肝硬化合并自发性细菌性腹膜炎患者外周血和腹水CD100水平和免疫调节作用

Ascites CD100 levels and immunomodulation effects in the peripheral blood of patients with liver cirrhosis combined with spontaneous bacterial peritonitis

目的观察肝硬化合并自发性细菌性腹膜炎(SBP)患者外周血和腹水中CD100水平,并在体外检测CD100对腹水中CD4^(+)和CD8^(+)T淋巴细胞活性的影响。方法入组肝硬化患者77例(肝硬化合并单纯腹水患者49例、肝硬化合并SBP患者28例),采集外周血和腹水,入组22例对照者采集外周血。酶联免疫吸附试验检测外周血和腹水中可溶型CD100(sCD100),流式细胞术检测CD4^(+)和CD8^(+)T淋巴细胞表面膜结合型CD100(mCD100)。分选腹水中CD4^(+)和CD8^(+)T淋巴细胞,CD100刺激后检测CD4^(+)T淋巴细胞增殖、关键转录因子mRNA和分泌细胞因子变化,检测CD8^(+)T淋巴细胞增殖、重要毒性分子mRNA和分泌细胞因子变化,利用直接接触和间接接触培养系统检测CD8^(+)T细胞的杀伤活性。符合正态分布的数据使用单因素方差分析、Student t检验或配对t检验进行比较。不符合正态分布的数据使用Krusal-Willis检验或Mann-Whitney检验进行比较。结果血浆sCD100水平在肝硬化合并单纯腹水患者(1415.0±434.1)pg/ml、肝硬化合并SBP患者(1465.0±386.8)pg/ml和对照者(1355.0±428.0)pg/ml之间的差异无统计学意义(P=0.655)。肝硬化合并SBP患者腹水sCD100水平低于合并单纯腹水患者[(2409.0±743.0)pg/ml比(2825.0±664.2)pg/ml,P=0.014]。外周血CD4^(+)和CD8^(+)T淋巴细胞中mCD100水平在3组间的差异无统计学意义(P>0.05)。肝硬化合并SBP患者腹水CD4^(+)和CD8^(+)T淋巴细胞中mCD100水平高于合并单纯腹水患者(P<0.05)。CD100刺激对肝硬化合并SBP患者腹水中CD4^(+)和CD8^(+)T淋巴细胞的增殖无明显影响(P>0.05),对效应性CD4^(+)T淋巴细胞中转录因子(T-bet、维甲酸相关孤独核受体γt、芳香烃受体)表达和细胞因子(干扰素-γ、白细胞介素-17、白细胞介素-22)分泌均无显著影响(P>0.05)。CD100刺激可增加肝硬化合并SBP患者腹水CD8^(+)T淋巴细胞穿孔素、颗粒酶B、颗粒溶素mRNA相对表达量及其分泌干扰素-γ和肿瘤坏死因子-α的水平(P<0.05),亦可增加CD8^(+)T淋巴细胞的杀伤活性。结论CD100的活性形式是sCD100而非mCD100,肝硬化合并SBP患者腹水中存在sCD100和mCD100表达失衡,sCD100可增强肝硬化合并SBP患者腹水CD8^(+)T淋巴细胞的功能,是潜在治疗靶点之一。

Objective To observe the level and detection of ascites CD100 on the activity of CD4^(+)and CD8^(+)T lymphocytes in vitro in the peripheral blood of patients with liver cirrhosis combined with spontaneous bacterial peritonitis.Methods Peripheral blood and ascites were collected from 77 cases of liver cirrhosis(49 patients with liver cirrhosis combined with simple ascites and 28 patients with liver cirrhosis combined with SBP),and peripheral blood was collected from 22 controls.Soluble CD100(sCD100)in peripheral blood and ascites was detected by an enzyme-linked immunosorbent assay.Flow cytometry was used to detect membrane-bound CD100(mCD100)on the surface of CD4^(+)and CD8^(+)T lymphocytes.CD4^(+)and CD8^(+)T lymphocytes in ascites were sorted.CD4^(+)T lymphocyte proliferation,key transcription factor mRNA,and secreted cytokine changes,as well as CD8^(+)T lymphocyte proliferation,important toxic molecule mRNA,and secreted cytokine changes,were detected after CD100 stimulation.The killing activity of CD8^(+)T cells was detected by direct contact and indirect contact culture systems.Data conforming to normality were compared using one-way ANOVA,a student's t-test,or a paired t-test.Data that did not conform to a normal distribution were compared using either the Krusal-Willis test or the Mann-Whitney test.Results There was no statistically significant difference in plasma sCD100 level between patients with liver cirrhosis combined simple ascites(1415±434.1)pg/ml,patients with liver cirrhosis combined with SBP(1465±386.8)pg/ml,and controls(1355±428.0)pg/ml(P=0.655).The ascites sCD100 level was lower in patients with liver cirrhosis combined with SBP than that of patients with simple ascites[(2409±743.0)pg/ml vs.(2825±664.2)pg/ml,P=0.014].There was no statistically significant difference in the level of mCD100 in peripheral blood CD4^(+)and CD8^(+)T lymphocytes among the three groups(P>0.05).The levels of mCD100 in ascites CD4^(+)and CD8^(+)T lymphocytes were higher in patients with liver cirrhosis combined with SBP than those in patients with simple ascites(P<0.05).CD100 stimulation had no significant effect on the proliferation of CD4^(+)and CD8^(+)T lymphocytes in the ascites of patients with liver cirrhosis combined with SBP(P>0.05).There were no significant effects on the expression of transcription factors in effector CD4^(+)T lymphocytes(T-bet,retinoic acid associated solitary nuclear receptorγt,aromatic hydrocarbon receptor)or secretion of cytokines(interferon-γ,17,and 22)(P>0.05).CD100 stimulation had increased the relative expression of perforin,granzyme B,and granlysin mRNA and the levels of secreted interferon-γand tumor necrosis factor-α,killing activity in ascites CD8^(+)T lymphocytes of patients with liver cirrhosis combined with SBP(P<0.05).Conclusion The active form of CD100 is sCD100 instead of mCD100.There is an imbalance between the expression of sCD100 and mCD100 in the ascites of patients with cirrhosis combined with SBP.sCD100 can enhance the function of CD8^(+)T lymphocytes in the ascites of patients with cirrhosis combined with SBP and thus is one of the potential therapeutic targets.