慢性乙型肝炎患者PB MC中H BV特异性IL-21表达及其对CD8＋T淋巴细胞功能的影响

摘要

目的：探讨慢性乙型肝炎患者外周血单个核细胞（PBMC）中 HBV特异性IL-21的表达及其对CD8＋T淋巴细胞功能的调节作用。方法以 HBVc18-27（10μg/mL）多肽刺激46例不同临床类型慢性乙型肝炎患者PBMC。ELISA检测上清液IL-21水平。在 HBVc18-27（10μg/mL）多肽和IL-21（50 U/mL）或IL-2（100 ng/mL）培养条件下，应用 MHC-肽五聚体结合流式细胞术检测PBMC中 HBV特异性CTL细胞频数变化；免疫磁珠分离纯化免疫耐受期和免疫激活期慢性乙型肝炎患者PBMC中CD8＋T淋巴细胞，以 HBcAg（10μg/mL）预刺激CD8-PBMC 5 h后，采用Transwell 小室共培养技术，在阻断IL-21通路条件下，应用实时PCR检测CD8＋T淋巴细胞IFN-γmRNA的表达。结果 HBV特异性IL-21在免疫控制期和免疫激活期慢性乙型肝炎患者P B MC中表达水平显著高于免疫耐受期患者，而在免疫控制期和免疫激活期两组之间无显著差异。与 PBS 对照组相比，IL-21组 HBV 特异性 CTL 细胞频数明显增高，差异有统计学意义（P＜0．01），而与IL-2组相比差异无统计学意义。以IL-21抗体阻断IL-21通路后，CD8＋T 淋巴细胞IFN-γmRNA 表达水平显著下降，差异有统计学意义。结论免疫控制期和免疫激活期慢性乙型肝炎患者PBMC 均可生成一定水平的 HBV 特异性IL-21，并能增强外周CD8＋T淋巴细胞的增殖和IFN-γ表达水平。%Objective To investigate the expression of HBV-specific IL-21 and its effect on the function of CD8+T cells from peripheral blood mononuclear cells (PBMCs)in patients with chronic hepatitis B.Methods PBMCs was collected from 46 chronic hepatitis B (CHB)patients of different clinical types and was stimulated by HBVc18-27 peptide (10μg/mL). IL-21 from supernatant was detected by ELISA. Stimulated by HBVc18-27 peptide (10μg/mL),or in combination with IL-2 (50U/mL)or IL-21 (100ng/mL),frequency of HBV specific CTL in CHB patients was evaluated by MHC-peptide pentamer in combination with flow cytometry assay. Pre-stimulated for 5 hours with HBcAg (10μg/mL),isolated CD8+T cells from PBMCs in immune-tolerant CHB patients co-cultured in transwell with isolated CD8-PBMCs derived from immune-active CHB patients. Level of IFN-γmRNA in CD8+T cells was measured by real-time PCR when using anti-IL-21 antibody blocked the IL-21 pathway or not.Results Compared to immune-tolerant CHB patients,levels of HBV-associated IL-21 was much higher in immune-active and immune-inactive CHB patients. There was no significant difference of HBV---associated IL-21 levels between immune-active and immune-inactive CHB patients. Compared to PBS control group, frequency of HBV specific CTL from CHB patients was significantly higher in IL-21 group (P<0.05);however,there was no significant difference of frequency of HBV specific CTL between IL-21 group and IL-2 group. Level of IFN-γmRNA in CD8+ T cells was significantly decreased when using anti-IL-21 antibody blocked the IL-21 pathway.Conclusions IL-21 , which was much higher in immune-active and immune-inactive CHB patients,could enhance the HBV- specific CD8+ T cell function in patients with chronic hepatitis B.