慢性乙肝患者树突状细胞对Th1/Th2分化的影响

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

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。

To investigate the functions of dendritic cells （DC） and its influences on the Th1/Th2 differentiation in patienis 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 CDla, 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 intra cellular fluorescent staining of the IFN 7 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 EI.ISA. It was found that the expression of CDla, CD83, CD80, CI）86 and HI.A-DR in the DC of the patients with CHB was markedly lower than that of health adults （P〈0.01）. On the 7^th 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.