纳米二氧化硅对斑马鱼血栓形成的影响及其分子机制研究

Epidemiological evidences confirm that the environmental nanoparticles can increase the morbidity and mortality of cardiovascular diseases. Our previous studies showed that silica nanoparticles induced microthrombi formation in the lungs of mice. Microarray analysis indicated that the genes expression (e.g., jak1, par1) related to inflammation and coagulation were significantly up-regulated. However, the underlying mechanism is still unclear. Combined with the various stains of zebrafish model, transgenic technology and reverse genetics technology, this study is aimed to investigate the toxic effect of silica nanoparticles on thrombosis formation and its related molecular mechanism. The vascular endothelial cells-labeled transgenic zebrafish strain is applied to explore the effect of silica nanoparticles on endothelial cells viability, inflammation response and vascular injury. Wild type zebrafish strain and platelet-labeled transgenic zebrafish strain are performed to investigate the change of blood coagulation function and platelet activation, and analyze the coagulation cascade and hypercoagulable state induced by silica nanoparticles. Utilized with transparent mutant zebrafish strain, the hemodynamic changes, erythrocyte aggregation and thrombosis formation are observed and measured directly in living embryos. For in-depth mechanism study, the JAK/STAT and PAR1 signaling pathways are detected to clarify the molecular mechanism of thrombosis formation induced by silica nanoparticles; the antisense morpholino oligonucleotides gene knock-down technology and in situ hybridization technique are conducted to verify the two pathways mentioned above. This study will provide the experimental evidences for cardiovascular toxicity and safety evaluation of silica nanoparticles. It will have a profound scientific significance.

流行病学研究证实环境纳米颗粒可致心血管疾病发病率和死亡率升高。课题组前期研究发现纳米SiO2可导致小鼠肺动脉微血栓形成，基因芯片分析显示与炎症及凝血相关的jak1、par1等基因表达明显上调，但毒作用机制尚不清楚。本研究拟采用不同品系斑马鱼模型，利用转基因和反向遗传学技术，系统研究纳米SiO2致血栓作用及其分子机制。采用血管内皮细胞标记转基因品系研究纳米SiO2对内皮细胞活性、炎症反应、血管损伤的影响；野生型品系和血小板标记转基因品系研究凝血功能改变和血小板活化，分析纳米SiO2诱发凝血级联反应及血液高凝状态；半透明突变品系活体观察血液动力学改变、红细胞聚集和血栓形成；深入研究JAK/STAT和PAR1信号通路对纳米SiO2致血栓形成的调控及分子机制，并采用吗啉代反义寡核苷酸基因沉默技术结合原位杂交技术进行验证。项目将为纳米SiO2心血管毒性研究及安全性评价提供实验依据，具有重要科学意义。

随着人群对纳米SiO2职业暴露、环境暴露和医源性暴露机会的日益增加，其对人群健康及环境的影响受到国内外学者越来越多的关注。研究纳米SiO2对人群健康和环境可能造成的危害并进行安全性评价具有重要科学意义和现实意义。然而，纳米SiO2对循环系统，尤其是对血栓形成的研究报道甚少。基于此，本研究探讨了纳米SiO2对血栓形成的影响及分子机制。研究发现，纳米SiO2可引起血管内皮细胞自噬激活和线粒体损伤，促进炎症因子和凝血因子的释放，造成血管内皮细胞损伤。纳米SiO2可引起Tg(mpo:GFP) 转基因斑马鱼品系血液中性粒细胞增多，激活炎症反应；抑制Tg(fli-1:EGFP) 转基因斑马鱼品系的血管内皮细胞表达水平，造成斑马鱼血管损伤，呈剂量-依赖关系；降低斑马鱼血流量和血流速，引起血流动力学参数改变；引起斑马鱼尾静脉红细胞增多和聚集，形成血液高凝状态，促进血栓形成，并呈剂量-依赖关系；纳米SiO2可上调斑马鱼GP130，JAK1，STAT3，TF，PAR1等基因和蛋白表达水平，激活JAK1/TF/PAR1信号通路；采用吗啉代反义寡核苷酸基因沉默技术，发现沉默il6st和f2r后，可有效降低纳米SiO2引起的JAK1/TF/PAR1信号通路相关基因蛋白表达水平。在此基础上，发现纳米二氧化硅与环境污染物苯并芘联合暴露可促进斑马鱼发生炎症-凝血级联反应。综上所述，纳米SiO2对斑马鱼具有心血管毒性和致血栓作用，通过激活JAK1/TF/PAR1信号通路引起斑马鱼炎症-凝血级联反应，造成血液动力学改变，形成血液高凝状态，进而促进血栓形成。本研究可为纳米SiO2安全性评价提供科学依据。