基于协同刺激分子表达量建立慢性HBV感染患者T淋巴细胞免疫功能评价新体系的研究

目的 探讨慢性乙型肝炎病毒（HBV）感染患者外周血T淋巴细胞CD28、细胞毒性T淋巴细胞相关抗原-4（CTLA-4）、程序性死亡分子-1（PD-1）和T淋巴细胞免疫球蛋白黏蛋白分子-3（Tim-3）的表达及意义.方法 连续收集2012年10月至2013年6月在苏州大学附属第一医院住院的慢性HBV感染患者102例,其中慢性乙型肝炎（CHB） 42例,乙型肝炎肝硬化30例,肝癌30例.另选30名健康体检者作为对照组.采用流式细胞术和定量聚合酶链反应（PCR）技术测定所有研究对象外周血T淋巴细胞表面CD28、CTLA-4、PD-1和Tim-3的表达量,并采用单因素方差分析和LSD-t检验比较各组间的差异.采用Spearman秩相关分析外周血T淋巴细胞表面CD28、CTLA-4、PD-1和Tim-3的表达量与HBV DNA载量、乙型肝炎e抗原（HBeAg）和丙氨酸转氨酶（ALT）之间的相关性.结果 慢性HBV感染患者CD4＋ CD28＋、CD8＋ CD28＋和CD4＋ CTLA-4＋的表达均低于健康对照人群,其中肝癌组的CD4＋ CD28＋表达量低于CHB组（t=2.373,P＜0.05）,肝硬化和肝癌组的CD8＋CD28＋表达量低于CHB组（t=4.324和4.088,P＜0.01）.CHB组CD8＋ PD-1＋、CD4＋ Tim-3＋和CD8＋ Tim-3＋的表达量高于肝硬化、肝癌以及健康对照组（t=3.051,3.130,3.121,3.254和3.723,P＜0.01）.相关性分析显示,CD8＋T淋巴细胞PD-1表达量与ALT水平、HBV DNA载量呈正相关（r=0.516和0.582,P＜0.01）;CD8＋T淋巴细胞Tim-3的表达量与ALT水平、HBV DNA载量也呈正相关（r=0.578和0.556,P＜0.01）.此外,CD8＋T淋巴细胞PD-1与Tim-3的表达量也呈一定的正相关（r =0.506,P＜0.01）.结论 HBV感染患者外周血T淋巴细胞表面分子异常表达与其免疫功能紊乱和疾病慢性化的发生和发展相关.

A novel evaluation system for T lymphocytes immune function based on the expression of costimulatory molecules in chronic HBV-infected patients

Objective To investigate the expression and significance of CD28,cytotoxic T-lymphocyte antigen-4 （CTLA-4）,programmed death-1 （PD-1） and T cell immunoglobulin mucin-3 （Tim3） on T lymphocytes in chronic HBV-infected patients.Methods A total of 102 chronic HBV-infected patients,including 42 patients with chronic hepatitis B （CHB）,30 patients with hepatitis B-induced liver cirrhosis （LC）,and 30 patients with hepatocellular carcinoma （HCC）,were enrolled from the First Affiliated Hospital to Soochow University during October 2012 and June 2013.Thirty healthy individuals were also enrolled as controls.Expression of CD28,CTLA-4,PD-1,Tim-3 on T lymphocytes in peripheral blood were determined by flow cytometry,and the differences among groups were analyzed using one-way ANOVA and LSD-t test.Spearman correlation test was performed to analyze the correlations of the expression of CD28,CTLA-4,PD-1,Tim-3 on T lymphocytes with HBV DNA loads,HBeAg and ALT.Results The expression of CD4 ＋ CD28 ＋,CD8 ＋ CD28 ＋,CD4 ＋ CTLA-4 ＋ in chronic HBV-infected patients were lower than those in healthy controls.CD4 ＋ CD28 ＋ expression in HCC group was lower than that in CHB group （t =2.373,P ＜ 0.05） ; CD8 ＋ CD28 ＋ expression in LC and HCC group was lower than that in CHB group （t =4.324 and 4.088,P ＜ 0.01） ; CD8 ＋ PD-1 ＋,CD4 ＋ Tim-3 ＋ and CD8 ＋ Tim-3 ＋ expressions in CHB group were higher than those in LC,HCC group and healthy controls （t =3.051,3.130,3.121,3.254 and 3.723,P ＜0.01）.CD8 ＋ PD-1 ＋ expression was positively correlated with ALT levels and HBV DNA loads （r =0.516 and 0.582,P ＜ 0.01） ; CD8 ＋ Tim-3 ＋ expression was also positively correlated with ALT levels andHBV DNA loads （r =0.578 and 0.556,P ＜0.01）; PD-1 and Tim-3 expressions on CD8 T lymphocytes were positively correlated with each other （r =0.578,P ＜ 0.01）.Conclusion The abnormal expression of the molecules on T lymphocytes in chronic HBV-infected patients is closely correlated with immune function disorder and the progression of the disease.