基于miRNA表达谱技术的纳米二氧化硅致肺毒性分子机制研究

Silica (SiO2) nanoparticles（SNPs）as a non-metal oxide is one of the large-scale industrialized production of nanometerials, which is almost being used in all field involving microscale SiO2,including industrial production as well as scientific, biological, medical research and so on.Potential pulmonary toxicity of SNPs has become hot topics due to its chamical composition being same to quartz as a representative of the free silica. The results of our early studies showed that mechanisms of cytotoxicity induced by SNPs was closely correlated to increase oxidative stress, decrease membrane fluidity, perturbation of intracellular calcium homeostasis, increase apoptosis and change of cell cycle. In vivo experiments revealed that SNPs could induce the high expression of cytokines (TNF-α, TGF-β1, IL-1β) and MMP-2, promote the fibroblast proliferation and collagen synthesis of CHL cells and lead to pulmonary fibrosis. The effect of fibrogenesis of SNPs might be milder than that of microscale SiO2 particles in rats, potentially resulting from nanoparticals tending to be diffused and easily translocated due to their ultrafine particle size compared to microsized particles. But at present the molechlar mechanism of SNPs induced lung toxicity was poorly understood.In recent years,microRNA(miRNA) was found to be an important regulation function of the non-coding strand small molecule RNA and to play an important role in the toxicity effect due to its speciality in space time. The research apply cDNA microarray to screen the specific expression of the miRNA related to pulmonary injury based on establishment of rat lung injury model indeced by SNPs after intratracheal instillation.Then the bioinformatics analysis and target gene sequencing of the specific expression of the miRNA are carried out and 1～2 specific expressions of miRNA are choosen.After over and cut its expression using molecular biology techniques, the effects on transfected cell lines and regulation of predicting target gene indeced by SNPs are analysed and biology function and its regulation mechanism of miRNA are probed into in SNPs induced lung toxicity.Finally,the mechanism of lung toxicity induced by SNPs and its biological markers will be revealed.

纳米二氧化硅（SiO2）与以石英为代表的游离SiO2的化学组成相同，其肺毒性效应成为当前关注的热点。我们早期的研究结果表明纳米SiO2可损伤RAW264.7细胞膜产生细胞毒性；通过诱导经呼吸道染尘大鼠肺组织脂质过氧化作用增强、细胞因子表达及肺细胞凋亡率增加、细胞周期改变而导致肺组织损伤。其毒性的分子机制尚不明了。鉴于miRNAs的表达具有时间和空间的特异性及可能在毒性效应产生过程中起着重要调节作用，本研究在前期工作的基础上，利用基因芯片技术筛选损伤相关的特异表达miRNA，对其进行生物信息学分析和靶基因测序，选择1～2个特异性表达miRNA，运用分子生物学技术过度和下调其表达，分析在有无纳米SiO2诱导作用下其对基因转染细胞株的影响及对预测靶基因的调节作用，结合分子生物学实验及生物信息学分析结果探讨miRNA在纳米SiO2致肺毒性中的生物学功能及调控机制，揭示其肺毒性作用机制及生物标志物。

随着纳米二氧化硅( SiO2)应用领域的扩大及生产量的增加，各类人群的暴露机会日益增多；其肺毒性效应及机制成为当前关注的热点。鉴于miRNAs 的表达具有时间和空间的特异性及可能在毒性效应产生过程中起着重要调节作用，本项目建立经气管灌注纳米SiO2致大鼠肺损伤模型，利用Illumina HiSeq 2000测序技术分析纳米SiO2对大鼠染尘7、15、30、60、90天后肺组织的miRNA表达谱变化，筛选并验证差异表达的miRNAs。研究发现纳米SiO2染尘早期（7、15、30天）引起的肺损伤以炎症为主，后期（60、90天）主要为肺间质纤维化， miR-208、miR-212、miR-18a可能在纳米SiO2致大鼠肺损伤过程中起调控作用，在靶基因PDCD4、LIN28B、CTGF的翻译水平参与纳米SiO2致大鼠肺损伤的调控作用；构建纳米SiO2染毒细胞株模型，以miR-208及miR-18a模拟物/抑制物分别转染NR8383大鼠肺泡巨噬细胞及CHL中国仓鼠肺成纤维细胞后发现纳米SiO2染毒NR8383细胞未引起细胞内miR-208表达水平的改变，过表达miR-208可导致PDCD4 mRNA、蛋白表达上调，抑制miR-208可致PDCD4 mRNA下调，miR-208有可能正调控或者间接调控靶基因PDCD4表达，纳米SiO2染毒NR8383细胞上清液刺激CHL细胞增殖且miR-18a表达量增加，通过与靶基因CTGF的3’UTR位点结合，经TGF-β1信号通路负调控CTGF的表达，进而可能对肺纤维化的发生发展进程起调控作用；整体动物及细胞两个水平的研究表明，纳米SiO2致大鼠肺纤维化中发生了EMT，miR-541通过直接调控TGF-β R Ⅲ、FGF7在EMT发生发展中有一定作用；TGF-β1可抑制miR-541的表达从而促进EMT的发生；过表达miR-541可通过负调控TGF-β R Ⅲ及FGF7的表达，减弱TGF-β及FGF通路从而抑制EMT的发生，具有拮抗肺纤维化的作用；miＲ-702-3p可通过与靶基因BMP-7 的3’UTR位点结合，调控TGF-β1/Smad通路在纳米SiO2致肺EMT的发生而促进肺纤维化。上述研究结果揭示了纳米SiO2致肺纤维化发生的分子机制，为其安全性评价及卫生标准的制订提供科学依据，也为其致肺纤维化作用提供早期敏感标志物及有效作用靶点。