The Study On A Disintegrin And Metalloproteinase33(ADAM33) In Asthmatic Airway Remodeling Process

Chapter1 Peripheral expression of ADAM33 in patients with different severity of asthma and the relation to airway chronic inflammationObjective To evaluate the peripheral expression of ADAM33 in patients with different severity of asthma and the relation to airway chronic inflammation.Methods Patients diganosed into mild-to-moderate asthma,svevre asthma and healthy control groups.Eosinophil(EOS)was counted in peripheral blood,the serum total IgE was detected by immunization,and the serum ADAM33,IL-4 and IL-13 1evel was detected by ELISA.The relation of ADAM33 and chronic inflammational markerts was assessed by Pearson analysis.Results The serum level of ADAM33 in mild-to-moderate asthma group?svevre asthma group and healthy control groups were 23.8±6.21pg/mL,64.8±12.8pg/m L and18.3±4.49pg/mL.The ADAM33 lever in mild-to-moderate asthma group and svevre asthma group were higer than in controls,and the svevre asthma was higer than mild-to-moderate asthma(P <0.05).The peripheral expression of ADAM33 was negatively correlated with FEV1%,and positively correlated with IL-4,IL-13 and TGF-?1,but had no correlation with EOS and IgE.Conclusions The peripheral expression of ADAM33 was up-regulated in ashtmatic patients along with the severity of asthma.The peripheral expression of ADAM33 was negatively correlated with FEV1%,and positively correlated with IL-4,IL-13 and TGF-?1,but had no correlation with EOS and IgE.Chapter2 The effect of house dust mite extracts on expresson of ADAM33 in 16-HBE cells and asthmatic airway epithelial-mesenchymal transitionObjective Epithelial-mesenchymal transition(EMT)exists in the prophase of asthmatic airway remodeling,but its mechanism remains unkown.This study was to investigate the effects of house dust mite extracts on asthmatic epithelial-mesenchymal transition with human bronchial epithelial cells(16-HBE).Methods 16-HBE cells divided into HDM,TGF-?1,HDM with TGF-?1 and control groups.After treatment for 72 h we investigated the morphology and proliferation capacity of 16-HBE cells with light microscope and CCK-8 method,and expression of E-cadherin,Vimentin and ?-SMA by western blot.Results HDM alone couldn't induced 16-HBE cells proliferating and changing to fibroblast-like morphology,nor the change of vimentin and ?-SMA expression,but could reduce cell-cell contact by down-regulating E-cadherin.HDM along with TGF-?1 could significantly induce 16-HBE cells proliferating and changing to fibroblast-like morphology,with down-regulation of E-cadherin and up-regulation of Vimentin and ?-SMA.Conclusions HDM alone could reduce cell-cell contact by down-regulating E-cadherin,while couldn't induce significant EMT process.But it could significantly enhance TGF-?1-induced EMT process.Chapter3 The expression of ADAM33 regulated by TGF-?1 and the relation between ADAM33 expression with the TGF-?1-induced epithelial-mesenchymal transition in 16-HBE cells.objective To observate the expression of a disintegrin and metalloproteinase33(ADAM33)regulated by transfer growth factor?-1 and the relation between the expression of ADAM33 with the TGF-?1-induced epithelial-mesenchymal transition in human bronchial epithelial cells(16-HBE cells),to explore the underlying mechanism of ADAM33 taking part in asthmatic airway remodeling process.Methords The 16-HBE cells were treated with TGF-?1(10 ? 20 and 30ng/mL respectively)for 72 h.Then we observated the morpho-change of cells and measured the expression of proteins and m RNA of ADAM33 and E-cadherin and Vimentin by western blot and RT-PCR.Results The 16-HBE cells were change from the adherence elliptic to spindle fibroblasts after TGF-?1 treatment,and the most changes in 20ng/m L.The expression of mRNA and proteins of ADAM33,Vimentin and ?-SMA were up-regulated,and mRNA and protein of E-cadherin were down-regulated.The expression of ADAM33 protein was positively correlated with Vimentin and negatively correlated with E-cadherin.Conclusions TGF-?1 could up-regulated expression of ADAM33 in 16-HBE cells to enhance the epithelial-mesenchymal transition in asthmatic airway remodeling.Chapter4 The effect of ADAM33 overpressed in human bronchial epithelial cells to TGF-?1-induced epithelial-mesenchymal transitionObjective Observing the effect of overpressed ADAM33 to TGF-?1-induced epithelial-mesenchymal transition by building recombinant adenovirus Ad-ADAM33 totransfect 16-HBE cells,in order to discover the possible mechanism of ADAM33 acted in asthmatic airway remodeling.Methods Building recombinant adenovirus Ad-ADAM33 and choosing the best MOI to transfect the 16-HBE cells.Cells divided into blank control group,Ad-EGFP group and Ad-ADAM33-EGFP group by their treatment.Then all groups were treated by TGF-?1(ng/mL)for 24 ? 48 and 72 h to measure the expression mRAN and Protein of E-cadherin and Vimentin by western blot and RT-PCR.Results The cell model overpressing ADAM33 was contructed successfully by transfecting recombinant adenovirus Ad-ADAM33 into 16-HBE cells in vitro with the best MOI(1500),which was supported by the ADAM33 protein being extremely upregulated in transfected 16-HBE cells.After treating by TGF-?1 for 24? 48 and 72 h,expression of Vimentin mRNA in Ad-ADAM33-EGFP group were significantly higer to blank control and Ad-EGFP groups(p < 0.05),and expression of E-cadherin mRNA in Ad-ADAM33-EGFP group were significantly lower to blank control and Ad-EGFP groups(p < 0.05)with RT-PCR.Expression of Vimentin protein in Ad-ADAM33-EGFP group were significantly higer to blank control and Ad-EGFP groups(p < 0.05),and expression of E-cadherin protein in Ad-ADAM33-EGFP group were significantly lower to blank control and Ad-EGFP groups(p<0.05)with Western blot.Conclusions Overpression of ADAM33 in 16-HBE cells could enhance the EMT process induced by TGF-?1.