TNF-α和IFN-γ对肾小管上皮细胞趋化因子表达的影响

The expression of CXCL9,CXCL10,and CXCL11 in renal tubular epithelial cells induced by IFN-γ and TNF-α

目的探讨细胞因子TNF-α和IFN-γ对肾小管上皮细胞趋化因子CXCL9、CXCL10和CXCL11表达的影响。方法分别以TNF-α以及IFN-γ和TNF-α联合作用肾小管上皮细胞HK-2不同时间后,用Real-time PCR和ELISA分别检测CXCL9、CXCL10和CXCL11 mRNA和蛋白水平。用流式细胞术检测淋巴细胞表面CXCR3表达情况,通过趋化实验检测细胞培养上清对淋巴细胞的趋化作用。结果肾小管上皮细胞在TNF-α作用12 h后能够诱导趋化因子CXCL9、CXCL10和CXCL11mRNA表达上调,CXCL9 mRNA的表达在48 h达到高峰,而CXCL10和CXCL11 mRNA表达高峰在12 h;CXCL9、CXCL10和CXCL11蛋白的基础分泌水平均较低,在TNF-α作用12 h后,分泌水平也逐渐升高,CXCL9、CXCL10在48 h时分泌达到高峰,CXCL11分泌高峰在72 h。与TNF-α单独作用组和IFN-γ单独作用组相比,TNF-α和IFN-γ联合作用使肾小管上皮细胞趋化因子CXCL9、CXCL10和CXCL11 mRNA表达和蛋白分泌明显增强(P<0.05)。与新鲜分离的淋巴细胞相比,活化的淋巴细胞表面的CXCR3表达明显升高(P<0.05)。TNF-α以及IFN-γ和TNF-α联合作用HK-2细胞培养上清对活化的淋巴细胞具有明显的趋化作用(P<0.05)且能被抗CXCR3抗体所阻断(P<0.05)。结论细胞因子TNF-α能诱导肾小管上皮细胞趋化因子CXCL9、CXCL10和CXCL11表达,且与IFN-γ联合作用对趋化因子的表达具有协同作用。

This study aimed to investigate the effects of TNF-α and TNF-α combined with IFN-γ on the release of CXCL9, CXCL10, and CXCL11 in renal tubular epithelial cells （HK-2）. After stimulation of various time of TNF-α andTNF-α combined with IFN-γ the HK-2 cells were analyzed by Real-time PCR to detect the expression of CXCL9, CXCL10, and CXCLll mRNA, and the supernatants were analyzed by ELISA to quantify the release of CXCL9,CXCL10, and CXCL11 from cells. Furthermore, the supernatant was used to activate lymphocytes, which were then analyzed by flow cytometry to evaluate the CXCR3 expression, and determined by chemotaxis assay to assesschemotactic activity of the culture supernatants of HK-2 cells. The mRNA level of CXCL9, CXCL10 and CXCL11 of HK-2 cells were significantly increased 12 h after TNF-α stimulation, and reached maximal response at 48 h, 24 h and24 h, respectively, while the protein level of CXCL9, CXCL10, and CXCL11 reached the peak at 48 b, 48 h and 72 h, respectively. Compared with IFN-γ alone or TNF-α alone, the combination treatment could significantly enhance themRNA expression and protein secretion of CXCL9, CXCL10 and CXCL11 （P 〈 0.05）. The supernatant-activated lymphoeytes expressed more CXCR3, as compared with freshly isolated lymphocytes. The activated lymphocytes can berecruited by the culture supernatants of HK-2 cells treated with TNF-α, IFN-γ, and combination of TNF-α and IFN-γ but pretreatment of CXCR3 antibody may inhibited this ehemotaetic effect. These results indicated that TNF-α andIFN-γ can significantly up-regulate the mRNA expression and protein excretion of chemokines such asCXCL9, CXCL10 and CXCL11, in which IFN-γ exerted synergic effect.