用于大气颗粒物对信号通路影响研究的无损采样及细胞暴露新方法

Environmental air pollution represented with frequent haze is a topic of great concern for the millions of Chinese people especially in urban areas. Numerous epidemiological and clinical studies have shown a relationship between exposure to airborne particulate matter and adverse effects of cardiopulmonary diseases and lung cancer morbidity and mortality. However, physical and chemical properties of airborne particles that are responsible for their toxicities and the mechanisms underlying airborne particle-induced carcinogenesis are still not fully known. Elucidation of signal transduction in cell upon exposure to airborne particulate matter is an effective solution to discover the association with a variety of adverse human health effects. In current studies, the aqueous or organic extracts of airborne particles were prepared before exposure on human and animal cells in vitro, in which, the contribution of particle morphological and mechanical properties to the adverse effects was neglected. Taking nondestructive air particle sampling and exposure on cell as breakthrough points, we will analyze and identify the differentially expressed proteins in cells exposed to typical airborne particulate matter with different sizes by iTRAQ-based proteomics technology to examine the signaling pathways, via which important physiological processes such as cell proliferation and apoptosis are influenced by airborne particles. This undisturbed and non-aggressive research strategy, comprehensively considering the effects of chemical composition, morphological feature and nanomechanical property of airborne particles on cell signal transduction, is promising to provide accurate and reliable experimental basis for revealing molecular mechanisms and screening biomarker candidates for toxicity of airborne particulate matter.

以雾霾频发为代表的空气污染是我国特别是许多城市居民非常关注的问题。流行病学和临床研究发现暴露大气颗粒物与心肺疾病和肺癌发病率和致死率相关，但是导致颗粒物毒性的物理和化学性质以及诱发癌变的内在机理尚不完全清楚。研究细胞受大气颗粒物刺激后信号转导过程是了解其对人体健康影响的一个有效手段。然而现有的人类和动物体外细胞实验通常采用大气颗粒物的水或有机溶剂提取物暴露细胞，忽视了颗粒物形貌和力学性质对毒性的影响。本课题以大气颗粒物的无损采样和细胞暴露为突破点，通过基于iTRAQ的蛋白质组学技术分析鉴定暴露不同尺寸典型大气颗粒物后细胞中的差异表达蛋白，探讨典型大气颗粒物影响细胞增殖和凋亡等重要生理过程的信号转导途径。这种无扰动、非破坏的研究策略综合考虑大气颗粒物的化学组成、形貌结构和纳米力学性质对细胞信号转导的影响，有望为揭示大气颗粒物毒性的分子机制、寻找颗粒物暴露的生物标志物提供准确可靠的实验依据。

现有流行病学调查、临床研究和动物实验发现大气颗粒物暴露与心肺疾病及肺癌发病率和致死率相关，但是导致颗粒物毒性的物理和化学性质以及诱发病变的内在机理尚不完全清楚。研究细胞受大气颗粒物刺激后信号转导过程是了解其对人体健康影响的一个有效手段，然而目前细胞实验通常采用水或有机溶剂破坏性地提取大气颗粒物然后暴露人和动物细胞，忽视了颗粒物形貌和力学性质对毒性的影响。本项目针对传统采样-提取-暴露方法的缺点，探索用生物兼容的琼脂制成的膜代替撞击式大气颗粒物采样器的常规滤膜，使颗粒物撞击到软性的琼脂膜表面，然后将含有大气颗粒物的琼脂膜悬浮在细胞培养液中直接暴露细胞。基于新型琼脂膜采样以及低扰动和少破坏的提取，我们利用体外细胞毒性实验和毒理蛋白质组学研究采集于琼脂膜上的大气细颗粒物对肺细胞、肝细胞和巨噬细胞的毒性作用，并以特氟隆膜采样-水提-冻干暴露方式的结果作为对照。在细胞活力、细胞凋亡和活性氧测定的基础上，通过基于iTRAQ的蛋白质组学技术分析鉴定暴露不同尺寸大气颗粒物后BEAS-2B人支气管上皮细胞和A549人肺上皮细胞中的差异表达蛋白，探讨大气颗粒物影响细胞增殖和凋亡等重要生理过程的信号转导途径。结果表明，不同采样膜采集的大气颗粒物及不同的细胞暴露方式有不同的毒性作用并对细胞蛋白质组有不同影响，细胞对于颗粒物刺激总的响应并不等于可溶部分和不可溶部分或无机物和有机物作用的简单加和，这种响应还与颗粒物尺寸、细胞系相关。因此，研究揭示了颗粒物采样-提取-暴露方式会造成不同细胞毒性和不同的信号通路变化，而琼脂膜采样及直接暴露细胞可以提供一种操作简单、而且结果可供比对的方法。这种低扰动、准原位、少破坏、颗粒物友好的研究策略综合考虑大气颗粒物化学组成、形貌结构和纳米力学性质对细胞信号转导的影响，为揭示大气颗粒物毒性的分子机制、寻找颗粒物暴露的生物标志物构建了更加精准、高效的实验研究平台。