甲苯二异氰酸盐对人支气管上皮细胞通透性的影响

摘要

目的 探讨甲苯二异氰酸盐(TDI)对正常人支气管上皮细胞(16HBE)活性氧(ROS)生成及通透性的影响.方法 应用改良的Son氏等方法制备TDI-人血清白蛋白(TDI-HSA).四唑盐(MTT)比色法检测不同浓度的TDI-HSA对正常人支气管上皮细胞株16HBE活力的影响.实验分4组:以未加任何处理因素的16HBE为对照组,20、60、100μg/ml的TDI-HSA处理16HBE 24 h,通过2',7'-二氢二氯荧光黄双乙酸钠(DCFH-DA)细胞内ROS荧光染色,收集细胞后以流式细胞仪检测荧光强度.选择对ROS水平影响较大的100 μg/ml的TDI-HSA处理16HBE 24 h.流式细胞仪定量检测抗氧化剂N-乙酰半胱氨酸(NAC)对TDI-HSA诱导ROS生成的影响,荧光显微镜观察照相.采用跨上皮电阻(TEER)法检测上皮单层细胞的通透性.结果 120μg/ml以下浓度的TDI-HSA对细胞活力无显著影响.ROS水平在对照组、20、60和100μg/ml组分别为:65.04±4.56,91.76±4.84,119.96±10.40,203.11±8.21.100μg/ml TDI-HSA组与对照组及20、60 μg/ml组相比,16HBE的ROS水平显著升高(P＜0.05).对照组、100 μg/ml TDI-HSA处理组、50 mmol/LNAC预处理组的细胞内ROS水平分别为:69.02±2.14,246.47±18.55,102.50±4.60;而TEER分别为:280.75±11.93,92.25±11.44,207.25±7.41.NAC可显著降低TDI-HSA诱导的ROS生成(P＜0.05),改善氧化应激对单层细胞通透性的影响(P＜0.05).结论 TDI显著增加HBEROS的产生,其诱导的氧化应激部分参与支气管上皮细胞通透性的增加.这可能是TDI诱发哮喘的发病机制之一.%Objective To investigate the effect of toluene diisocyanate (TDI) on the production of reactive oxygen species (ROS) and the permeability of human bronchial epithelial (HBE) cells. Methods TDI-human serum albumin (TDI-HAS) conjugate was prepared using a modified Son's method. MTT assay was used to assess HBE cell viability after exposure to different concentrations of TDI-HAS. The level of intracellular ROS of HBE cells was detected by flow cytometry with an oxidation-sensitive fluorescent probe 2',7'-dichIorofluorescein diacetate (DCFH-DA) uploading, and the permeability of cell monolayer was assessed by detecting the transepithelial electrical resistance (TEER). Results The exposure to 120 ug/ml TDI-HAS did not obviously affect the cell viability. Compared with the control group, the intracellular fluorescent intensity increased significantly in the cells exposed to 20, 60, and 100 μg/ ml TDI-HAS (P0.05). The intracellular ROS production increased significantly after 100 μg/ml TDI-HAS treatment (P< 0.05), but the increment in ROS production was significantly suppressed by pretreatment of the cells with N-acetylcysteine (NAC) (P<0.05), which also enhanced the TEER decreased by TDI-HAS treatment (P<0.05). Conclusion TDI enhances the permeability of HBE cell monolayer partially through a ROS-mediated pathway, suggesting the importance of oxidative stress in TDI-induced pulmonary diseases.