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目的 探讨肝素体外诱导的树突状细胞(DC)促进慢性乙型肝炎(CHB)患者CD4+T细胞亚群由Th0向Th1分化的机制. 方法 分离CHB患者外周血单个核细胞,以重组人白细胞介素(rhIL)-4、重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)和肝素诱导培养DC.流式细胞仪和定量PCR检测肝素处理及未处理的DC中Toll样受体(TLRs)分子表达水平.对高表达的TLRs,用TLRs-siRNA干扰处理后检测DC的功能变化.免疫磁珠分离外周血CD4+T细胞亚群.酶联免疫吸附(ELISA)法检测Poly(I∶C)和R848刺激后DC上清液中IL-12及DC和CD4+T细胞混合培养上清液中干扰素(IFN)v和IL-4的含量.均数的比较采用配对t检验.结果 肝素处理的DC (heparin-DC)其TLR3表达水平明显高于未处理的DC (t=2.849,P＜0.05;t=3.027,P＜ 0.05);而两组间TLR7和TLR8的表达水平差异无统计学意义;Heparin-DC在Poly(I∶C)刺激下分泌IL-12水平明显高于未处理的DC(t=8.68,P＜0.01),同时高于R848刺激的heparin-DC(t=19.01,P＜0.01);经TLR3-siRNA干扰后,heparin-DC分泌的IL-12水平明显降低(t=31.49,P＜0.01).TLR3-siRNA干扰后的heparin-DC与同种异体的CD4+T细胞共培养,其CD4+IFN Y +T细胞比例与未干扰组相比明显降低(1.64％±0.57％对比6.31％±0.88％,P＜0.01),同时其上清液中Thl型细胞因子IFNY的水平也显著降低(t=20.83,P＜0.01),而IL-4分泌水平变化无统计学意义.结论 肝素在体外可能通过上调CHB患者树突状细胞TLR3的表达水平而促进外周血Th0向Th1细胞的分化.“,”Objective To investigate the mechanisms underlying the ability ofheparin-treated dendritic cells (DCs) to promote Th0 to Th1 differentiation in chronic hepatitis B (CHB).Methods Peripheral blood mononuclear cells (PBMCs) were isolated from CHB patients and cultured in RPMI-1640 with recombinant GM-CSF and IL-4 with or without heparin to obtain DCs for study.The levels of Toll-like receptors (TLRs) on the DCs were measured using FACS and qPCR techniques.DC subsets with high expression of TLRs were selected for analysis of functional changes by treatment with the corresponding TLR-siRNA.The CD4+ T cell subpopulation was purified from peripheral blood by Dynal immunomagnetic beads,and then the production of IL-12 by DCs in the presence of poly(I∶C) or R848 and ofIFN and IL-4 by Th cells co-cultured with DCs was evaluated by ELISA.The t-test was used for statistical analysis.Results TLR3 expression,and not expression of TLR 7 or TLR8,was significantly increased in heparin-treated DCs as compared to levels detected in the DCs without heparin treatment (t =2.849,P ＜ 0.05;t =3.027,P ＜ 0.05).The level of IL-12 produced by heparin-treated DCs stimulated with poly(I∶C) was obviously higher than that produced by DCs without heparin treatment and stimulated with poly(I∶C) (t =8.68,P ＜ 0.01) or with R848 (t =19.01,P ＜ 0.01).However,the IL-12 production by TLR3-siRNA transfected-DCs was significantly reduced (t =31.49,P ＜ 0.01).When Th cells from allogenic patients with CHB were co-cultured with the TLR3-siRNA transfectedDCs,the frequency ofCD4+ IFN+ cells was significantly reduced (1.64±0.57％ vs.6.31±0.88％,P ＜ 0.01),as was the capability of Thl to generate IFNg (t =20.83,P＜ 0.01).Conclusion Heparin may have up-regulated the TLR3 expression level of DCs,and sequentially promoted Th0 to Th1 differentiation.