【作者】 李萍；

【导师】 倪春辉；

【作者基本信息】 南京医科大学 ， 公共卫生（专业学位）， 2020， 硕士

【摘要】 矽肺病是由于长期暴露于二氧化硅粉尘作业环境引起的,以肺纤维化为主的肺部疾病。矽肺病是尘肺病中的主要类型之一,具有肺泡上皮细胞受损,肺成纤维细胞异常增殖和活化,细胞外基质大量沉积以及肺部广泛纤维化的特征。在停止接触二氧化硅粉尘后,纤维化进程仍在继续,严重影响了粉尘接触工人的健康并造成了巨大的社会负担。现有的与矽肺病相关的研究仍未能提供有效的治疗方法来阻断或逆转肺纤维化,因此进一步深入研究矽肺病的发病机制具有重大的意义。肺成纤维细胞是纤维化疾病中的重要效应细胞之一。二氧化硅粉尘进入肺组织后可激活巨噬细胞,损伤肺泡上皮细胞,导致两者分泌释放大量炎性因子和促纤维化因子,其中TGF-β1是一种强效的促纤维化因子,能够诱导肺成纤维细胞的异常增殖并转化为肺肌成纤维细胞,获得更强的合成细胞外基质的能力。大量的细胞外基质无法得到有效的降解,在肺泡间隙中过度沉积,逐渐形成瘢痕和结节性病变,从而导致肺组织结构异常重塑。Micro RNAs(miRNAs)是一类小的,非编码RNA,其长度不超过25个核苷酸,主要通过与靶基因mRNA的3’-UTR区结合,在转录后水平调控靶基因的表达。现有的研究发现多种疾病中存在异常表达的miRNAs,与人类疾病的发生发展关系密切。肺纤维化进程中也有多种失调的miRNAs。在本课题组前期的矽尘诱导的小鼠纤维化mi RNA芯片结果中,miR-122的表达在肺组织中是降低的。后期我们进一步在小鼠矽肺模型、TGF-β1诱导的肺成纤维细胞活化模型中进行验证,同样得到了miR-122的表达水平是降低的结果。因此我们作出假设:miR-122在矽尘诱导的肺纤维化中可能发挥重要的作用。目的将整体动物水平表型观察和细胞水平机制研究相结合,通过构建矽尘诱导的小鼠肺纤维化模型、TGF-β1诱导的肺成纤维细胞活化模型以及在小鼠体内高表达miR-122的干预模型,来探究miR-122参与调控矽肺进程的分子机制,为矽肺病的防治策略提供新的依据。方法(1)动物水平:构建矽尘诱导的小鼠肺纤维化模型和miR-122 agomir干预的肺纤维化模型,肺组织的病理改变通过HE染色进行观察,并进行纤维化评分,小鼠肺组织中miR-122和纤维化标志物的表达情况分别使用qRT-PCR、western blot来检测,羟脯氨酸含量测定检测肺组织纤维化程度,验证体内升高miR-122表达对矽肺的干预作用。(2)细胞水平:使用TGF-β1处理肺成纤维细胞(MRC-5和NIH/3T3细胞),构建肺成纤维细胞活化模型,miR-122和纤维化标志物的表达情况分别通过qRT-PCR、western blot进行检测;在生物信息学网站上预测ADAM10与miR-122之间的结合位点,随后使用双荧光素酶报告基因实验验证两者的结合;在肺成纤维细胞中分别转染miR-122 mimic,ADAM10 si RNA,qRT-PCR检测两者的表达,下游分子及纤维化指标的表达通过western blot和免疫荧光法进行检测,Ed U细胞增殖法检测肺成纤维细胞的增殖况;在TGF-β1处理的肺成纤维细胞中共同转染miR-122 mimic和ADAM10 plasmid进行功能回复实验,证明miR-122通过ADAM10调控肺成纤维细胞的活化;使用Ephrin-B2si RNA和ADAM10过表质粒,Ephrin-B2胞外域重组蛋白、EPHB3/4 si RNA处理肺成纤维细胞,进一步对ADAM10的下游分子机制进行验证。结果1.miR-122在矽尘诱导的小鼠肺纤维化模型中表达降低小鼠矽肺模型中肺组织病理切片的HE染色结果显示,随染尘时间延长,小鼠肺组织的纤维化严重程度逐渐加重。染尘28d,在小鼠肺组织中能够观察到矽结节的形成;纤维化标志物Fibronectin、Collagen I、α-SMA、Vimentin的蛋白表达水平升高;而肺组织中miR-122的表达水平逐渐降低。2.miR-122减轻了矽尘诱导的肺纤维化升高小鼠体内miR-122的表达后,观察肺纤维化的严重程度。小鼠肺组织病理切片HE染色结果以及纤维化评分表明,矽尘+miR-122 agomir组的小鼠肺组织纤维化严重程度比矽尘+agomir-NC组的更轻,肺纤维化的分布范围也更小;western blot和羟脯氨酸含量检测结果也提示干预组的纤维化标志物表达明显降低,纤维化程度减轻。这些结果说明高表达miR-122能减轻矽尘诱导的肺纤维化。3.miR-122抑制TGF-β1诱导的肺成纤维细胞增殖与分化使用TGF-β1处理肺成纤维细胞后,纤维化标志物Fibronectin、Collagen I、α-SMA、Vimentin的蛋白表达水平升高,miR-122的表达水平降低。在活化的肺成纤维细胞中转染miR-122 mimic后,纤维化标志物表达水平和肺成纤维细胞的增殖能力受到明显抑制,表明miR-122能够抑制TGF-β1诱导的肺成纤维细胞增殖与分化。4.ADAM10是miR-122的靶基因通过在生物信息学网站(Targetscan)的预测和文献检索发现ADAM10(A disintegrin and metallopeptidase domain 10)是miR-122的靶基因,我们使用双荧光素酶报告基因实验在人胚肺成纤维细胞(MRC-5)和小鼠胚胎成纤维细胞(NIH/3T3)中证实了两者的结合。在小鼠矽肺组织以及TGF-β1处理的肺成纤维细胞中,ADAM10的表达水平均升高,而在体内和体外升高miR-122的表达后,抑制了ADAM10的表达,提示miR-122与ADAM10之间存在调控关系。5.miR-122通过调控ADAM10影响肺成纤维细胞增殖与分化在证实miR-122与ADAM10结合的基础上,我们进一步探究miR-122是否通过调控ADAM10影响肺成纤维细胞活化。首先,我们在肺成纤维细胞活化模型中单独转染ADAM10 si RNA,发现纤维化标志物蛋白表达水平和细胞增殖水平均受到抑制。接着我们在肺成纤维细胞中联合转染miR-122 mimic和ADAM10 plasmid进行回复实验,发现过表达ADAM10后能够回复miR-122对纤维化标志物的抑制作用,说明miR-122通过调控其靶基因ADAM10影响肺成纤维细胞的增殖与分化。6.ADAM10通过Ephrin-B2/EPHB3/EPHB4/STAT3信号轴影响肺成纤维细胞的活化文献报道ADAM10能够剪切肺成纤维细胞表面跨膜蛋白Ephrin-B2的胞外域,产生可溶性的s Ephrin-B2,并通过其受体EPHB3/4促进肺成纤维细胞的活化。另外EPHB3/4均能促进STAT3发生磷酸化。因此我们进一步探究了ADAM10是否通过对Ephrin-B2的调控来影响STAT3的磷酸化。首先,我们使用Ephrin-B2胞外域重组蛋白(Ephrin-B2-Fc)处理肺成纤维细胞,观察到纤维化标志物和STAT3磷酸化水平的升高,而加入EPHB3/4 si RNA则抑制了这一变化。接着我们在肺成纤维细胞中共同转染ADAM10过表达质粒和Ephrin-B2 si RNA,结果显示敲低Ephrin-B2的表达后明显抑制了过表达ADAM10诱导的p-STAT3和纤维化标志物的蛋白表达水平。进一步通过功能回复实验发现,Ephrin-B2-Fc能够逆转ADAM10 si RNA对p-STAT3和纤维化标志物的蛋白表达的抑制作用。最后在肺成纤维细胞中联合转染miR-122 mimic和ADAM10过表达质粒后,我们观察到ADAM10能够回复miR-122对p-STAT3的抑制作用。这些结果表明miR-122调控ADAM10抑制肺成纤维细胞活化可能是通过Ephrin-B2/EPHB3/EPHB4/STAT3途径实现的。结论本研究通过体内和体外实验证明,在矽尘诱导的肺纤维化中,miR-122通过靶向ADAM10调控Ephrin-B2,影响EPHB3/EPHB4/STAT3通路,进而参与调控肺成纤维细胞活化,发挥抗纤维化的作用。更多还原

【Abstract】 Silicosis,a kind of pneumoconiosis,is a worldwide occupational lung disease caused by long-term inhalation of silica dusts.It is characterized by damaged alveolar epithelial cells,abnormal proliferation of pulmonary fibroblasts,massive deposition of extracellular matrix and eventually extensive fibrosis in the lungs.Even the exposure of silica dust ceases,the process of fibrosis will still continue which seriously affects the health of dust exposed workers causing a huge social burden.The existing studies on silicosis could not provide effective treatment methods to block or reverse pulmonary fibrosis,so it is of great significance to further study the pathogenesis of silicosis.Fibroblasts are key effector cells in fibrotic disease.Silica activates macrophages and damages alveolar epithelial cells after entering lung tissue,resulting in the secretion and release of a large number of inflammatory factors and profibrotic factor.Transforming growth factor-β1(TGF-β1)is a critical profibrotic factor,which can induce activation of pulmonary fibroblasts.Upon activation,resting fibroblasts obtain a myofifibroblast phenotype,which is characterized by expression of contractile proteins and enhanced release of extracellular matrix.Excessive deposition of a large amount of extracellular matrix in the alveolar space gradually leads to the formation of scarring and nodular lesions,resulting in abnormal remodeling of the lung tissue structure.Micro RNAs(miRNAs)are a class of small,non-coding RNAs with within 25 nucleotides in length.They could bind to the 3’-UTR region of target m RNA through base pairing,regulate gene expression post-transcriptionally.Growing evidences have found multiple dysregulated miRNAs in a variety of diseases and play an important role in the occurrence and development of human diseases,including pulmonary fibrosis.In our previous miRNA microarray analysis study,the expression of miR-122 was decreased in fibrotic murine lung tissues induced by silica.This observation was further demonstrated by the finding that miR-122 was down-regulated in two fibroblast lines treated with TGF-β1 in vitro and silica-induced pulmonary fibrosis mouse model in vivo.Therefore,we hypothesize that miR-122 may play an important regulatory role in TGF-β1-medicated fibroblast activation and silica-induced pulmonary fibrosis.Objectivewe constructed mice silicosis model,TGF-β1-induced pulmonary fibroblast activation model and an intervention mice model with high expression of miR-122,to investigated the molecular mechanisms of miR-122 in the regulation of silicosis process,aimed to provide a new basis for prevention and treatment prevention and treatment of silicosis.Methods(1)Animal level: the model of silica-induced pulmonary fibrosis in mice and the model of miR-122 agomir intervention in pulmonary fibrosis were constructed,HE staining was used to observe the pathological changes of lung tissue and to score the severity of fibrosis.the expression of miR-122 in mouse lung tissue was verified by q RT-PCR and the expression of fibrosis markers was detected by western blot,hydroxyproline content was used to detect the degree of pulmonary fibrosis,to verify the intervention effect on silicosis of miR-122 in vivo.(2)Cell level: fibroblast activation models were constructed by treating lung fibroblasts(MRC-5 and NIH/3T3cells)with TGF-β1,the expression level of miR-122 was detected by q RT-PCR,fibrosis markers were detected by western blot;bioinformatics software was used to predict that ADAM10 was a potential target gene of miR-122,and the combination of them was verified by double luciferase reporter assay;lung fibroblasts were transfected with miR-122 mimic,ADAM10 si RNA,respectively.Then the levels of downstream molecules and fibrosis markers were detected by western blot and immunofluorescence.Besides,the Ed U cell proliferation assay kit was used to detect the proliferation of lung fibroblasts;miR-122 mimic and ADAM10 plasmid were co-transfected into TGF-β1 treated MRC-5 cells,to demonstrate that miR-122 regulates the activation of lung fibroblasts by ADAM10;the downstream molecular mechanism of ADAM10 was further validated by Ephrin-B2 extracellular recombinant protein treating the MRC-5 cells with ADAM10 si RNA or plasmids.Results1.miR-122 was decreased in silica-induced mouse pulmonary fibrosis modelHE staining results of pathological sections showed that the severity of pulmonary fibrosis increased gradually with the prolonged exposure time.The formation of silica nodules was observed in the lung tissue of the dust-stained 28 d mice.The protein expression levels of fibrosis markers including Fibronectin 、Collagen I、α-SMA、Vimentin increased gradually,while miR-122 expression levels in lung tissue decreased.2.miR-122 alleviates silica-induced pulmonary fibrosisThe intervention effect of miR-122 on pulmonary fibrosis was observed after increasing the expression of miR-122 in mice by injecting miR-122 agomir through tail vein.HE staining and fibrosis score of mice showed that the severity and distribution of pulmonary fibrosis in the intervention group were significantly reduced compared with that in the miR-NC agomir group;Western blot and hydroxyproline content test results also indicated that the expression of fibrosis markers in the intervention group decreased significantly.Above results indicate that elevated miR-122 expression can alleviate the pulmonary fibrosis induced by silica dust.3.miR-122 inhibits TGF-β1-induced activation of fibroblastsThe expression level of miR-122 decreased and the protein expression level of fibrosis markers(Fibronectin、Collagen I、α-SMA、Vimentin)increased in TGF-β1treated lung fibroblasts,which was consistent with results of mice lung tissue.After the transfection of miR-122 mimic into lung fibroblasts,the expression level of fibrosis markers and the proliferation ability of lung fibroblasts were significantly inhibited.These results showed that miR-122 could inhibit TGF-β1 induced pulmonary fibroblast activation.4.ADAM10 is the target gene of miR-122Through prediction in bioinformatics website(Targetscan),we found ADAM10 might be a potential target gene of miR-122 which was proved in existing studies.We further confirmed the combination in activated fibroblasts induced with TGF-β1 by double luciferase reporter gene experiments.ADAM10 expression levels were elevated in mouse silicosis and TGF-β1-treated pulmonary fibroblasts,while ADAM10 expression was inhibited in vivo and in vitro,further suggesting a regulatory relationship between miR-122 and ADAM10.5.miR-122 affects the activation of lung fibroblasts by regulating ADAM10On the basis of confirmed combination between miR-122 and ADAM10,we further explored whether miR-122 could affect lung fibroblast activation through regulating ADAM10.Firstly,we transfected ADAM10 si RNA into lung fibroblast,the results showed that both fibrotic markers protein expression and cell proliferation were inhibited.Then we co-transfected miR-122 mimic and ADAM10 plasmid into lung fibroblast finding that overexpression of ADAM10 could restore the inhibitory effect of miR-122 on fibrosis markers.6.ADAM10 affects pulmonary fibroblasts activation via Ephrin-B2/EPHB3/EPHB4/STAT3 signaling axisPrevious studies have confirmed that ADAM10 could shear the transmembrane proteins Ephrin-B2 extracellular domain on the surface of lung fibroblasts,producing soluble s Ephrin-B2,and promote the activation of lung fibroblasts through its receptors EPHB3/4.Interestingly,studies also found that both EPHB3 and EPHB4 promoted STAT3 phosphorylation.Therefore,we further explored whether ADAM10 affected the phosphorylation of STAT3 by regulating Ephrin-B2.We observed increased protein levels of fibrosis markers and STAT3 phosphorylation after treatment of MRC-5 cells using Ephrin-B2 extracellular recombinant protein,however,it could be inhibited by EPHB3/4 si RNA.Then we co-transfected ADAM10 plasmid and Ephrin-B2 si RNA into MRC-5 cells,results showed that knockdown of Ephrin-B2 expression significantly inhibited ADAM10 induced elevated protein expression levels of both p-STAT3 and fibrosis markers.In addition,the results of functional recovery experiments showed that Ephrin-B2-Fc could reverse the inhibitory effect of ADAM10 si RNA on protein expression of p-STAT3 and fibrosis markers.Finally,after combined transfection of miR-122 mimic and ADAM10 plasmids into MRC-5 cells,we observed that ADAM10 could revert the inhibition of miR-122 to p-STAT3.These results suggest that miR-122 regulated ADAM10 inhibition of pulmonary fibroblast activation,at least,partly depend on Ephrin-B2/EPHB3/EPHB4/STAT3 pathways.ConclusionIn this study,in vivo and in vitro experiments demonstrated that miR-122 played an anti-fibrotic role by targeting ADAM10 to regulate Ephrin-B2,influencing pulmonary fibroblast activation through EPHB3/EPHB4/STAT3 pathways in silica-induced pulmonary fibrosis.更多还原