目的:观察ESAT-6对人外周血γδT细胞表达IL-17的影响,并对其信号转导机制进行初步探讨.方法:用Ficoll密度梯度离心法从健康人外周血中分离单个核细胞,培养3天后用流式细胞仪分离纯化γδT细胞,并用唑来磷酸及IL-2刺激扩增γδT细胞,12天后用ESAT-6刺激γδT细胞,并给予信号传导及转录激活因子3(STAT3)抑制剂S3I-201,设置未加入ESAT-6及不加任何抑制剂的对照组,用PCR法检测IL-17、STAT-3的基因转录水平,用ELISA法检测细胞培养液中IL-17的含量,用Western blot检测STAT3蛋白的表达.结果:ESAT-6能显著提高STAT3及IL-17的基因转录及蛋白表达(P〈0.01),且S3I-201能显著抑制ESAT-6所致的γδT细胞IL-17mRNA的转录及淋巴因子的增加(P〈0.01).结论:ESAT-6能提高外周血γδT细胞IL-17、STAT3的转录及表达,ESAT-6增强γδT细胞IL-17表达的机制可能与STAT3信号通路有关.
Objective:To study the effect of ESAT-6 on expression of interleukin-17 in γδT cells and explore its mechanism of signaling pathway. Methods: Peripheral blood mononuclear cells (PBMC) were collected from health human blood with the ficoll density gradient centrifugation methods, and the T cells were gained by the nylon wool column method, and then the γδT cells were separated from the T cells with Flow cytometry. The zoledronie acid (ZA) and interleukiu-2 (IL-2) were used to stimulate and expand the γδT cells. The γδT cells were treated and stimulated by ESAT-6 after 12 days cultured, and the inhibitor of STAT3 signaling pathway ( S3I-201 ) was administered, besides control groups without ESAT-6 and stimulator were established. The change of IL-17 and STAT3 were determined by methods of PCR, and the expression of STAT3 protein was analyzed by the method of Western blot, and then the change of IL-17 was tested by ELISA. Results: ESAT-6 could increased the expression of IL-17 and STAT3 (P 〈 0.01 ) , and the inhibitor of STAT3 ( S3I-201 ) decreased expression of IL-17 ( P 〈 0.01 ). Conclusion : ESAT-6 can enhance the expression of IL-17 produced by γδT cells, and the mechanism of which maybe concerned with STAT3 signaling pathway.