慢性乙肝患者树突状细胞对Th1/Th2分化的影响

探讨慢性乙肝患者树突状细胞(dendritic cells,DC)对CD4+Th细胞亚群分化的影响。分离慢性乙肝患者外周血单个核细胞(PBMC),以rhIL-4(50 ng/ml)、rhGM-CSF(10 ng/ml)和rhTNF-α(100 u/ml)诱导培养DC。以流式细胞仪检测DC表面CD1a、CD83、CD80、CD86、HLA-DR分子表达情况。MTT法检测DC刺激同种异体淋巴细胞增殖能力。免疫磁珠分离外周血CD4+T细胞亚群,PMA+Ionomycin刺激后胞内荧光染色,流式细胞仪检测辅助性T细胞(helper T cell,Th)内特征性细胞因子IFN-γ/IL-4以判断Th1/Th2分化。ELISA法检测DC或Th细胞培养上清中IL-6、IL-12、IFN-γ和IL-4的含量。结果:慢性乙肝患者的DC表达CD1a、CD83、CD80、CD86、HLA-DR分子水平明显低于正常人(P<0.01);培养至第7天,慢性乙肝患者DC分泌的IL-12水平低于正常人(P<0.01),而分泌的IL-6水平增高(P<0.05)。与正常人相比,慢性乙肝患者外周血中Th1细胞占CD4+T细胞的百分比较低(P<0.01),其Th细胞培养上清中IFN-γ的量也较低(P<0.01)。患者DC与同种异体的健康人Th细胞共培养,刺激Th1型细胞因子IFN-γ产生的能力低于正常人(P<0.01)。慢性乙肝患者体内DC功能的异常可能导致了外周血Th1细胞分化不足。

Influence of dendritic cells on Th1/Th2 differentiation in patients with chronic hepatitis B

To investigate the functions of dendritic cells(DC) and its influences on the Th1/Th2 differentiation in patients with chronic hepatitis B infection(CHB).The peripheral blood mononuclear cells(PBMC) were isolated and then cultured in RPMI 1640 with recombinant GM-CSF(10 ng/ml) and IL-4(50 ng/ml) to obtain DC,which was further matured by stimulation with recombinant TNF-α(100 ng/ml).The functions of DC stimulating proliferation of allogenic T lymphocytes was detected through MTT method,and the phenotypes of DC,including CD1a,CD83,CD80、CD86,HLA-DR,were detected by flow cytometer(FCM).The CD4+ Th cell subpopulation in peripheral blood was separated by immunomagnetic beads,followed by stimulation with PMA+Ionomycin and then,the differentiation of Th1/Th2 cell was detected by flow cytometry using intracellular fluorescent staining of the IFN-γ and IL-4.The levels of IL-6,IL-12,IFN-γ and IL-4 in culture supernatants of DC or Th cell were detected with ELISA.It was found that the expression of CD1a,CD83,CD80,CD86 and HLA-DR in the DC of the patients with CHB was markedly lower than that of health adults(P<0.01).On the 7th culture day,the concentration of IL-12 in patients was also lower,while the IL-6 production was higher than that of control group(P<0.05).In comparison with health adults,the percentages of Th1 cells in CD4+ T cells in peripheral blood of CHB patients and the contents of IFN-γ in Th1 cell supernatants of them were both lower(P<0.01).When co-cultured with Th cells from allogenic health adults,the capability of the CHB patients' DC to make Th1 cells,characterized with IFN-γ secreation,was remarkably weaker than that of health adults' cells(P<0.01).It is evident that Th1 cell differentiation is impaired in patients with CHB,which may be correlated with the dysfunctions of DC.