摘要
目的探讨HBeAg对慢性乙型肝炎（CHB）患者外周血单个核细胞（PBMC）功能的调节作用。方法以重组的HBeAg体外刺激CHB患者和健康志愿者的PBMC，用流式细胞术和酶联免疫吸附试验法检测其刺激前后Th1／Th2型细胞因子的变化情况，并观察HBeAg对CHB患者PBMC表面细胞程序性死亡受体（PD）1及其配体（PD—L）l表达的影响。两组间资料比较采用独立样本t检验IPD—I／PD—L1表达水平与HBVDNA拷贝数的相关性采用Spearman相关分析。结果HBeAg刺激后可使HBeAg阴性CHB患者和健康志愿者CD3＋CD4＋T淋巴细胞内干扰素（IFN）Y表达水平（0．17％±0．08％与0．17％±0．04％）明显低于未刺激组（0．30％±0．16％与0．32％±0．12％），t值分别为-2．382和-4．190，P值均〈0．01;培养上清液中白细胞介素（IL）-6、IL—10和肿瘤坏死因子α含量明显高于未刺激组（HBeAg阴性CHB患者的t值分别为2．504，3．583和4．324，健康志愿者t值分别为3．542，6．246和5．273，P值均〈0．01）。HBeAg刺激PBMC后，HBeAg阴性CHB患者和健康志愿者CD14＋细胞表面PD—L1表达水平分别为13．02％±4．98％和3．10％±2．47％，明显高于未刺激组的5．89％±1．56％和0．97％±0．83％，t值分别为4．815和3．454，P值均〈0．05。基础状态下在HBeAg阳性CHB患者外周血中，CD3＋CD4＋T淋巴细胞内IFNY表达水平为0．23％±0．09％，明显低于HBeAg阴性CHB患者和健康志愿者的0．34％±0．15％和0．35％±0．09％（t=-3．177，P〈0．01；t=-4．541，P〈0．01）；而IL-4表达水平为0．39％±0．16％，明显高于HBeAg阴性CHB患者和健康志愿者的0．26％±0．12%和0．23％±0．12％，t值分别为3．382和4．393，P值均〈0．01。基础状态下在HBeAg阳性CHB患者外周血中，CD3＋T淋巴细胞表面PD-1和PD—L1表达水平明显高于HBeAg阴性CHB患者及健康志愿者（P值均〈0．01），CD14＋T淋巴细胞表面PD-L1表达水平显著高于HBeAg阴性患者和健康志愿者，t值分别为5．092和5．473，P值均〈0．01}HBeAg阴性CHB患者外周血中CD3＋T淋巴细胞表面PD-L1表达水平明显高于健康志愿者（t=3．214，P〈0．01）。结论HBeAg可以明显抑制Th1型细胞因子IFNγ的产生，促进Th2型细胞因子IL-6和IL-10分泌，上调外周血PBMC表面PD-1／PD-L1的表达，从而有利于形成对HBV感染的免疫耐受。因此，HBeAg可能是造成慢性HBV感染者体内免疫耐受的重要因素之一。
Objective To study the immunoregulatory effect of hepatitis B virus (HBV) e antigen (HBeAg) on peripheral blood monocytes (PBMCs).Methods PBMCs were isolated from patients with chronic hepatitis B (CHB; both HBeAg-and HBeAg+) and healthy controls,and cultured with recombinant HBeAg.The HBeAg-induced changes in expression of PD-1/PD-L1 were measured by flow cytometry of the cells and in secreted cytokines were measured by enzyme-linked immunosorbent assay of the supernatants.Comparisons between two groups were made by the independent-samples t-test;the relationship between PD-1/B7-H1 level and HBV DNA copy number was evaluated by Spcarman's correlation analysis.Results Exposure to HBeAg led to a significant decrease in CD3+CD4+ T lymphocyte-specific expression of IFNα for both the CHB patients' and healthy controls' samples (t =2.382 and-4.190 respectively,P ＜ 0.01).For the HBeAg-CHB patients' and healthy controls' samples,the HBeAg exposure led to increased levels of secreted cytokines IL-6,IL-10 and TNFα (t =2.504,3.583 and 4.324,P ＜ 0.01 and t =3.542,6.246 and 5.273,P ＜ 0.01 respectively) and of CD14+ PBMC-specific expression of PD-L1 (t =4.815 and 3.454,P ＜ 0.05 respectively).Compared to the HBeAg-negative CHB patients' and healthy controls' samples,the HBeAg+ CHB patients' samples had significantly lower CD3+CD4+ T cell-specific expression of IFNα (t =-3.177 and-4.541,P ＜ 0.01 respectively),but significantly higher levels of secreted IL-4 (t =3.382 and 4.393,P ＜ 0.01 respectively),ofCD3+ T cells-specific expression of PD-1/PD-L1 (t =4.755,2.942 and 4.518,4.595,P ＜ 0.01 respectively),and of CD14+ T cells-specific expression of PD-L1 (t =5.092 and 5.473,P ＜ 0.01 respectively).The CD3+ T cells-specific expression of PD-L1 was significantly higher in the samples from HBeAg-CHB patients than from the healthy controls (t =3.214,P ＜ 0.01).Conclusion HBeAg was able to down-regulate the production ofTh1-type eytokines (IFNγ),and up-regulate the secretion of Th2-type cytokines (IL-6,IL-10) and the expression of PD-1/PD-L1on monocytes.These changes are conducive to the formation of immune tolerance to HBV.Therefore,HBeAg may play an important role in immune tolerance to chronic HBV infection.
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2013年第8期584-589,共6页
Chinese Journal of Hepatology
基金
基金项目：国家自然科学基金（30972618）,国家科技重大专项（2011ZX10004-002）
江苏省卫生厅指导性科研课题（Z201001）I江苏省医学创新团队与领军人才资助项目（LJ201121）
