The Impact And Relative Signal Mechanism Of HCMV To Expression Of Fibrosis Media By Human Lung Fibroblast And Epithelial A549 Transdifferentiation

Background:Progressive pulmonary fibrosis is an important cause of death in patients with connective tissue diaeases. A large number of research demonstrated viral infection plays a role in the development of fibrosis diseases. On the one hand,a variety of gene production encoded by HCMV disorder normal human immunity resulted in persistent infection.On the other hand,various genes expressed in order to promote cell proliferation leading to fibrosis. Connective Tissue Dieases associated Interstitial lung Disease(CTD-ILD) is inflammatory fibrosis based on autoimmune disorders.At present,the mechanism of pulmonary fibrosis in patients with autoimmune diseases has not been elaborated.The treatments are limited.Evidences had been found that Epstein-Barr virus(EBV)and human herpesvirus6(HHV-6) in the herpes virus family play an important role in the pathogenesis of autoimmune disorder.Furthermore,research reported that HCMV specific infected CD4+T cell abound in peripheral blood of patients with vascular autoimmune diseases.On the background of that,we hypothesis that HCMV participated the development of CTD-ILD via profibrotic effect.Purpose:Progressive pulmonary fibrosis is one of the main causes of death in patients with connective tissue diseases. This paper observed the impact of HCMV infected PBMCs to expressions of key profibrotic media by human lung fibroblasts,and the role of US28 as a key structure protein to expression of profibrotic media and EMT marker.It aims to explore the role of US28, expressed by HCMV promotes,and the possible mechanisms and finally provide more theoretical basis for future treatment of fibrosis.Methods:Part1 Human peripheral blood mononuclear cells infected with virus strain AD169 co-cultured with fibroblasts,testing the changes of associated cytokines.Quantitative the cultured virus strain AD169 with TCID50,then infected Peripheral blood mononuclear cells in MOI 0.1.1hour later,abandoned the supernatant and washded the cells with PBS twice.Finally,co-cultured the Peripheral blood mononuclear cells and fibroblast.Harvested the fibroblast after 6h,12 h,24h,respectively.Extracted RNA and reversed transcription.Quantitative the expression of IL-10,IFN-γ,TNF-α,IP-10,IL-17 A,IL-6,TGF-βby Fluorescent quantitative PCR. Part2 US28 as one of the key structure protein impacts the cytokines expression and phenotype of A549 via IL-6/STAT3 signal pathway Planted A549 cells in 6 well cell culture cluster.24 hours later,transfected p XLSN-control and p XLSN-us28 into A549 cells with lipofectin transfection,respectively.Another 24 hours later,harvested A549 cells, extracted RNA and reversed transcription.Quantitative the expression of IL-10,Fibronectin,Ecadherin, IL-6,TGF-βby Fluorescent quantitative PCR and test expression of protein Fibronectin,Ecadherin,p-stat3.Results:Part1 Peripheral blood mononuclear cells infected with virus strain AD169 co-cultured with fibroblasts,testing the changes of associated cytokines. Screen several cytokines associated with immunosupression and fibrosis after viral infection.Immunosupressive cytokine IL-10 up-regulated at all three timing,the same as fibrosis related cytokine IL-6. TNF-α,IP-10,IL-17 A up-regulated at 24 h,while IFN-γexpression reduced gradually.Surprisingly,classical fibrosis related cytokine TGF-βwas stablely expressed without obvious changes. Part2 US28 as one of the key structure protein impacts the cytokines expression and phenotype of A549 via IL-6/STAT3 signal pathway Based on the results of Part1,we continued to study the role of protein US28 in the process of pulmonary fibrosis progression.The results demonstrated that IL-10 and IL-6expression significantly increased,instead TGF-βexpression reduced.Meanwhile,the epithelial phenotype Ecadherin down-regulated and mesenchymal phenotype Fibronectin up-regulated.At the protein expression level, the epithelial phenotype Ecadherin and mesenchymal phenotype Fibronectin expression were corresponded with the level of gene expression.Even,protein p-stat3 expression was associated with EMT.Conclusion:1.HCMV infected human lung fibroblast highly expressed profibrotic media,expecially IL-6.2.US28 expressed by HCMV promotes epithelial-mesenchymal transition via IL-6/STAT3 signal pathway...