【摘要】 目的研究4-羟基-3-甲基-2-烯基焦磷酸(HMBPP)和多肽E7对结核患者胸水及脐带血中γδT细胞分泌白介素6(IL-6)的影响和机制。方法收集自广州市胸科医院临床诊断结核性胸膜炎患者胸水标本18例,自中山大学附属第三医院正常健康产妇脐带血标本17例。使用流式分选技术分选出结核性胸水和脐带血中的γδT细胞与CD277+CD14+单核-巨噬细胞混合培养,分别用HMBPP或E7刺激后,运用流式细胞术检测分泌IL-6的γδT细胞百分率及表达程序性死亡配体1(PD-L1)的CD277+CD14+单核-巨噬细胞百分率;利用RT-PCR技术检测细胞中IL-6、PD-L1的mRNA表达水平;用ELISA方法检测细胞培养上清中IL-6的含量;用PD-L1特异性小分子干扰RNA(siRNA)沉默PD-L1的表达以检测PD-L1是否参与分泌IL-6;在分选于结核性胸水和脐带血的共培养细胞中,HMBPP或E7刺激后上述指标在每一时间点对照组和实验组间分别采用独立样本t检验比较其差异,分析其统计学意义。结果在结核性胸水和脐带血中,给予HMBPP或E7刺激24 h之后,与对照组相比IL-6+γδ+T细胞占比显著升高,给予HMBPP或E7刺激12 h之后γδT细胞中IL-6在mRNA水平的相对表达量显著上调,以及在蛋白质水平的表达显著上调;在分选于脐带血的共培养细胞中,给予HMBPP或E7刺激,CD277+CD14+单核-巨噬细胞中PD-L1在mRNA水平的相对表达量显著上调[(3.70±0.33)vs(4.20±0.34)vs 1.00];在沉默PD-L1的表达后,γδT细胞中IL-6的表达显著降低[(0.53±0.03)vs(0.55±0.01) vs 1.00],差异均具有统计学意义(P均<0.05)。结论 HMBPP和多肽E7能够促进CD277+CD14+单核-巨噬细胞PD-L1表达上调,后者参与刺激与其共培养的γδT细胞分泌更多的IL-6,这为γδT细胞的抗结核免疫机制的研究以及结核患者的免疫治疗提供依据。更多还原

【Abstract】 Objective To investigate the effect of(E)-4-hydroxy-3-methyl-but-2-enyl diphosphate(HMBPP) and peptide E7 on the production of interleukin-6(IL-6) by γδ T cells in the pleural fluid of tuberculosis patients and umbilical cord blood. Methods Eighteen samples of pleural fluid were collected from patients who were clinically diagnosed with tuberculous pleurisy at Guangzhou Chest Hospital and 17 samples of umbilical cord blood were collected at the Third Affiliated Hospital of Sun Yat-sen University. γδT cells and CD277+CD14+ monocyte-macrophages were sorted from the pleural fluid and umbilical cord blood samples by fluorescence-activated cell sorting. They were co-cultured and stimulated with HMBPP or E7. The percentages of γδ T cells secreting IL-6 and CD277+CD14+ monocyte-macrophages expressing programmed cell death ligand-1(PD-L1) were detected by flow cytometry. Expression of IL-6 and PDL1 mRNA in cells was detected by RT-PCR. Secretion of IL-6 was measured by ELISA. PD-L1 specific small interfering RNA(siRNA) was used to knock down the expression of PD-L1 to detect whether PDL1 is involved in the production of IL-6. After stimulation with HMBPP or E7, the difference in the above mean values was compared between the control group and the treatment group by independent sample t-test. Results In the pleural fluid of the tuberculosis patients and umbilical cord blood, after 24 hours of stimulation with HMBPP or E7, the percentages of IL-6+ γδ T cells were significantly higher than that of the control group. After 12 hours of stimulation with HMBPP or E7, the relative expression of IL-6 in γδ T cells was significantly increased at the mRNA and protein levels. After HMBPP or E7 stimulation, the relative expression of PD-L1 in CD277+CD14+ monocyte-macrophages were significantly up-regulated [(3.70±0.33) and(4.20±0.34) vs 1.00]. When the expression of PD-L1 was knocked down, the expression of IL-6 in γδ T cells was significantly decreased [(0.53±0.03) and(0.55±0.01) vs 1.00]. All the above differences were statistically significant(all P < 0.05). Conclusion HMBPP and peptide E7 up-regulate PD-L1 expression in CD277+CD14+ mononuclear-macrophages, which are involved in stimulating co-cultured γδ T cells to produce more IL-6. This finding provides a basis for the research of the anti-tuberculosis immune mechanism of γδ T cells and the immunotherapy of tuberculosis patients.更多还原