机车排放颗粒物（DEPs）胁迫下的致敏花粉的抗原性及其协同诱导的氧化应激研究

Ambient allergenic pollen and airborne fine partilces(PM2.5) has been taken has done great harm to human health and substantially deteriorated urban air quality in China. Allergencity of pollens under the stress of the DEPs(one of main composition in the urban airborne particles),and sygernetic oxidative stress caused by DEPs and pollens (containing NADPH oxidase) have not been reported. Fresh Platanus pollens will be collected from Platanus tree which were widely planted in Shanghai urban area,and will be exposuered to the DEPs in the exposure chamber which has been designed by our research group. Mass concentration of DEPs,NO2 and O3 in the chamber could be stimulated compared with their mass level in the urban atmophere.After exposure, the pollens will be collected (polluted pollens).Then,multiple technologies,such as polyacrylamide gel electrophoresis (SDS-PAGE),two-dimensional gel electrophoresis (2-DE), mass spectrometry will be employed to investigate protein expression released from fresh Platanus pollens and polluted pollens. Immunobolotting will be used to study allergenicity of allergenic protein. Taking human epithelial cells ( A549 cells) (in vitro)and allergenic mouse (in vivo)as the model,the study intends respectively to measure intracellular reactive oxygen species (ROS) by using of DCFH assay and inflammation cells （including macrophage,eosinophils, neutrophils and lymphocytes） and chemokines (such as Interleukin-4, Interferon-γ) in the bronchoalveolar lavage fluid(BALF), also pathological evaluation of the lung tissue will be examined. Based on the above experimental data, sygernetic biological effects caused by allergenic pollen and DEPs will be evaluated. This experimental data will set up a solid base for evaluating sygernetic biological effects caused by allergenic pollen and DEPs. Further more, it is highly expected that our study can provide new scientific data to elucidate mechanism of allergenicity caused by pollen, and help promote development of cross-over studies among atmospheric sciences, environmental chemistry, environmental toxicology and immunology.

飞散致敏花粉与大气细颗粒物(PM2.5)已经对人群健康和城市空气质量产生了较为严重的影响, 作为城市大气细颗粒物主要组分的机车尾气排放颗粒物（DEPs）对致敏花粉变应原蛋白抗原性的影响以及DEPs与花粉颗粒间所产生的协同氧化应激的研究还没见报道。本项目以致敏悬铃木花粉为对象，构建花粉暴露装置，利用二维凝胶电泳和质谱技术解析花粉暴露DEPs前后其变应原蛋白表达的差异，用免疫印迹方法研究花粉变应原蛋白的致敏特性；以肺上皮细胞(A549)和致敏小鼠为模式，分析致敏花粉中的NADPH氧化酶（烟酰胺腺嘌呤二核苷酸磷酸）与DEPs协同诱导所产生的细胞间活性氧水平及小鼠肺液中炎症细胞数量、细胞因子、肺部病理学特征的变化，以此探究致敏花粉与DEPs协同诱导的氧化应激；研究的成果不仅能够为完善现有花粉的致敏机理提供新的科学数据，也必将促进大气环境科学、环境化学、环境毒理学、免疫学等不同学科间交叉研究的发展。

在自然基金的支持下，课题组选择在上海分别广泛的悬铃木花粉和葎草花粉为研究对象，在查明致敏花粉时空分布规律的基础上，利用分子生物学的技术成功获取重组致敏花粉蛋白（Plaa3, Huuu），通过诱导表达富集并纯化了该类蛋白，利用纯化后的蛋白对SD大鼠进行免疫处理，制备出高效价和优良特异性的大鼠来源的IgG和IgE抗体。以此为基础，对上海市春季大气颗粒物样品中的花粉致敏蛋白的分布特征进行了研究；构建了花粉暴露大气污染物实验装置，对葎草花粉暴露污染物后的蛋白结构变化及致敏特性进行了探讨，研究结果表明.大气颗粒物中的致敏蛋白Pla a3主要集中在>7.0μm的粒径段内，其质量浓度平均为9.6 pg/m3，在此粒径段内，其质量浓度呈先增后降的时间变化趋势，与悬铃木的花期相吻合；在3.3~7.0μm、2.0~3.3μm、1.1~2.0μm等三个粒径段内，致敏蛋白Pla a3的质量浓度分别为1.3、1.9、2.1 pg/m3；在<1.1μm的粒径段内，致敏蛋白Pla a3的含量最少，约0.5 pg/m3。致敏蛋白Pla a3含量随着粒径的减小而减少；颗粒物中的Ca2+, NH4+等离子对悬铃木花粉蛋白的释放有促进作用；致敏悬铃木花粉蛋白暴露O3/NO2后，其致敏特性有明显的增强，葎草花粉蛋白暴露O3后，其profilin 蛋白结构的变化，而单独暴露NO2并没有导致该类蛋白结构的变化，蛋白结构的变化导致其致敏性有明显的增强。此外，课题组还利用制备的高效抗体与取自致敏人群的血清之间的免疫反应进行了分析，结果显示该抗体可以用来对花粉过敏症进行诊断，显示了研究成果具备潜在应用前景。