In Vitro Study And Bioinformatics Analysis Of Sirt6-modified Bone Marrow Mesenchymal Stem Cells In Idiopathic Pulmonary Fibrosis Microenvironment

Background and purpose:Idiopathic pulmonary fibrosis(idiopathic pulmonary fibrosis,IPF)is a common respiratory medicine disease,usually characterized by lung function decline,progressive exertional dyspnea,cough,sputum less,cyanosis,chest pain and clubbing,patients with more severe illness further development may eventually died of respiratory failure,usually after diagnosis of IPF patients survival time were less than 5 years.In recent years,the study points out that the occurrence and development of IPF disease has some characteristics of epidemiology,susceptibility of the family,related to gene mutation and so on,at present most of the studies have shown that the mechanism of the occurrence of IPF and progress is abnormal epithelial cell injury and damage caused by the activation of repair pathways of this abnormal epithelial repair after injury sustained made mesenchymal cells(epithelial cells to change,in the process of the transition of the cell also secrete other growth factors,such as transforming growth factor beta 1-(transforming growth factor beta 1,TGF-?1),platelet-derived cytokines(PDGF)and vascular endothelial Growth factor(VEGF),etc.increase in the number of signaling pathways,resulting in over-activation of signaling pathways,resulting in over-deposition of myofibroblasts in the stroma and accelerating the progression of disease.Other studies have confirmed that the development of IPF disease process with epithelial cell aging phenotype also exists certain relevance,in the alveolar epithelial cells of patients with IPF,age-related factor secretion to increase,and value-added/apoptosis related factor expression decreased,compared with the same age matched control group also showed telomeres get shorter and the phenotype of mitochondrial dysfunction.SIRT6 is a member of the NAD+ dependent enzyme Sirtuin family and plays an important role in chromatin signaling and genome maintenance.SIRT6 prevents age-related diseases,including metabolic diseases and cancer,and extends the lifespan of mice through these functions.Bone marrow Mesenchymal stem cells(BMSCs)is a kind of stem cells with self-renewal and multidirectional differentiation potential derived from the mesoderm.Some studies have shown that BMSCs can be induced to differentiate into alveolar epithelial cells in vitro.This study hypothesized that bone marrow mesenchymal stem cells modified by SIRT6 simulate the epithelial environment of IPF in vitro,to observe whether SIRT6 gene has a role in slowing or improving the development and progression of IPF disease,and to explore its pathogenesis.Materials and methods:Start with IPF patient samples,evaluate the correlation between SIRT6 and IPF,then extract 6-8 weeks of SPF rat bone marrow mesenchymal stem cells,perform flow cytometry and induce adipogenesis to identify BMSCs;Ask the company to construct a lentiviral vector that over-expresses the SIRT6 gene and transfect it into the extracted BMSCs cells to obtain BMSCs-SIRT6 cells.BMSCs and BMSCs-SIRT6 cells were treated with TGF-?1 to simulate fibrosis in vitro;Western blot was used to detect the expression of P16,SIRT6,type ? collagen and ?-SMA protein.RT-PCR was used to detect the expression of P16,SIRT6,fibrin,type I collagen and ?-SMA genes.Results:1.Before the start of the experiment,gene chip screening was used in GEO database to analyze the decreased expression of SIRT6 gene in IPF patients and bleomycin-induced pulmonary fibrosis model.2.Plasmids containing SIRT6 target genes encapsulated by lentiviruses can be successfully infected into BMSCs and their high expression can be detected3.TGF-?1 aging treatment after BMSCs cells expressed and fibrosis indexes P16,collagen ? and alpha SMA rise4.TGF-?1 deal with BMSCs after SIRT6 cell aging and fibrosis indexes P16,collagen ? and alpha SMA BMSCs group5.SIRT6 gene expression may improve the fibrosis induced by TGF-?1.Conclusion:1.The IPF-related genes were analyzed with the information of GEO database,and SIRT6 gene was found to be down-regulated in IPF.2.BMSCs were extracted successfully and SIRT6 gene was introduced into BMSCs successfully.3.The concentration of TGF-?1 in pulmonary fibrosis microenvironment was 5ng/ml,which could down-regulate SIRT6 gene expression.4.After TGF-?1 stimulation,the expressions of fibros-related proteins and genes were up-regulated in the GFP-BMSC group,while the expressions of related proteins and genes in the SIRT6-bmscs group were down-regulated than those in the GFPBMSC group,demonstrating that the up-regulated SIRT6 genes can counter the development and progression of FIBROSIS induced by TGF-?1.5.Through the screening of differential genes and the analysis of protein interaction sub-network modules,it was concluded that the expressions of HSP90AA1,SPP1 TPR,COL1A1,COL3A1,RPS13,MMP1,CP,SMC3,RANBP2,VCAN,CXCL10 and NCL were significantly changed in IPF patients,among which the expressions of RPS13 and CXCL10 were increased in IPF patients,while the expressions of other genes were decreased.