论文部分内容阅读
目的研究结核杆菌耐热抗原(MTB-HAg)激活的人外周血γδT细胞Th2极性分化特点和表达于T细胞中的T盒(T-bet)和GATA结合蛋白3(GATA-3)转录因子的调控作用。方法用MTB-HAg刺激人外周血单个核细胞(PBMC)获得MTB-HAg激活的T细胞(MTBAT),分别在中性条件和Th2极化条件培养后,再经10 ng/m L佛波醇酯(PMA)、500 ng/m L离子霉素(ionomycin)和2.5μmol/L莫能菌素(monensin)刺激6 h,四色荧光抗体染色流式细胞术检测γδT细胞和αβT细胞内细胞因子γ干扰素(IFN-γ)和白细胞介素4(IL-4)的表达。流式细胞术分选28 d MTBAT中的γδT细胞和CD4+T细胞,反转录PCR(RT-PCR)检测T-bet和GATA-3 mRNA的表达。结果在中性条件和Th2极化条件下培养的MTBAT中,γδT细胞一直优势表达IFN-γ,而Th2极化条件下,随培养时间增加IFN-γ+αβT细胞百分率显著下降。与中性条件培养比较,Th2极化条件下MTBAT培养28 d,Th0型(IFN-γ+IL-4+)γδT细胞显著增加;而Th2型(IFN-γ-IL-4+)αβT细胞显著增加。RT-PCR结果显示,Th2极化的γδT细胞仍高表达T-bet mRNA,而在CD4+T细胞T-bet mRNA表达被显著下调;同时,GATA-3 mRNA在γδT细胞和CD4+T细胞的表达均显著上调。结论 Th2极化条件下,大部分γδT细胞仍为Th1型,仅部分极性分化为Th0型细胞;Th2极性分化的γδT细胞转录因子T-bet和GATA-3未表现出交互调节功能。
Objective To investigate the Th2 polarity differentiation of human peripheral blood γδT cells activated by Mycobacterium tuberculosis heat-tolerance antigen (MTB-HAg) and the expression of T-bet and GATA-3 transcription factor The regulatory role. Methods MTB-HAg-stimulated T cells (MTBAT) were stimulated with MTB-HAg in human peripheral blood mononuclear cells (PBMCs) and cultured under neutral and Th2 polarization conditions respectively. After stimulation with 10 ng / (PMA), 500 ng / mL ionomycin and 2.5 μmol / L monensin for 6 h, and the cytotoxicity of γδT cells and αβT cells by flow cytometry γ interferon (IFN-γ) and interleukin-4 (IL-4) expression. The γδT cells and CD4 + T cells in MTBAT were sorted by flow cytometry. The expressions of T-bet and GATA-3 mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR). Results γδT cells predominantly expressed IFN-γ in MTBAT cultured under neutral conditions and Th2 polarization. However, under the conditions of Th2 polarization, the percentage of IFN-γ + αβ T cells decreased significantly with increasing incubation time. Compared with the control group, Th2 (IFN-γ + IL-4 +) γδT cells were significantly increased after MTBAT was cultured for 28 days under Th2 polarization, while αβT cells in Th2 type (IFN-γ-IL-4 + increase. RT-PCR results showed that Th2-polarized γδT cells were still highly expressed T-bet mRNA, while T-bet mRNA expression was significantly down-regulated in CD4 + T cells; meanwhile, GATA-3 mRNA expression in γδT cells and CD4 + T cells Expression were significantly increased. Conclusion Under Th2 polarization condition, most of γδT cells are still Th1-type cells and only partially differentiate into Th0-type cells. The transcriptional factors T-bet and GATA-3 of γδT cells differentiated by Th2 polarization did not show any interactive regulation.