| Idiopathic pulmonary fibrosis(IPF)is a kind of pulmonary fibrosis diseases without an accurate reason,which could result in a high mortality rate.Currently,there is no effective drug treatment and patients of clinical diagnosis almost have a median survival period less than three years.Research has shown that lung mesenchymal stem cells plays an important role in the development of the occurrence of IPF.Lung mesenchymal stem cells influenced by the microenvironment will transform into the fibroblast,producing a large amount of extracellular matrix,such as collagen,fibrin,and affect the occurrence of IPF.Literature has suggested that microRNAs in differentiation of stem cells plays a very important role in the process,and the expression of microRNAs in lung tissue of IPF patients has spectral anomalies,revealing the microRNAs play an important role in pulmonary fibrosis development.So this topic will be discussed about the occurrence and development of pulmonary fibrosis molecular mechanism,from the angle of microRNAs and try to make the key miRNA targets to intervene the occurrence of IPF.In addition,the early stage of the laboratory has confirmed the Wnt signaling pathways affected lung mesenchymal stem cells differentiation direction,so in this study,we will also explore the regulation of Wnt signaling pathways of intervention of IPF in IPF animal model.Part1:Establishment of the model of idiopathic pulmonary fibrosis in mice.Objective:To confirm that idiopathic pulmonary fibrosis in mice model was established and confirm the success of the model through the detection of lung function,lung tissue structure and the lungs into fiber related protein expression.Methods:1.C57BL/6 mice were narcotized by abdominal cavity and bleomycin was atomized through weasand to establish animal model of IPF.2.At the 7th,14th,21st,28th day after the establishment of the IPF model,arterial blood oxygen partial pressure was measured.Then broke the necks of mice to death.The damage of lung tissue was observed by mason dyeing and determination of hydroxyproline content was detected for judging the collagen deposition in lung tissue.Testing fibrosis related protein expression by western blotting to confirm the success of the IPF model.Results:1.Bleomycin distributed in the lung tissue of mice uniformly and the mice lived in good condition after waking up.2.Blood gas analysis showed that lung function of model group mice declined;Pathological observation of the tissue structure of lung in model mice is damaged,gradually thickening of alveolar walls and eventually developing into the pulmonary fibrosis.Collagen content in the lung tissue increased gradually and fibrosis related protein expression elevated.Conclusion:The mice model of idiopathic pulmonary fibrosis established by atomizating bleomycin through airway could be observed that alveolar structure disordered,the pulmonary interstitial collagen accumulated,the arterial blood oxygen partial pressure drop,epithelial markers in the lung tissue decreased and the fiber marked protein increased.All the above changes are similar to the process of human idiopathic pulmonary fibrosis.Compared with conventional airway drip into the airway,the airway atomization dosing mode is good with focal uniform,high success rate of modeling,more suitable for the basic research of idiopathic pulmonary fibrosis.Part2:The research of the molecular mechanisms in the development of IPF(I)Research of the changes of miRNA expression in the process of IPF Objective:To confirm pulmonary interstitial stem cells can differentiate into fibroblasts,explore the change of the expression of microRNAs in the process of the differentiation by the spectrum and screen of microRNAs associated with fibrosis.Methods:1.The lung mesenchymal stem cells were isolated by the method of magnetic bead separation extraction in mice and the cells were identified by flow cytometry.2.The lung mesenchymal stem cells were induced into fibroblast by TGF-beta and the results were confirmed by western blotting and immunofluorescence.3.Using the low density of microRNAs chip to find the change of microRNAs during the process of lung mesenchymal stem cells differentiation to fibroblast.Using bioinformatics software,such as miWalk,to predict differences of miRNA change target genes and filter out the miRNAs associated with fibrosis.4.Using the technology of Q-PCR to verify the reliability of the chip.Results:1.Observed by light microscopy,pulmonary interstitial stem cells present a long spindle,spindle shape and spiral configuration.Via flow cytometry appraisal,the cells express and CD29,SC A-1 and did not express CD31,CD34 and CD45.2.Induced by TGF-beta induced for 7 days in a row,through western blotting and immunofluorescence test,we found that lung mesenchymal stem cells increasingly express the markers of fiber cells,such as alpha-SMA,Collagen I and fibronectin significantly.3.miRNA chip testing found that during the process of mesenchymal stem cells in the lungs differentiated to fibroblast,there are 299 of microRNAs significant changes have taken place in higher(or lower ratio greater than 2).In 252 of these significant changes of microRNAs rise significantly,47 lowered significantly.The most obviously change of miRNA higher multiples largest is miR-877-3p.Through a database to predict,we found Smad7 for miR-877-3p target genes.4.The results of the Q-PCR consisted with the results of the chip.Conclusion:1.Successfully isolating and training primary lung mesenchymal stem cells,and prompting it to fibroblast differentiation through the TGF-beta.2.There are 299 kinds of miRNA expressing changeling significantly in the differentiation of lung mesenchymal stem cells to fibroblast,of which 252 kinds of microRNAs rising significantly,47 microRNAs lowered significantly.Database to predict found that higher expression of the most significant miR-877-3p,target genes for Smad7,might have close relationship with the occurrence of pulmonary fibrosis.(Ⅱ)The research of the role of miR-877-3p played in the process of lung mesenchymal stem cells differentiation to fibroblastsObjective:Find out the role of miR-877-3p played in the process of lung mesenchymal stem cells differentiation to fibroblasts.Methods:1.Building up the luciferase reporter gene recombinant plasmid contains the Smad7 and miR-877-3p complementary pairing base sequence.Using liposome transfection exogenous miR-877-3p or microRNAs negative control and recombinant plasmid or empty plasmid into 293 t cells to find out the relations between the miR-877-3p and Smad7.2.Building slow virus(LV-miR-877-3p,LV-miR-877-3p inhibitor)containing miR-877-3p or its antisense sequence can be transferred to the conventional slow virus transfection methods in pulmonary interstitial stem cells.Adopt the method of Q-PCR detection of mesenchymal stem cells in the lungs of miR-877-3p expression level.Using western blotting experimental technology to detect Smad7 protein expression level,validating the relations between miR-877-3p and Smad7 and the regulation effect of Smad7.3.Adopt the method of Q-PCR detection in the lungs to fibroblast differentiation of mesenchymal stem cells in miR-877-3p and Smad7 mRNA expression level.Using western blotting experimental technology to detect Smad7 protein expression level,confirm that relationship between miR-877-3p and expression of Smad7.4.During the differentiation of lung mesenchymal stem cells to fibroblast in the lungs,respectively,transfecting the LV-control,LV-miR-877-3p or the TGF-beta cultivation for 7 days to detect lung mesenchymal stem cells express fibroblasts tag(alpha SMA,Collagen and fibronectin).5.During the differentiation of lung mesenchymal stem cells to fibroblast in the lungs,respectively,transfecting miR-877-3p inhibitor to detect the expression of SMA,Collagen I and the expression of fibronectin,and downstream protein to represent the target genes of miR-877-3p,Smad7 by western blotting and immunofluorescencealpha.6.During the process of the differentiation of lung mesenchymal stem cells to fibroblast,transfecting the LV-Smad7 to observe expression Smad7 affect the differentiation process.7.At the 7th and 14th day after the establishment of pulmonary fibrosis model,through the Q-PCR and immunohistochemical techniques,detecting miR-877-3p and changes the expression of Smad7 in the lung tissue of mice.Results:1.Compared with transfection negative controls,the plasmid group and transfection miR-877-3p after plasmid can significantly reduce the loading of wild type Smad7 sequence of fluorescein enzyme activity,and the negative control luciferin enzyme plasmid and load mutant Smad7 sequence of fluorescein had no effect on enzyme activity.2.Q-PCR found,after transfection LV-miR-877-3p,the expression of miR-877-3p can significantly increase and after the transfection miR-877-3p inhibitor,the expression of miR-877-3p can obviously reduce.By western blotting test,we also found that overexpression of miR-877-3p can significantly inhibit the expression of Smad7,and cut the expression of miR-877-3p can make the expression of Smad7 increased.3.During the differentiation of lung mesenchymal stem cells to fibroblast,there is no significant change of Smad7 in RNA level,but a significant decrease in protein levels.4.Lung mesenchymal stem cells of overexpression of miR-877-3p can promote the expression of alpha SMA,Collagen I and fibronectin,and consistent with the TGF-beta induced effect.5.Western blotting and immunofluorescence test showed that,compared with single TGF-beta induced group,the expression of alpha SMA,Collagen I and fibronectin lung mesenchymal stem cells transfected by miR-877-3p inhibitor decreased significantly.As miR-877-3p target genes,Smad7,compared with the TGF-beta induced group,the expression of Smad7 in TGF-beta+miR-877-3p group inhibitor increased significantly and its downstream protein phosphorylation of Smad2 and Smad3 level decreased obviously.6.Western blotting test found that LV-Smad7 can effectively reverse the protein caused by TGF-beta Smad7 and suppress the expression of alpha SMA fiber related protein,Collagen I,fibronectin and FSP-1.7.Compared with the control group,the miR-877-3p in lung tissue of pulmonary fibrosis increased consistent with the degree of the fibrosis and there is no significant change of Smad7 in RNA level,but a significant decrease in protein levels.Conclusion:1.The Smad7 is the target genes of miR-877-3p,and express the miR-877-3p can inhibit the expression of Smad7.2.miR-877-3p does not affect Smad7 in transcription level,but in the translation level inhibits the expression of Smad7,in accordance with the mechanism of action of microRNAs.3.Overexpression of miR-877-3p can induce differentiation to fibroblast of lung mesenchymal stem cells.Inhibition of miR-877-3p can significantly increase the Smad7 protein expression,decrease differentiation of lung mesenchymal stem cells to fibroblast.miR-877-3p may play an important role in development of pulmonary fibrosis.Part3:Exploring the function of miR-877-3p on the occurrence and development of IPF pulmonary fibrosis in mice.Objective:Exploring the function of miR-877-3p on the occurrence and development of IPF pulmonary fibrosis in mice and making a new point for the treatment of IPF.Methods:1.Observing the virus GFP protein expression by fluorescence microscopy after giving the slow virus to the mice.2.The experimental mice were divided into three groups:negative control slow virus control group(LV-NC),negative lentivirus+bleomycin group(LV-NC+BLM),miR-877-3p+bleomycin group inhibitor slow virus(LV-miR-877-3p inhibitor+BLM)and the expression of miR-877-3p in each group was tested by Q-PCR.3.Western blotting,immunofluorescence and immunohistochemical were used to detect the expression of Smad7 and its related protein in downstream in each group.Results:1.Control group had little green fluorescence and transfection virus group had bright green fluorescence.2.Compared with the control,the expression of miR-877-3p in LV-NC+BLM group was increased obviously,and LV-miR-877-3p inhibitor+BLM group did not change significantly.3.Downregulated expression of miR-877-3p in the lung tissue can effectively improve the blood oxygen partial pressure of pulmonary fibrosis mice;the fiber hyperplasia area was significantly reduced,alveolar structure tend to be normal,collagen deposition decreased significantly,as well as the fibrosis related protein.Conclusion:Downregulated the expression of miR-877-3p can inhibit the development of pulmonary fibrosis.Part4:To explore the intervention effect of NSC668036,one of the Wnt/β-catenin signaling pathway inhibitor,on occurence and development of IPF in miceObjective:To confirm NSC668036,an inhibitor of Wnt/β-catenin signaling pathway,can inhibit the development of pulmonary fibrosis in mice,as well as restrain the activation of the fibroblasts.Methods:1.The experimental mice were divided into three groups:control group,the group bleomycin(BLM),NSC668036+bleomycin group(NSC668036+BLM).The Wnt signaling pathways related protein expression in mice lung tissue was tested by western blotting.2.The repair of damaged lung structure was evaluated using HE staining;The collagen deposition within the lung tissue was detected by Mason dyeing and hydroxyproline content determination;The expression of fibrosis related protein and β-catenin was tested by western blotting and immunofluorescence.3.NIH-3T3 cells were treated with different concentrations of NSC668036.The cell proliferation was measured by CCK 8 test;The cell migration ability was tested by the scratches experiment;The levels of cell activation were measured by Q-PCR,western blotting and immunofluorescence.Results:1.Compared with control,the expression of β-catenin and p-GS-3βwas increased in pulmonary fibrosis.2.After inhibition of Wnt signaling pathways in the lung tissue,hyperplasia of fibre area decreased significantly,the alveolar structure tends to be normal,collagen deposition was decreased significantly,the expression of fibrosis related protein in lung tissue were significantly suppressed.3.NSC668036 can inhibit the proliferation of NIH-3T3 cells when the concentration was more than 10 μM.Compared with control group,the TGF-β and Wnt3α can significantly promote cell migration,NSC668036 can inhibit cell migration.The expression of S100A4,MMP-7,β-catenin,α-SMA and Collagen I rised significantly under the action of TGF-β or Wnt3α,while NSC668036 can significantly inhibit the expression of these proteins.Conclusion:1.Wnt signaling pathway was activated in the IPF lung tissue.2.NSC668036 can effectively inhibit the occurrence of pulmonary fibrosis.3.NSC668036 can effectively restrain the proliferation,migration and activation levels of NIH-3T3 fibroblasts,Innovative outcome obtained in this study1.We discovered the miRNA expression change in the fibroblast differentiation of lung resident mesenchymal stem cells.2.This is the First study showed that miR-877-3p was increased significantly during the fibroblast differentiation of lung resident mesenchymal stem cells.In addition,through in vitro and in vivo study,we proved that miR-877-3p can inhibite the expression of its target genes Smad7,lead to the downstream Smad2 and Smad3 protein phosphorylation,promoted the fibroblast differentiation of lung resident mesenchymal stem cells,then accelerate the development of IPF,which can provide a new idea for the clinical diagnosis and development of IPF clinical drugs.3.We proved that NSC668036,an inhibitor of Wnt/β-catenin signaling pathway,can inhibit the development of pulmonary fibrosis in mice,as well as restrain the activation of the fibroblasts.This can provide the experimental basis and theoretical basis for clinical treatment of IPF. |
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