基于转录组测序及微流控芯片技术的PM2.5诱导人细胞促癌信号通路分子机制研究

The current air quality around the world, especially developing countries continued to deteriorate. Aerosol haze frequents occurrence. WHO announced in 2013 that PM2.5 was carcinogen for humans, but limited to existing relavent research about epidemiology and hypothesis in carcinogenic mechanism, Signal transduction of human Cells in response to carcinogenic PM2.5 is still blank. This study try to combine the RNA seq technology and microfluidic chip technology, to solve the technical difficulties that real quantity of collected PM2.5 cannot meet the multicomponent chemical analysis and multiple biological analysis of PM2.5 . In this study, integration with 3D cell culture and protein analysis unit of microfluidic chip system will be designed and fabricated. By the design of thick channel branch into thin channel to simulate the pulmonary vessels and bronchi, and then perform "lungs on chip" chip and the lytic cell in situ. According to the result of the RNA seq, the protein array chip will be designed with protein probe response to carcinogenic PM2.5 Combined with the Western, Elisa and flow cytometry technology for validation of chip function. Further reveal the molecular mechanism of signal transduction of human cells in response to carcinogenic PM2.5, and to get the maps of related transcriptional pathway and the key protein localization in cells. For the prevention and treatment of public health hazards caused by atmospheric particulates provide reliable theory basis.

当前世界各地特别是发展中国家空气质量持续恶化，雾霾频发。2013年WHO宣布PM2.5为人类致癌物，但现有研究仅局限于流行病学及致癌机制假说方面，其致癌基因信号通路分子机制研究尚属空白。本研究拟结合转录组测序技术和微流控芯片技术，解决PM2.5实采量不能同时满足PM2.5多组分化学分析与多重样本生物分析的技术难点。设计制造整合有3D细胞培养单元和蛋白分析单元的微流控芯片系统，通过由粗腔道逐步分支成细腔道的设计模拟肺部血管和支气管，以实现“芯片上的仿生肺”，完成培养细胞的原位裂解；根据转录组测序结果，设计促癌各相关蛋白的芯片微阵列分析，并结合Western、Elisa、流式细胞等技术对芯片功能进行验证。深入揭示PM2.5促癌基因信号通路分子机制，绘制相关蛋白转录调控途径图谱，并进行促癌关键蛋白细胞内定位，为预防和治疗大气颗粒物所造成的公共健康危害提供可靠的理论依据。

流行病学证明PM2.5能引发肺器官产生炎性反应，甚至癌症。PM2.5诱导的细胞癌变毒性分子机制对疾病诊断和治疗具有重要意义。本研究成功制备了基于微流控的气液界面（ALI）类肺器官芯片系统，通过转录组和蛋白质组学技术（RNA-seq，iTRAQ）揭示了PM2.5诱导人肺细胞BEAS-2B癌变相关信号转导通路的分子机制。结合Elisa、TEM、细胞凋亡检测对类肺器官芯片上多组学分析癌相关蛋白进行验证。比较气液暴露和浸没暴露两种体系下细胞分泌IL6和AKT1表达量的变化，证实了ALI系统检测毒性效应更优。并对PM2.5致癌组分和其诱导的细胞内差异表达的癌相关蛋白和细胞因子进行了相关性分析。.RNA-seq结果证明：BEAS-2B细胞内共有473个基因表达具有显著差异，iTRAQ结果有250个蛋白表达具有显著差异。多重组学结果证明了在基因和蛋白水平上，BEAS-2B细胞内共同有25条信号通路被启动，其中包括癌相关信号通路（P53, JAK-STAT和PI3K-Akt信号通路）。ELISA表明：随着PM2.5浓度升高，IL6，AKT1，VEGFA，P53和STAT等癌蛋白及细胞因子也随之升高，而MDM2的表达量下降。TEM表明：PM2.5能够进入细胞破坏细胞膜等结构，对线粒体和细胞核造成难以恢复的损伤。皮尔森相关指数表明：PM2.5中的组分(OC、EC、16PAHs、Alkane和重金属)与IL6，AKT1，VEGFA，P53，STAT等蛋白显著相关。.致癌PM2.5引发人肺细胞毒性分子机制：PM2.5破坏细胞微结构，诱导线粒体内的ROS表达量上升及NF-κB家族表达紊乱，介导促炎性因子下游癌蛋白如AKT1和P53等的表达，启动癌相关信号通路PI3K-Akt和P53。24hPM2.5气液暴露后，BEAS-2B内Akt表达量上升，抑制了下游基因MDM2的表达，直接引起细胞凋亡紊乱，最终诱发细胞损伤，甚至产生癌变。PM2.5诱导STAT表达量上升，启动了JAK-STAT信号通路，同时促进MYC和VEGFA等癌基因表达上调，导致细胞内凋亡通路紊乱、癌标志信号通路的启动最终引发细胞癌变。.本研究成功构建了ALI类肺器官芯片，为致癌污染物的体外细胞毒性机制研究提供了快速检测平台，揭示了PM2.5致癌的复杂分子机制，为大气污染及人类健康和相关疾病的预防和治疗策略提供了基础性资料。