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100μg/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、60μg/ml组相比,16HBE的ROS水平显著升高(P<0.05)。对照组、100μg/ml TDI-HSA处理组、50 mmol/L NAC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-HSA） 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-HSA.The level of intracellular ROS of HBE cells was detected by flow cytometry with an oxidation-sensitive fluorescent probe 2＇,7＇-dichlorofluorescein diacetate（DCFH-DA） uploading,and the permeability of cell monolayer was assessed by detecting the transepithelial electrical resistance（TEER）.Results The exposure to 120 μg/ml TDI-HSA 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-HSA（P0.05）.The intracellular ROS production increased significantly after 100 μg/ml TDI-HSA treatment（P0.05）,but the increment in ROS production was significantly suppressed by pretreatment of the cells with N-acetylcysteine（NAC）（P0.05）,which also enhanced the TEER decreased by TDI-HSA treatment（P0.05）.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.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2011年第2期239-243,共5页
Journal of Southern Medical University
基金
国家自然科学基金(30270593)
中华医学会慢性呼吸道疾病专项基金(07010130021
08020460124)
广东省科技计划基金(2005207007)~~
