CAJ | 학술논문

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12 d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。
목적：관찰결핵분지간균조기분비파항원（ESAT-6）대γδT세포산생림파인자적영향，병대기신호전도궤제진행초보탐토。방법사용 Ficoll밀도제도리심법분리건강인외주혈단개핵세포（PBMC），용류식세포술분선순화γδT세포；Toll양수체-4（TLR-4）특이성억제제 E5564처리γδT세포이억제 TLR-4적공능，병용단백질면역인적법（Western blot）급역전록-PCR （RT-PCR）법검측γδT세포TLR-4단백급mRNA적표체，이불가임하자격인자적γδT세포작공백대조，용（ESAT-6）자격γδT세포，병재제0、1、3、6、9화12 d취세포상청액，용매련면역흡부시험（ELISA）시제합검측 IL-17、종류배사인자-α（TNF-α）급간우소-γ（IFN-γ）적분비수평。결과 Western blot급PCR결과현시 ESAT-6능현저제고γδT세포TLR-4기인적전록여단백적표체（P＜0．01）；ESAT-6능현저제고γδT세포 IL-17、TNF-α、IFN-γ적표체（P＜0．01），이TLR-4특이성억제제 E5564능현저적억제ESAT-6소치적γδT세포 IL-17、TNF-α、IFN-γ림파인자적증가（P＜0．01）。결론 ESAT-6능현저상조γδT세포 IL-17、TNF-α급 IFN-γ적표체，기증강γδT세포 IL-17、TNF-α급 IFN-γ표체적신호전도궤제가능여TLR-4신호통로유관。
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.