纳米二氧化硅与微米二氧化硅致大鼠肺纤维化miRNAs差异表达比较研究

Comparison study on the differential expression of miRNAs in rat pulmonary fibrosis induced by nanosized SiO2 and microsized SiO2

目的比较纳米二氧化硅(SiO2)与微米二氧化硅(SiO2)致大鼠肺纤维化过程中miRNAs差异表达的变化,分析miRNAs表达水平与肺纤维化的相关性。方法将36只体重为180~220 g的SPF级健康雄性SD大鼠随机分为对照组、纳米SiO2染尘组和微米SiO2染尘组,每组12只,用一次性气管灌注染毒方法分别经气管灌注1ml生理盐水、25mg/ml纳米SiO2溶液和25mg/ml微米SiO2溶液。分别于染尘60、90 d各处死6只大鼠,用光学显微镜和透射电镜观察大鼠肺组织病理学及超微结构改变;用IlluminaHiSeq 2000高通量测序技术分析大鼠肺组织miRNA表达谱;用Targetscan对miRNAs进行靶基因预测,通过基因本体论Gene Ontology(GO)和京都基因与基因组百科全书(KEGG)分别对靶基因进行功能分类和pathway分析,筛选与肺纤维化相关的靶基因。结果光学显微镜下可见,染尘60、90 d时,纳米SiO2染尘组大鼠肺组织支气管和血管周围、肺泡壁和小叶间隔增宽,纤维结缔组织轻度增生。染尘60 d时,微米SiO2染尘组大鼠肺内弥漫性分布细胞性结节,多数结节融合,纤维母细胞增生明显,结节周围肺泡代偿性肺气肿;染尘90 d时,微米SiO2染尘组大鼠肺内弥漫性分布的细胞纤维性结节内纤维成分增加,融合的大结节周围肺泡代偿性肺气肿。透射电镜下可见,染尘60、90 d时,纳米SiO2组大鼠肺组织Ⅱ型肺泡上皮细胞嗜锇板层小体增多、肿胀,空泡样变,间质中胶原纤维及弹力纤维增生;微米SiO2染尘组大鼠肺组织Ⅱ型肺泡上皮细胞胞质内嗜锇板层小体增多,间质中胶原纤维及弹力纤维增生,嗜锇板层小体空泡样变。与对照组比较,染尘60、90d时,纳米SiO2染尘组大鼠肺组织中差异上调表达miRNA分别为50、72个,下调表达miRNA为22、24个;微米SiO2染尘组差异上调表达miRNA分别为91、70个,下调表达miRNA为34、78个。两者共同差异上调表达的miRNA为miRNA-18a、miRNA-702-3p,下调表达的miRNA为miRNA-541、miRNA-127、miRNA-379,与肺纤维化有关的靶基因分别为CTGF、IGF、BMP7、FGF7、TGF-βRIII、IGF1R与TGF-β1,其生物学功能为参与TGF-β、MAPK及Wnt信号通路、成纤维细胞活化等调控。结论相同剂量纳米SiO2长时间经呼吸道染尘可致大鼠肺间质纤维化,不同于微米SiO2所致的以硅结节为主的病理改变;miRNA-18a、miRNA-702-3p、miRNA-541、miRNA-127、miRNA-379可能通过调控其靶基因在纳米SiO2与微米SiO2致大鼠肺纤维化过程中发挥一定作用。

Objective To explore the correlation between expression level of miRNAs and pulmonary fibrosis on the basis of comparison the differential expression of miRNAs in rat pulmonary fibrosis induced by nano SiO2 and micron SiO2.Methods Thirty-six healthy male SD rats weighting 180-220 g were randomly divided into 3 groups.They were instilled intratracheally with 1 ml suspension of saline,25 mg/ml nanosized SiO2 and microsized SiO2 particles and sacrificed at 60 d and 90 d postexposure from each group with six rats.The change of pathological morphology and ultrastructure of lung were observed by optical and transmission electron microscopy.The differentially expressed microRNAs in lung tissue of the rats after instilled intrachcally nanosized SiO2 and microsized SiO2 particles at 60 d and 90 d were determined by Illumina HiSeq 2000 sequencing technique.Target prediction for miRNAs was conducted by databases of Target-scan.Function-significant enrichment analysis and signal pathway analysis for predicted target genes were respectively conducted by the GO and the KEGG,then target genes related to pulmonary fibrosis were screened out.Results Light microscope examination showed that wide bronchi,vessels,interlobular septa and slight fibrous connective tissue proliferation at 60 d and 90 d postexposure in 25 mg/ml nanosized SiO2 group.A few fused nodules at 30 d postexposure,a lot of fused nodules at 60 d postexposure,fibrous cell nodules and compensatory emphysema around alveolar at 90 d postexposure in 25 mg/mL microsized SiO2 group were observed.Electron microscopy demonstrated swelling and vacuolar degeneration of osmiophilic lamellar bodies in typeⅡalveolar epithelial cells,collagen fiber and elastic fiber hyperplasia in pulmonary interstitial at 60 d,90 d postexposure in 25 mg/ml nanosized SiO2 group.Increased and vacuoloid changed osmiophilic lamellar bodies in typeⅡalveolar epithelial cells,collagen fiber and elastic fiber hyperplasia in the interstitial at 60 d,90 d postexposure in 25 mg/ml microsized SiO2 group were observed.Comparing to saline control group,the number of miRNA up-regulated expression was 50,70,and down-regulated expression was 22 and 24 at 60 d,90 d postexposure in 25 mg/ml nanosized SiO2 group respectively.There were 91,70 miRNAs up-regulated expression and 34,78 miRNAs down-regulated expression at 60 d,90 d postexposure in 25 mg/ml microscale SiO2 group.The common miRNA of differential up-regulated expression are miRNA-18a and miRNA-702-3p,down-regulated expression are miRNA-541,miRNA-127 and miRNA-379 both in nanosized SiO2 and microscale SiO2 group.The target genes related to pulmonary fibrosis were CTGF,IGF,BMP7,FGF7,TGF-βRIII,IGF1R and TGF-β1 respectively.Their biologic functions are to regulate signal pathway of TGF-β,MAPK and Wnt,and activation of fibroblast.Conclusion These findings suggested that same dose of nanosized SiO2 particles could cause mainly characterized by pulmonary interstitial fibrosis differing from silicotic nodule caused by microsized SiO2.miRNA-18a,miRNA-702-3p,miRNA-541,miRNA-127 and miRNA-379 may play a role in the process of pulmonary fibrosis in nanosized SiO2 and microscale SiO2 by regulating its target genes.