肝癌恶性腹水患者Th9细胞的分布及募集至腹腔的机制研究

Distribution and recruitment of Th9 cells in malignant ascites of hepatocellular carcinoma

目的检测肝癌所致的恶性腹水(MA)患者Th9细胞的分布情况,阐明Th9细胞从外周血募集浸润至腹腔的机制以及MA中Th9细胞比例与患者生存期的关系。方法采用酶联免疫吸附试验检测30例肝癌患者MA和外周血中,以及15例健康对照者外周血中细胞因子IL-9和趋化因子CCL20、CCL22的质量浓度。流式细胞术检测腹水及外周血中Th9细胞比例以及Th9细胞表面CCR4、CCR5、CCR6和CCR7的表达水平。使用Transwell迁移实验检测MA对Th9细胞的趋化效应。Kaplan-Meier法分析MA中Th9细胞比例与患者生存期的关系。结果肝癌患者MA中Th9细胞比例和IL-9质量浓度明显高于相应外周血以及健康对照者外周血中的水平(P<0.01)。MA中趋化因子CCL20质量浓度明显高于相应外周血清(P<0.01)。体外趋化迁移实验表明,MA上清液具有诱导Th9细胞趋化的能力,当用抗体中和CCL20时,这种趋化效应明显减弱。MA中Th9细胞比例与患者生存期负相关,Th9细胞高比例组的死亡风险比是Th9细胞低比例组的2倍。结论 MA中Th9细胞数量较外周血显著增多;CCL20/CCR6趋化轴可能参与Th9细胞募集至MA的过程;MA中Th9细胞比例升高与患者预后不良有关。

This study was performed to investigate the distribution of Th9 cells in malignant ascites （MA） of hepatocellular carcinoma, the mechanism of Th9 cells recruitment into MA and the relationship between Th9 cells and the survival time of patients with MA. The levels of IL-9 and chemokines CCL20 and CCL22 in 30 hepatic carcinoma patients with MA and 15 healthy controls percentage of Th9 cells and the expression of CCR4, were examined by enzyme-linked immunosorbent assay. The CCR5, CCR6 and CCR7 on Th9 cells were examined by flow cytometry. Then the chemoattractant activity of chemokine for Th9 cells was detected in vitro. The relationship between the percent of Th9 cells and the survival time of patients with MA was analyzed by Kaplan-Meier method. Data showed that comparing with those in the corresponding blood and healthy controls, the percentage of Th9 cells and IL-9 level in MA were higher （P〈0.01）. The level of chemokine CCL20 in MA was significantly higher than that in the corresponding peripheral serum （P〈0.01）. In vitro migration assay confirmed that MA superuatant could induce the migration of Th9 cells and anti-CCL20 mAb could significantly block the ability of MA to stimulate Th9 cell chemotaxis. We also found that the accumulation of Th9 cells in MA predicted shorter patient survival time. conclusions, the number of Th9 cells in MA is significantly increased compared with the peripheral blood; the recruitment of Th9 cells into MA could be induced by CCL20/CCR6 axis; and the accumulation of Th9 cells in MA predicted poor patient survival.