in the cells after pulsing with HBcAg protein. HBcAg loading will not influence the DC maturation process and their functions, in the other word, stimulating autogenic T cells producing IFN-γ and inducing Thl immune response.