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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。
Objective To investigate the distribution of Th9 cells in malignant ascites (MA) patients induced by hepatocellular carcinoma (HCC) and to elucidate the mechanism of Th9 cells recruitment and infiltration into peritoneal cavity from peripheral blood and the relationship between the proportion of Th9 cells in MA and patient survival. Methods The concentrations of cytokines IL-9, chemokines CCL20 and CCL22 in the peripheral blood of MA and peripheral blood and 15 healthy controls were detected by enzyme-linked immunosorbent assay (ELISA). The proportion of Th9 cells in ascites and peripheral blood and the expression of CCR4, CCR5, CCR6 and CCR7 on Th9 cells were detected by flow cytometry. Transwell migration assay was used to detect the chemotactic effect of MA on Th9 cells. Kaplan-Meier analysis of the relationship between the proportion of Th9 cells in patients with the survival of patients. Results The proportion of Th9 cells and the concentration of IL-9 in MA patients were significantly higher than those in corresponding peripheral blood and healthy controls (P <0.01). The concentration of chemokine CCL20 in MA was significantly higher than that of corresponding peripheral serum (P <0.01). In vitro chemotactic migration experiments showed that the MA supernatant has the ability to induce chemotaxis of Th9 cells, and this chemotactic effect is significantly diminished when the CCL20 is neutralized with the antibody. The proportion of Th9 cells in MA was negatively correlated with the survival of patients, and the risk of death in the high proportion of Th9 cells was 2 times than the low proportion of Th9 cells. Conclusion The number of Th9 cells in MA is significantly higher than that in peripheral blood. The chemotactic axis of CCL20 / CCR6 may be involved in the recruitment of Th9 cells to MA. The increased proportion of Th9 cells in MA is associated with poor prognosis.