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γ链细胞因子对慢性乙型肝炎患者T淋巴细胞免疫球蛋白黏蛋白分子3表达调控的诱导机制
DOI: 10.3969/j.issn.1001-5256.2021.05.017
利益冲突声明：本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突。
作者贡献声明：杨晓飞、董杰、胡海峰、毕占虎负责实验操作和论文撰写；杨晓飞、王临旭、黄长形、连建奇、张野负责入组患者和数据分析；连建奇、张野负责课题设计，资料分析，拟定写作思路，指导撰写论文并最后定稿。
Mechanism of gamma-chain cytokines in regulating the expression of T-cell immunoglobulin and mucin domain-containing molecule 3 in CD8+ T cells of chronic hepatitis B patients
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摘要:
目的 探讨γ链(γC)细胞因子对慢性乙型肝炎(CHB)患者CD8+T淋巴细胞中T淋巴细胞免疫球蛋白黏蛋白分子3(TIM-3)表达调控的机制。 方法 选取2017年1月—5月在第四军医大学唐都医院就诊的CHB患者23例，采集外周血，利用Ficoll密度梯度离心法分离外周血单个核细胞，分别使用IL-7、IL-15和IL-21刺激培养，同时向培养液中加入抗γC和/或抗IL-7Rα、抗IL-2Rβ、抗IL-21R。培养96 h后，流式细胞术检测CD8+T淋巴细胞中TIM-3表达水平以及IL-2、IL-10、IFNγ和相关磷酸化信号传导及转录激活蛋白(STAT)磷酸化水平。计量资料多组间比较采用单因素方差分析，进一步两两比较采用LSD-t检验。 结果 IL-7和IL-15刺激后，CD8+T淋巴细胞中TIM-3阳性细胞比例较无刺激组升高(t值分别为9.966、9.074，P值均＜0.05)，IL-2、IL-10、IFNγ水平以及STAT-5、pSTAT-1水平亦较无刺激组升高(P值均＜0.05)。抗IL-7Rα+抗γC联合刺激后可降低IL-7刺激组TIM-3、IL-2和IL-10表达升高(t值分别为5.537、6.224和4.500，P值均＜0.05)。抗IL-2Rβ单独刺激、抗IL-2Rβ+抗γC刺激均可降低IL-15刺激组的TIM-3、IL-2、pSTAT-1水平(P值均＜0.05)。 结论 IL-7和IL-15可能主要通过γC受体介导的STAT-细胞因子信号通路上调CHB患者CD8+T淋巴细胞中TIM-3表达。 -
关键词:
- 乙型肝炎, 慢性 /
- 细胞因子类 /
- CD8阳性T淋巴细胞 /
- T淋巴细胞免疫球蛋白黏蛋白分子3
Abstract:Objective To investigate the mechanism of gamma-chain (γC) cytokines in regulating the expression of T-cell immunoglobulin and mucin domain-containing molecule 3 (TIM-3) in CD8+ T cells of chronic hepatitis B (CHB) patients. Methods A total of 23 CHB patients who attended Tangdu Hospital, Fourth Military Medical University, from January to May, 2017, were enrolled. Peripheral blood was collected from all patients, and Ficoll density gradient centrifugation was used to isolate peripheral blood mononuclear cells (PBMCs). PBMCs were stimulated with interleukin-7 (IL-7), interleukin-15 (IL-15), and interleukin-21, respectively, and then anti-γC antibody and/or anti-IL-7Rα, anti-IL-2Rβ, and anti-IL-21R were added to the culture solution. After 96 hours of culture, flow cytometry was used to measure the expression of TIM-3, interleukin-2 (IL-2), interleukin-10 (IL-10), and interferon-γ (IFNγ) and the phosphorylation level of signal transducer and activator of transcription (STAT) in CD8+ T cells. A one-way analysis of variance and the least significant difference t-test were used for comparison of continuous data. Results The CD8+ T cells stimulated by IL-7 and IL-15 had a significantly higher percentage of TIM-3-positive CD8+ T cells than those without stimulation (t=9.966 and 9.074, P < 0.05), as well as significantly higher expression levels of IL-2, IL-10, and IFN-γ and phosphorylation levels of STAT-5 and STAT-1 (all P < 0.05). Stimulation with anti-IL-7Rα and anti-γC antibody significantly reduced the elevated expression levels of TIM-3, IL-2, and IL-10 in the IL-7 stimulation group (t=5.537, 6.224, and 4.500, P < 0.05). Stimulation with anti-IL-2Rβ alone or in combination with anti-γC antibody significantly reduced the expression levels of TIM-3 and IL-2 and the phosphorylation level of STAT-1 in the IL-15 stimulation group (P < 0.05). Conclusion IL-7 and IL-15 can upregulate the expression of TIM-3 in CD8+ T cells of CHB patients, possibly via the γC receptor-mediated STAT-cytokine signaling pathway. -
图 2 IL-7对CHB患者CD8+T淋巴细胞中TIM-3、IL-2、IL-10、IFNγ和pSTAT-5表达的影响
注：a，TIM-3+比例；b，IL-2+TIM-3+比例；c，IL-10+TIM-3+比例；d，IFNγ+TIM-3+比例；e，pSTAT-5+TIM-3+比例。
图 3 IL-15对CHB患者CD8+T淋巴细胞中TIM-3、IL-2、IL-10、IFNγ和pSTAT-1表达的影响
注：a，TIM-3+比例；b，IL-2+TIM-3+比例；c，IL-10+TIM-3+比例；d，IFNγ+TIM-3+比例；e，pSTAT-1+TIM-3+比例。
表 1 IL-21对CHB患者CD8+T淋巴细胞中TIM-3、IL-2、IL-10、IFNγ、pSTAT-1和pSTAT-3表达的影响
项目 无刺激 IL-21 IL-21+抗IL-21R IL-21+抗IL-21R+抗γC F值 P值 TIM-3+CD8+/CD8+(%) 11.90±2.32 12.92±2.94 10.62±4.53 10.67±6.23 1.520 0.215 IL-2+TIM-3+CD8+/TIM-3+CD8+ (%) 17.89±6.22 15.37±2.92 14.57±4.01 14.54±4.09 2.401 0.073 IL-10+TIM-3+CD8+/TIM-3+CD8+ (%) 51.14±8.31 49.17±9.70 50.46±9.25 50.41±9.77 0.180 0.909 IFNγ+TIM-3+CD8+/TIM-3+CD8+ (%) 20.90±5.78 22.11±5.02 24.60±9.01 25.42±9.31 1.753 0.162 pSTAT-1+TIM-3+CD8+/TIM-3+CD8+ (%) 17.30±3.66 20.83±3.81 20.71±6.45 19.92±6.84 2.137 0.101 pSTAT-3+TIM-3+CD8+/TIM-3+CD8+ (%) 8.15±2.37 8.68±2.19 8.36±3.18 8.41±3.09 0.145 0.932 
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