摘要目的 通过检测染矽尘大鼠肺组织和A549细胞中微RNA-29b-3p (miRNA-29b-3p)和微RNA-34c-3p(miRNA-34c-3p)的表达水平,探讨miRNA-29b-3p和miRNA-34c-3p在肺纤维化进程中的作用.方法 SPF级雄性Wistar大鼠60只,按体重随机分为对照组和二氧化硅(SiO2)染尘组,每组30只,用一次性非暴露式气管滴注法灌注SiO2染尘组大鼠1 ml SiO2悬浊液,对照组大鼠相同方法气管内注入1 ml灭菌生理盐水.于染尘后1、7、14、21、28 d两组分别采集6只大鼠肺组织,其中3只大鼠取出肺组织进行病理学观察,另外3只采用miRNA微阵列芯片技术筛选肺组织中差异表达的miRNA.在体外细胞水平中培养A549细胞,分为对照组、SiO2刺激组和TGF-β1刺激组,在处理后的12、24和48 h收取细胞,通过荧光定量PCR(qRT-PCR)方法验证miRNA-29b-3p和miRNA-34c-3p在各组大鼠肺组织和A549细胞中的表达水平,对miRNA-29b-3p和miRNA-34c-3p进行靶基因预测并进行GO富集分析和KEGG通路分析.结果 对照组大鼠体重增长速度较SiO2染尘组大鼠明显;与对照组比较,SiO2染尘组大鼠的肺脏质量和肺系数明显升高,差异有统计学意义(P＜0.05).对照组大鼠肺组织21、28 d炎症反应较1、7、14d明显减轻,SiO2染尘组大鼠肺组织有持续性炎性细胞浸润;对照组大鼠肺组织21、28 d有少量胶原分布,SiO2染尘组大鼠肺组织21d后开始出现大量胶原纤维沉积.与对照组比较,SiO2染尘组中miRNA-29b-3p和miRNA-34c-3p表达水平均明显下调,差异均有统计学意义(P.＜0.05).A549细胞在SiOz和人重组TGF-β1处理后,与对照组比较,24 h和48 h处理组中miRNA-29b-3p和miRNA-34c-3p表达水平均明显下调,差异有统计学意义(P＜0.05).结论 大鼠肺组织和A549细胞中miRNA-29b-3p和miRNA-34c-3p的下调可能与早期矽肺的发生发展有关,有望成为早期矽肺诊断以及预后的指标.

abstractsObjective To investigate the role of microRNA-29b-3p(miRNA-29b-3p) and miRNA-34c-3p in the process of pulmonary fibrosis,we detected the expression levels of miRNA-29b-3p and miRNA-34c-3p in the lung tissue of rats exposed to silica and A549 cells.Methods SPF male Wistar rats were randomly divided into 1,7,14,21,28 d control group and silica (SiO2) dusting group,with 6 rats in each group.Onetime non-exposure method was used to infuse 1ml SiO2 suspension.The rat SiO2 dusting group was established in the liquid,and the control rats were intratracheally injected with 1 ml of sterile physiological saline in the same manner.The lung tissues of each group were collected at the corresponding time points after dusting.Three of the rats were taken out for pathological observation,and the other three were used to screen differentially expressed miRNAs in lung tissueby miRNA microarray technology.A549 cells were cultured at the in vitro cell level and divided into control group,SiO2 stimulation group and TGF-β1 stimulation group,and cells were collected at 12,24 and 48 h after treatment.The expression levels of miRNA-29b-3p and miRNA-34c-3p in rat lung tissue and A549 cells were verified by real-time PCR (qRT-PCR),target gene prediction of miRNA-29b-3p and miRNA-34c-3p and perform GO enrichment analysis and KEGG pathway analysis.Results The weight growth rate of the control group was significantly higher than that of the SiO2 dusting group.Compared with the control group,the lung mass and lung coefficient of the SiO2 dusting group were significantly increased (P＜0.05).The inflammatory response of the lungs in the control group was significantly reduced at 21 and 28 days,and the inflammatory cells infiltrated in the lung tissue of the SiO2 group.The rats in the control group had a small amount of collagen at 21 and 28 days.A large amount of collagen fiber deposition began to appear in the lung tissue of rats exposed to SiO2 for 21 days.Compared with the control group,the expression levels of miRNA-29b-3p and miRNA-34c-3p in the SiO2 dusting group were significantly down-regulated,and there was significant difference compared with the control group (P＜0.05).The expression levels of miRNA-29b-3p and miRNA-34c-3p in A549 cells treated with SiO2 and human recombinant TGF-β1 were significantly lower than those in the control group at 24 h and 48 h,and the difference was statistically significant (P＜0.05).Conclusion Down-regulation of miRNA-29b-3p and miR-34c-3p in rat lung tissue A549 cells may be associated with the development of early silicosis and is expected to be an indicator of early silicosis diagnosis and orognosis.