甲苯二异氰酸盐对人支气管上皮细胞血管内皮生长因子表达的影响

Toluene diisocyanate increases vascular endothelial growth factor expression in human bronchial epithelial cells

目的探讨甲苯二异氰酸盐(TDI)对正常人支气管上皮细胞(HBE)血管内皮生长因子(VEGF)表达的影响。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和HSA对正常人支气管上皮细胞株HBE135-E6E7活力的影响;分别用浓度为10、20、30、40μg/ml的HSA和TDI-HSA处理HBE135-E6E7,以未加任何处理因素的HBE135-E6E7为对照组,用半定量RT-PCR同时扩增各组HBE135-E6E7VEGF和β-actin基因片段,从基因水平上比较各组VEGF的相对表达情况。结果40μg/ml以下浓度的HSA和TDI-HSA不影响HBE135-E6E7活力;HBE135-E6E7组成性表达VEGF189和VEGF165;与10μg/mlHSA组以及对照组相比,10μg/mlTDI-HSA组HBE135-E6E7 VEGF189和VEGF165的表达增加,但无显著差异(P>0.05),20、30、40μg/ml的TDI-HSA组与相应浓度的HSA组以及对照组相比,HBE135-E6E7 VEGF189和VEGF165的表达均显著增加(P<0.05),且随着TDI-HSA浓度的增加,HBE135-E6E7 VEGF189和VEGF165的表达亦随之增加(P<0.05)。结论TDI显著增加HBE VEGF表达,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 expression of vascular endothelial growth factor （VEGF） in 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 examine the viability of HBE135-E6E7 cells cultured in serum-free medium after treatment with HSA or TDI-HSA at different concentrations. VEGF mRNA expression of the HBE cells treated with HSA or TDI-HSA at 10, 20, 30 and 40 μg/ml, respectively, was detected using semi-quantitative RT-PCR. Results Treatment with 40μg/ml HSA and 40 μg/ml TDI-HSA did not result in significant changes in the viability of HBE135-E6E7 cells. RT-PCR revealed the constitutive expression of two VEGF isoforms, namely VEGF189 and VEGF 165, in cultured HBE135-E6E7 cells. After exposure to TDI-HSA at the different concentrations （except for 10μg/ml）, a significant increase occurred in both VEGF189 and VEGF165 mRNA expressions in HBE135-E6E7 cells as compared with the expressions in the control group and the HSA-treated cells （P〈0.05）, and significant dose dependence was noted in the effect of TDI-HSA （P〈0.05）. No significant difference was found in the expressions between the control cells and the HAS-treated cells （P〉0.05）. Conclusion TDI induces significant increase in VEGF expression in HBE cells, and VEGF overexpression may play an important role in the pathogenesis of TDI-induced asthma.