The Molecular Mechanism Of Phenotypic Changes Of Vascular Endothelial Cells In Rats During The Pulmonary Fibrosis

Objective:The aim of this project is to clarify the effect on endothelial-mesenchymal transition(EnMT)during the development of pulmonary fibrosis,and study the detail of pulmonary microvascular endothelial cells.Therefore,we are going to discuss the function of signal pathway of EnMT during pulmonary fibrosis,and confirm the correlation between different signal pathway and EnMT while pulmonary fibrosis happened.Then,to explore the newly signal transduction pathway which working on epithelial-mesenchymal transition(EMT).Methods:1.Establish animal model: 60 SD rats were purchased from the Center of Experimental animals in Hebei province,which were separated into two groups randomly,30 per group.The model of pulmonary fibrosis was established by intratracheal instillation of Bleomycin,and normal saline for control instillation.Rats were killed in 7,14,and 28 days,respectively.Histomorphology was observed by hematoxylin and eosin(H & E)stain,and the expression of collogen type I was determined by Masson’s stain.2.In vitro: pulmonary microvascular endothelial cells were separated and cultrued.The expression of E-cadherin was measured by immunohistochemistry.The transcription level of Smad and β-catenin were detected by Q-PCR.3.Transfection: Smad-si RNA,β-catenin-siRNA,and Notch1-siRNA were transfected in primary cells in experimental group.The protein and mRNA level of E-cadherin,α-smooth muscle actin,and collogen type I were examined by Western blot and Q-PCR,respectively.Results:1.H & E stain: Obvious hydroncus and multi-hemorrhagic spots were found in bilateral pulmonary in the experiment group on 7d,and decreased on 14 d.On 28 d,the decresed volume,increased solidity,reduced elasticity,irregular surface,scattered bleeding spots and consolidated focus were found.2.Masson’s stain: lots of blue-green fibrosis tissues were observed in the experimental group.In detail,destruction of alveolar structure,thickening of interlobular septa,collagen deposition,and consolidated focus.3.Q-PCR: mRNA level of Smad and β-catenin in pulmonary microvascular endothelial cells was significantly increased in experimental group than control group after Bleomycin treatment 7 days later(P<0.05),and decreased gradually.However,they all higher than control(P<0.05).4.SncRNA transfection: After transfected with Smad-siRNA,β-catenin-siRNA,and Notch1-siRNA,both the expression of α-SMA and collogen type I were increased,otherwise,E-cadherin was decreased significantly(P<0.05).No significant difference was observed between the transfected cells by different sncRNA(P>0.05).Conclusion:1.Bleomycin could induce morphology changes and dysfunction of rats’ lung.The endothelial-mesenchymal transition(EnMT)might be the key point of the development of pulmonary fibrosis.2.Genes involved both TGF-β / Smad and Wnt / β-catenin pathways,which were up-regulated during bleomycin-induced pulmonary fibrosis,may play a role to promote pulmonary fibrosis.