无定形纳米SiO2诱导细胞恶性转化及其相关机制研究

Crystalline silica has already been designated as a human carcinogen by IARC. Due to the limited epidemiology and laboratory research evidence of carcinogenicity on animals and humans, and also its mechanism still unclear, the amorphous silica is classified as Group 3, and thus it is necessary to investigate its correlation with tumor. Researches showed that amorphous silica nanoparticle (nano-silica) could cause oxidative stress, induce DNA injury, and thus increase the risk of tumor occurrence. Furthermore, pevious study in our research group showed that amorphous nano-silica could induce the formation of multinucleation, which is also closely related to malignant transformation and carcinogenesis. Our project aims to investigate the malignant transformation of human bronchial epithelial cell, 16HBE induced by nano-silica and its related mechanisms. The 16HBE cells were exposed to nano-silica repeatedly, and the nano-silica-induced malignant transformation cell model was established eventually under the validation through soft agar colony formation, concanavealin A(ConA) agglutination and tumorigenecity in nude mice. The change of ROS content before and after the malignant transformation was determined by flow cytometry, and also the protein expressions of the signal molecules in the ROS-mediated MAPK/ERK and PI3K/Akt pathways, and those of the oncogenes and tumor suppressor genes were detected by western blot. Furthermore, the ROS inhibitor NAC, H2O2, and ERK, Akt over-expression or gene silence were applied to elucidate the role of ROS-mediated MAPK/ERK and PI3K/Akt pathways in the nano-silica-induced malignant transformation. The research achievement would provide a theoretical basis for nano-silica safety evaluation.

结晶形二氧化硅（SiO2）被IARC划定为人类致癌物，而无定形SiO2为Group3，对动物和人类致癌的流行病学、实验室证据均有限、机制不清，深入探讨其与肿瘤关系很有必要。研究发现，无定形纳米SiO2可引发氧化应激，造成DNA损伤，增加肿瘤发生风险，且课题组研究发现纳米SiO2有致多核作用，而多核形成与细胞恶性转化及肿瘤发生密切相关。本项目研究纳米SiO2诱导人支气管上皮细胞16HBE恶性转化及相关作用机制，细胞多次纳米SiO2染毒，采用软琼脂集落形成、刀豆蛋白A凝集和裸鼠成瘤试验验证恶转发生，流式细胞术检测恶转前后ROS含量，Western blot检测ROS介导的MAPK/ERK和PI3K/Akt信号通路分子以及癌基因、抑癌基因表达，并应用ROS抑制剂NAC、H2O2及过表达/基因沉默ERK和Akt阐明上述信号通路在纳米SiO2诱导细胞恶转中的作用，为纳米SiO2安全性评价提供理论依据

结晶形二氧化硅（silica dioxide，SiO2）被IARC划定为人类致癌物，而无定形SiO2为Group 3，对动物和人类致癌的流行病学、实验室证据均有限、机制不清，深入探讨其与肿瘤关系很有必要。研究发现，无定形纳米SiO2可引发氧化应激，造成DNA损伤，增加肿瘤发生风险，且课题组研究发现无定形纳米SiO2有致多核作用，而多核形成与细胞恶性转化及肿瘤发生密切相关。本项目研究无定形纳米SiO2诱导人肺支气管上皮细胞恶性转化及相关作用机制，分别采用单次染毒和多次无定形纳米SiO2染毒，单次染毒检测无定形纳米SiO2的细胞毒性情况，包括细胞通透性、氧化应激水平、线粒体损伤、细胞凋亡和周期等的检测，多次染毒主要检测细胞恶性转化，采用软琼脂集落形成和裸鼠成瘤试验验证细胞恶性转化的发生，流式细胞术检测恶转前后细胞中活性氧（reactive oxygen species，ROS）含量变化，western blot检测ROS介导的MAPK和PI3K/Akt信号通路分子以及癌基因、抑癌基因的表达，并应用ROS抑制剂NAC验证氧化应激在无定形纳米SiO2诱导细胞恶性转化中的作用，为无定形纳米SiO2安全性评价提供理论依据。结果显示：单次无定形纳米SiO2染毒可引起细胞活力下降、细胞通透性增加、ROS释放、线粒体受损、细胞凋亡增加以及细胞周期G2/M阻滞，并呈剂量依赖性关系，还促进细胞炎症因子和氧化应激相关因子的表达，呈一定的剂量依赖关系；多次无定形纳米SiO2染毒后细胞的增殖能力增强，软琼脂集落形成能力增加，裸鼠荷瘤实验阳性，证实细胞发生恶性转化。同时，恶转细胞MAPK和PI3K/Akt信号通路发生改变，p38MAPK和MEK的磷酸化水平显著升高，Akt的磷酸化水平显著降低，细胞癌基因c-jun、c-fos表达升高，抑癌基因p53表达降低。给予NAC组细胞显示裸鼠荷瘤实验阴性，说明抑制氧化应激可遏制细胞发生恶性转化。现已发表中文核心期刊论文1篇， SCI论文1篇（international journal of nanomedicine, IF=4.383），参加国内学术会议交流五次，发表会议摘要4篇。通过本课题的实施，课题负责人李艳博晋升副教授职称，并作为项目负责人获批国家自然科学基金面上项目1项、北京市教委面上项目1项和北京市自然科学基金面上项目1项，2015年度获批“首都