摘要
目的观察结核分枝杆菌早期分泌靶抗原(ESAT-6)对γδT细胞产生淋巴因子的影响,并对其信号转导机制进行初步探讨。方法使用Ficoll密度梯度离心法分离健康人外周血单个核细胞(PBMC),用流式细胞术分选纯化γδT细胞;Toll样受体-4(TLR-4)特异性抑制剂E5564处理γδT细胞以抑制TLR-4的功能,并用蛋白质免疫印迹法(Western blot)及逆转录-PCR(RT-PCR)法检测γδT细胞TLR-4蛋白及mRNA的表达,以不加任何刺激因子的γδT细胞作空白对照,用(ESAT-6)刺激γδT细胞,并在第0、1、3、6、9和12d取细胞上清液,用酶联免疫吸附试验(ELISA)试剂盒检测IL-17、肿瘤坏死因子-α(TNF-α)及干扰素-γ(IFN-γ)的分泌水平。结果 Western blot及PCR结果显示ESAT-6能显著提高γδT细胞TLR-4基因的转录与蛋白的表达(P<0.01);ESAT-6能显著提高γδT细胞IL-17、TNF-α、IFN-γ的表达(P<0.01),而TLR-4特异性抑制剂E5564能显著的抑制ESAT-6所致的γδT细胞IL-17、TNF-α、IFN-γ淋巴因子的增加(P<0.01)。结论 ESAT-6能显著上调γδT细胞IL-17、TNF-α及IFN-γ的表达,其增强γδT细胞IL-17、TNF-α及IFN-γ表达的信号转导机制可能与TLR-4信号通路有关。
Objective To observe the expression of immune substances secreted by peripheral bloodγδT cells after stimulated by early secreted antigenic target-6（ESAT-6）,and explore its mechanism of signaling pathway.Methods Peripheral blood mononucle-ar cells（PBMC）were collected from health human blood with the ficoll density gradient centrifugation method,and theγδT cells were separated from the PBMC with flow cytometry;the inhibitor of TLR-4 signaling pathway（E5564）was used to cocultured withγδT cells to inhibit the function of TLR-4,and the change of TLR-4 was analyzed by the methods of PCR and Western blot.ESAT-6 were used to stimulate theγδT cells,and the control group without any stimulating factor was established,then the expression levels of IL-17,TNF-α,IFN-γwere determined by ELISA method after 0、1、3、6、9 and 12 days.Results The results of PCR and Western blot showed that ESAT-6 could increase the expression of TLR-4（P〈0.01）;The results of ELISA showed that ESAT-6 could enhance the expression of IL-17,TNF-αand IFN-γ（P〈0.01）,and the inhibitor of TLR-4（E5564）could decrease the expres-sion of IL-17,INF-α,IFN-γ（P〈0.01）.Conclusion ESAT-6 can induceγδT cells to produce more IL-17,TNF-α,IFN-γ,and the mechanism of which maybe concerned with TLR-4 signaling pathway.
出处
《重庆医学》
CAS
CSCD
北大核心
2014年第13期1537-1539,1542,共4页
Chongqing medicine
基金
国家自然科学基金资助项目(30872261)
