The Effect Of 3D Acellular Scaffold Of Pulmonary Fibrosis On The Epithelial-mesenchymal Transition And The Possible Mechanism

Idiopathic pulmonary fibrosis is a kind of pulmonary interstitial disease with unknown etiology,lack of effective treatment and high mortality rate.It is now reported that the formation of fibroblast foci with fibroblast / muscle is an important pathological feature in IPF.The myofibroblasts are the major effector cells.The present study found that epithelial-mesenchymal transition(EMT)is one of the main source of myofibroblasts.Therefore,it is of great significance to study the epithelial-mesenchymal transition.Recent studies have found that the accumulation of ECM is not only the final result of the multiple factors in the pathogenesis of pulmonary fibrosis,but also affects the process of pulmonary fibrosis.Preparation of acellular scaffold through enzymatic and chemical detergent twelve sodium dodecyl sulfate(SDS)and Triton-X100 methods to remove the organ parenchymal cells and soluble protein,and insoluble ECM with good ultrastructure was remained.A recent study found that the fibroblasts were transformed to myofibroblasts when co-cultured with 3D acellular scaffold acellular scaffold of pulmonary fibrosis in vitro.but it is unkown that the effects of the 3D acellular scaffold acellular scaffold of pulmonary fibrosis in alveolar type II epithelial cells.In this study,normal and pulmonary fibrosis of the acellular scaffold will be prepared,the scaffold will be co-cultured with type II alveolar epithelial cell.The effects of the acellular scaffold of pulmonary fibrosis on phenotype of type II alveolar epithelial cell and the possible mechanisms will be studied.Part one The preparation of lung acellular scaffoldObjective: To prepare an ideal acellular scaffold through pulmonary artery perfusionMethods: The method of Triton X-100 and SDS through pulmonary artery perfusion was used to prepare the acellular scaffold.General view,HE,Masson staining,electron microscopy,lung cast was detectd for the assessment of the formation.The DNA content was detected.The acellular scaffold components(collange I and fibronectin)were detectedby immunofluorescence staining.biomechanics of scaffold was detected.Cell culture and cell activity was observed.Results: 1.The general view shows that the whole lung into milky white translucent after lung perfusion finished;2.The neclei components were rare and extracellular matrix structure was complete and continuous showed by HE staining,Masson staining,electron microscope and lung cast.3.DNA of acellular scaffold was extremely low <50ng/ml.4.Immunofluorescence staining showed that collange I and fibronectin were remained.5.Lung acellular scaffold was co-cultured with A549,the cell activity was well.Conclusion: The morphology,composition and mechanics were well maintained and the cell removal was good in the acellular scaffold we acquired.Part two Preparation of pulmonary fibrosis model and the effect of 3D pulmonary fibrosis acellular scaffold on the epithelial-mesenchymal transitionObjective: To study the effect of 3D pulmonary fibrosis acellular scaffold on epithelial-mesenchymal transition.Methods: Pulmonary fibrosis in rats induced by bleomycin.HE staining and Masson staining were used to determine the fibrosis.Normal and fibrotic acellular scaffold were prepared.Primary culture of type II alveolar epithelial cell was made and co-culture with acellular scaffold.It was divided into 5 groups: non scaffold group,2D normal scaffold group,2D pulmonary fibrosis acellular scaffold group,3D normal scaffold group,3D pulmonary fibrosis acellular scaffold group.The protein and m RNA expression of E-cadherin,?-SMA of type II alveolar epithelial cell were detected by immunofluorescence staining,westen bloting and real-time PCR.Result: 1.The rat model of pulmonary fibrosis induced by bleomycin was successfully prepared.2.Immunofluorescence staining and real-time PCR showed that the protein and gene expression of E-cadherin in the 3D group were down-regulated,and the expression of ?-SMA protein and gene expression were up-regulated in the 3D group compared with that of the normal group and 2D group.Conclusion: 1.2D normal and fibrotic lung acellular scaffold had no significant EMT effect on alveolar type II epithelial cells.2.3D normal and fibrotic lung acellular scaffold could induce EMT in type II alveolar epithelial cells,particularly in 3D fibrotic lung acellular scaffold 3.The changes of ECM and its three-dimensional microenvironment in pulmonary fibrosis may promote EMT in type II alveolar epithelial cell,which probably further aggravates the pulmonary fibrosis.Part three The role of integrin beta 1 in EMT promoted by 3D fibrotic lung acellular scaffoldObjective: To investigate the effects of integrin beta 1 on EMT promoted by 3D fibrotic lung acellular scaffold.Methods: The protein and gene expression of integrin beta 1 in normal and fibrotic lung acellular scaffold were detected by western and real-time PCR.It was divided into 3 groups: 1.the normal control group.2.3D pulmonary fibrosis acellular scaffold group.3.the integrin beta 1 blocking group.The protein and gene expression of E-cadherin and ?-SMA were detected by immunofluorescence,western bloting and real-time PCR.Result: 1.Compared with 3D group,the expression of E-cadherin protein and gene were higher in the integrin beta 1 blocking group.2.The protein and gene expression of ?-SMA was decreased in the integrin beta 1 blocking group compared with the 3D group.Conclusion: integrin beta 1 may mediate EMT induced by 3D pulmonary fibrosis acellular scaffold.