LAG-3抑制性共刺激分子和慢性HBV感染关系的研究

发布时间：2018-09-08 09:03

【摘要】：背景和目的淋巴细胞活化基因-3分子(lymphocyte activation gene-3, LAG-3)是一种独立免疫负调节分子,具有维持内环境稳定和参与免疫调节的功能,与慢性病毒感染导致的CD8+T细胞耗竭密切相关。慢性乙型肝炎的发病机理主要是一种免疫病理反应,HBV持续感染与特异性CD8+T淋巴细胞应答功能低下有关。本文主要探讨LAG-3与慢性乙肝疾病进展的关系,为寻求新的抗病毒治疗策略提供新的思路。对象和方法 1研究对象筛选2012年2月～2013年6月在浙江大学附属第一医院感染科门诊和住院HBV感染者90例,同时以18例正常健康人作为对照组,结合临床资料将其分为三组,分别为：慢性乙型肝炎患者组、慢性无症状HBV携带者组及正常对照组。 2实验方法 1)外周血单个核细胞(peripheral blood mononuclear cells,PBMC)分离空腹静脉采集全血标本,采用Ficoll淋巴细胞分离液分离PBMC. 2)CD8+T淋巴细胞CD223的测定采用流式细胞技术测CD8+T淋巴细胞CD223(LAG-3)表达。 3)细胞内IFN-γ的测定采用流式细胞技术测定,通过破膜、染色,测定表达或不表达LAG-3的CD8+T细胞内细胞因子IFN-γ的分泌差异。 4)LAG-3抗体阻断对淋巴细胞分泌IFN-γ的影响(ELISPOT法)。 5)HBV病毒学指标的检测HBsAg、抗HBs、HBeAg、抗HBe及抗HBc采用化学发光微粒子免疫法(CMIA)检测；HBV-DNA定量采用荧光定量聚合酶链反应(PCR)检测,截止值为103拷贝/m1。 6)血清丙氨酸氨基转移酶(ALT)测定使用HITACHI7600-110型全自动生化分析仪测定。 7)数据的统计学处理采用SPSS13.0统计软件包及GraphPad Prism5软件进行统计学分析。结果 1)慢乙肝患者组淋巴细胞表面CD8+CD223+分布频数明显高于正常对照组和慢性HBV携带者组,有统计学意义(P0.01)。 2)慢乙肝患者中ALT高DNA低组和ALT高DNA高组淋巴细胞表面CD8+CD223+分布频数明显高于正常对照组(P0.05)。而慢乙肝患者中ALT低DNA低组、ALT低DNA高组淋巴细胞表面CD8+CD223+分布频数与正常对照组比,均无统计学意义(P0.05)。 3)慢乙肝患者组及慢性HBV携带者组表面CD8+CD223分布频数与相应的ALT水平有明显的正相关性。(P=0.03,r=0.23)。 4)慢乙肝患者PBMC中,IFN-γ+在CD8+CD223-细胞的分布频数明显高于其在CD8+CD223+细胞的分布频数(P=0.02)。 5)慢乙肝患者PBMC在乙肝核心抗原及CD223抗体的作用下斑点形成细胞数明显高于乙肝核心抗原刺激组(P0.01)、乙肝核心抗原加同型IgG组(P0.01)及乙肝核心抗原加PD-L1组(P=0.01)；慢乙肝患者PBMC在乙肝核心抗原加PD-L1及CD223抗体作用下斑点形成细胞数明显高于乙肝核心抗原加PD-L1组(P=0.03)而其与乙肝核心抗原加CD223抗体组无显著性差异(P0.05) 结论 1)慢性乙肝患者外周血CD8+T细胞表面LAG-3表达明显增加,且与肝脏炎症程度成正相关,而与HBV DNA载量无关。 2)慢性乙肝患者中,高表达LAG-3的CD8+T细胞功能受损,表现为其分泌细胞因子IFN-γ的水平下降。 3)加入LAG-3抗体阻断,可恢复慢性乙肝患者外周血CD8+T细胞功能,并且LAG-3抗体联合PD-L1阻断,对CD8+T细胞功能的恢复优于单一PD-L1阻断。
[Abstract]:Background and purpose
Lymphocyte activation gene-3 (LAG-3) is an independent negative immunoregulatory molecule, which can maintain the stability of the internal environment and participate in immune regulation. It is closely related to the depletion of CD8 + T cells caused by chronic virus infection. Continued infection is associated with low specific CD8 + T lymphocyte responsiveness. This article mainly discusses the relationship between LAG-3 and the progression of chronic hepatitis B, and provides new ideas for seeking new antiviral treatment strategies.
Objects and methods
1 object of study
Ninety outpatients and inpatients with HBV infection from February 2012 to June 2013 in the Department of Infections of the First Affiliated Hospital of Zhejiang University were selected and divided into three groups: chronic hepatitis B patients group, chronic asymptomatic HBV carriers group and normal control group.
2 experimental method
1) Peripheral blood mononuclear cells (PBMC) were isolated from fasting veins to collect whole blood samples, and then separated from PBMC by Ficoll lymphocyte isolation solution.
2) CD8+T lymphocyte CD223 was measured by flow cytometry to detect the expression of CD223 (LAG-3) in CD8+T lymphocytes.
3) Intracellular IFN-gamma was measured by flow cytometry. The secretion of cytokine IFN-gamma in CD8+T cells expressing or not expressing LAG-3 was determined by membrane breaking and staining.
4) the effect of LAG-3 antibody blocking on the secretion of IFN- gamma by lymphocytes (ELISPOT method).
5) HBsAg, anti-HBs, anti-HBeAg, anti-HBe and anti-HBc were detected by chemiluminescent microparticle immunoassay (CMIA), and HBV-DNA was detected by fluorescence quantitative polymerase chain reaction (PCR) with a cut-off value of 103 copies/m1.
6) serum alanine aminotransferase (ALT) was determined by HITACHI7600-110 automatic biochemical analyzer.
7) Data were analyzed by SPSS 13.0 and GraphPad Prism5.
Result
1) The frequency of CD8 + CD223 + distribution on lymphocyte surface in patients with chronic hepatitis B was significantly higher than that in normal control group and chronic HBV carrier group (P 0.01).
2) The frequency of CD8+CD223+ distribution on lymphocyte surface in patients with chronic hepatitis B was significantly higher than that in normal control group (P 0.05) in patients with ALT high DNA and ALT high DNA. However, the frequency of CD8+CD223+ distribution on lymphocyte surface in patients with chronic hepatitis B was significantly higher than that in normal control group (P 0.05).
3) The frequency of CD8 + CD223 distribution on the surface of chronic hepatitis B patients and chronic HBV carriers was positively correlated with the corresponding ALT level (P = 0.03, r = 0.23).
4) In PBMC of patients with chronic hepatitis B, the frequency of IFN-gamma distribution in CD8+CD223-cells was significantly higher than that in CD8+CD223+ cells (P=0.02).
5) The number of dot forming cells of PBMC in patients with chronic hepatitis B was significantly higher than that in patients with hepatitis B core antigen and CD223 antibody stimulation group (P 0.01), hepatitis B core antigen plus homotype IgG group (P 0.01) and hepatitis B core antigen plus PD-L1 group (P = 0.01); PBMC in patients with chronic hepatitis B was dot-shaped in patients with hepatitis B core antigen plus PD-L1 and CD223 antibody. The number of adult cells was significantly higher than that of hepatitis B core antigen plus PD-L1 group (P=0.03), but there was no significant difference between hepatitis B core antigen plus CD223 antibody group (P 0.05).
conclusion
1) The expression of LAG-3 on peripheral blood CD8 + T cells in patients with chronic hepatitis B was significantly increased, and was positively correlated with the degree of hepatitis, but not with HBV DNA load.
2) In patients with chronic hepatitis B, the function of CD8 + T cells with high LAG-3 expression is impaired, and the level of IFN-gamma secreted by CD8 + T cells is decreased.
3) The function of CD8+T cells in peripheral blood of patients with chronic hepatitis B could be restored by adding LAG-3 antibody, and the function of CD8+T cells could be restored by LAG-3 antibody combined with PD-L1 blockade.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R512.62

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