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目的通过体外研究白细胞介素(IL)-10对慢性乙型重型肝炎患者树突状细胞(DC)活性的抑制作用特点,探讨IL-10抑制慢性乙型重型肝炎患者产生成熟DC的作用。方法选取2012年3月-9月16例慢性乙型重型肝炎住院患者,采集外周静脉血,经密度梯度离心法分离获得外周血淋巴细胞(PBMC),然后采用重组人IL-4和重组人粒-巨噬细胞集落刺激因子体外刺激PBMC诱导产生DC,再分别用IL-10或(及)用乙型肝炎核心抗原(HBc Ag)处理DC;用流式细胞术检测各组DC表面抗原;用酶联免疫法检测各组细胞分泌的细胞因子;采用细胞杀伤实验检测体外诱导的淋巴细胞毒活性;由此分为4组,A组为阴性对照组,B组为IL-10处理组,C组为HBc Ag处理对照组,D组为IL-10预处理后再用HBc Ag刺激组。结果与未做处理的A组相比,B组的DC表面抗原HLA-DR、CD 83、CD 86的阳性表达率显著降低(P<0.01),DC分泌的干扰素(IFN)-γ和IL-12 p 70亦显著降低(P<0.05);C组的DC表面抗原HLA-DR、CD 83、CD 86的阳性表达率及DC分泌的IFN-γ和IL-12 p 70表达量较A组显著升高(P<0.01);D组与C组相比,D组DC的HLA-DR、CD 83、CD 86的阳性表达率则显著降低(P<0.05),其DC分泌的IFN-γ和IL-12 p 70表达量亦显著降低(P<0.01)。在40︰1的靶效比条件下,与未做处理的A组相比,B组的DC诱导淋巴细胞对Hep G 2的杀伤率显著降低(P<0.01);C组的DC诱导淋巴细胞对Hep G 2的杀伤率较A组显著升高(P<0.05);D组DC诱导淋巴细胞对Hep G 2的杀伤率较C组显著降低(P<0.01)。结论 IL-10可抑制慢性乙型重型肝炎患者的DC的活性,使其诱导淋巴细胞毒活性减弱,从而导致自体免疫耐受。
Objective To investigate the inhibitory effect of interleukin-10 (IL-10) on the dendritic cell (DC) activity in patients with chronic severe hepatitis B in vitro and to explore the role of IL-10 in inhibiting the production of mature DC in patients with chronic severe hepatitis B. Methods From March to September in 2012, 16 patients with chronic severe hepatitis B were enrolled in this study. Peripheral venous blood was collected and peripheral blood lymphocytes (PBMCs) were isolated by density gradient centrifugation. Then the recombinant human IL-4 and recombinant human granulocyte DCs induced by PBMC were stimulated by macrophage colony-stimulating factor in vitro, then DCs were treated with IL-10 or HBcAg respectively; DC surface antigens were detected by flow cytometry; The cytokines secreted by cells in each group were detected by enzyme-linked immunosorbent assay. The cytotoxicity induced by in vitro cytotoxicity was detected by cell killing assay. The cells were divided into 4 groups: A group, negative control group, B group, IL-10 treatment group, C The control group was treated with HBc Ag, the D group was treated with IL-10 pretreatment and then stimulated with HBc Ag. Results Compared with untreated group A, the positive expression rates of HLA-DR, CD 83 and CD 86 of group B were significantly decreased (P <0.01), and that of IFN-γ and IL secreted by DC -12 p70 also decreased significantly (P <0.05). The positive expression rates of HLA-DR, CD 83, CD 86 and the levels of IFN-γ and IL-12 p 70 secreted by DC in group C were significantly higher than those in group A (P <0.01). Compared with group C, the expression of HLA-DR, CD83 and CD86 in group D was significantly lower than that in group C (P <0.05) And IL-12 p 70 expression was also significantly decreased (P <0.01). Compared with untreated group A, the killing rate of Hep G2 by DCs induced by DCs in group B was significantly decreased (P <0.01) at the target ratio of 40: 1. DCs in group C induced lymphocytes The killing rate of Hep G2 was significantly higher than that of group A (P <0.05). The killing rate of Hep G2 induced by DC induced by DC in group D was significantly lower than that of group C (P <0.01). Conclusion IL-10 can inhibit the activity of DC in patients with chronic severe hepatitis B, leading to weakened lymphocyte cytotoxicity, leading to autoimmune tolerance.