| Pulmonary fibrosis is a major involvement of pulmonary interstitial and alveolar or bronchioles diffuse lung disease,its pathogenic mechanism is not yet clear,Pulmonary fibrosis of men is usually more than women,and mostly middle-aged,especially the air pollution in our country in recent years,PM2.5 pollutants is serious damage to lung health among urban residents and the incidence of pulmonary fibrosis increased year by year.Pulmonary fibrosis pathogenic process is relatively complicated,alveolitis segment is the first stage,flammatory cells release is main pathogenic factor activated,lung injury after repair of alveolar process and fibrosis is the final stage of the disease,mainly including mesenchymal cells increased,collagen metabolism,continue to progress and excessive pulmonary fibrosis process.The prevention of Pulmonary fibrosis is more difficult,it is mainly due to the unknown etiology.Transforming growth factor beta(TGF-beta)is believed to be caused by Smad3 signal through express fiber proliferative diseases and fibrosis,especially pulmonary fibrosis.Test show that can block TGF-beta signaling pathways of medicines that can reduce the tissue fibrosis,and how to block TGF-beta signal pathways that inhibition of pulmonary fibrosis specific mechanism remains is unclear.Kruppel-like factors(KLFs)are a family of zinc-finger transcription factors which are widely expressed in multiple human organs or tissues.KLFs pose regulatory roles in a variety of cellular processes such as proliferation,inflammation,differentiation,and migration.The tumor suppressive KLF-4 has been identified to be decreased or lost in lung cancer and other types of cancers.And the inhibited KLF-4 results in epithelial-to-mesenchymal transition(EMT)by regulating the expression of EMT-related markers,such as E-cadherin,vimentin,β-catenin,vascular endothelial growth factor-(VEGF-)A,endothelin-1,and JNK1,or through crosstalk with the TGF-β,Notch,and Wnt signaling pathways.And a recent study has demonstrated that KLF-4 is downregulated during EMT in renal fibrosis and functions as a suppressor of renal fibrogenesis.And the anti-EMT effect of KLF-4 has also been recognized in hepatocellular carcinoma cells.Plasminogen activator inhibitor type-1(PAI-1)could efficiently induce epithelial to mesenchymal cells,PAI-1 often localizes in tumor cells and myofibroblasts,implying involvement as a positive modulator of cellular invasive potential.PAI-1 is a target gene of TGF-β and Smads(Smad3 and Smad4).And PAI-1 is regulated by TGF-β in different kinds of cells,such as macrophages,renal tubular cells,trophoblast cells,and hepatocytes.PAI-1 has been recognized to be increased in pulmonary fibrosis.However,it is not clear whether there is an interaction between KLF-4 and PAI-1 in the pulmonary fibrosis.Therefore,in the present study,we investigated the regulation on KLF-4 and PAI-1 by TGF-β in mouse lung epithelial LA-4 cells,then examined the expression of EMT-associated markers,such as Collagen I,α-SMA and E-cadherin in the TGF-P treated LA-4 cells,and determined the influence on the TGF-P induced EMT by the PAI-1 knockdown or the KLF-4 overexpression.Our study indicated the inhibitory role of KLF-4 in the TGF-β promoted EMT via downregulating PAI-1 in the mouse lung epithelial cells.Materials and methodsPart one1.1 Cell cultureMouse lung epithelial LA-4 cells line was purchased from American Type Culture Collection(ATCC),and was grown in the F-12K Medium,which was supplemented with 10%Fetal Bovine Serum and with 100 U/ml penicillin and 100 mg/ml streptomycin at 37℃ in a humid incubator.1.2 The Kriippel-like factor-4 of protein and mRNA change for transforming growth factor beta disturbing mouse lung epithelial LA-4 cellsTo model the TGF-β-induced EMT in LA-4 cells,LA-4 cells with approximately 90%confluence were incubated with the F-12K Medium,adding 2%FBS,and the TGF-β with a final concentration of 0,1,2,5,or 10ng/mL for 1,3,6,12,24,or 48 hours.Using Trizol reagent extraction cell total mRNA in LA-4 cells.Real-time quantitative reverse transcription polymerase chain reaction measurement Kruppel-like factor-4 and internal beta actin mRNA level.The primer by Sangon biotech companies.The results as A delta express Ct method with multiple changes.Quantitative analysis using western blot method Kruppel-like factor-4 protein content,protein samples by 10%gel electrophoresis separation and transferred to nitrocellulose membrane,5%skim milk sealing membrane 4 degrees Celsius for the night,and then film and forgive Kruppel-like factor-4 in mice and mice resistant beta actin at 4℃ training 2 hours at room temperature.1.3 Plasminogen activator inhibitor-1 and transformation of epithelial mesenchymal related protein were detected after transforming growth factor beta interference mouse lung epithelial LA-4 cellsTransforming growth factor beta interference mouse lung epithelial LA-4 cells is the main way through 90%concentration of LA-4 cells in F-12 k culture medium adding 10%fetal bovine serum,at 37℃,respectively,to join the 0,1,2,5 or 10 ng/mL concentration of transforming growth factor beta train for 24 hours,using western blot method to the quantitative analysis of PAI-1.Transformation of epithelial mesenchymal detection we use calcium mucin epithelial markers of epithelial cell,fibrosis markers of alpha SMA and Collagen I,at 37℃,respectively,to join the 0,1,2,5 or 10 ng/mL concentration of transforming growth factor beta train for 24 hours,using western blot method of quantitative analysis of calcium mucin epithelial cell,fibrosis markers of alpha SMA and Collagen I protein content.Part two2.1 Experimental cells:mouse lung epithelial LA-4 cells culture2.2 siRNA-PAI-1 transfection mouse lung epithelial LA-4 cellsSi-RNA design mainly adopts literature retrieval gene,entrust Invitrogen company for design synthesis,using liposome transfection original activators RNAiMax fibrinolytic enzyme inhibitors-1 synthetic specific siRNA(siRNA-PAI-1)and negative control siRNA(siRNA-Con).Reference manual,the transfection reagents respectively by 20 and 40 nM and LA-4 lung epithelial cells in mice,inhibit experimental mouse lung epithelial LA-4 cells plasminogen activator inhibitor type-1.2.3 The siRNA-PAI-1 transfection of mouse lung epithelial LA-4 cells successful validationThe transfection reagents respectively by 20 and 40 nM and LA-4 lung epithelial cells in mice,thus inhibiting experimental mice lung epithelial LA-4 cells plasminogen activator inhibitor type-1.Using western blot method to the quantitative analysis of Plasminogen activator inhibitor type-1 using Trizol reagent extraction cell total mRNA in LA-4 cells.Real-time quantitative reverse transcription polymerase chain reaction to measure mouse Plasminogen activator inhibitor-1 and internal beta actin mRNA level.As △ delta express Ct method with multiple enzyme the change of the Plasminogen activator inhibitor type-1 mRNA content results.2.4 Transforming growth factor beta transfection induced siRNA-PAI-1 LA-4 cells,the change of the plasminogen activator inhibitor type-1,the E-cadherin markers of epithelial cells,fibrosis markers of alpha SMA and Collagen I contentWe Respectively j ion 0,5 ng/ml concentration of Transforming growth factor beta interference 40 nM siRNA-PAI-1 transfection LA-4 cells,using western blot method to measure the Plasminogen activator inhibitor type-1 the transformation of epithelial mesenchymal markers of epithelial cell adhesion protein,calcium fibrosis markers of alpha SMA and the change of Collagen I content.In beta actin,for internal,calculation and relative value of the proteins under test.Part three3.1 Experimental cells:mice lung epithelial LA-4 cells culture3.2 Adenovirus-Mediated Overexpression of Kruppel-like factor-4 in LA-4 cells establishment and validation of the modelRat Kruppel-like factor-4 open reading frame use delete the termination codon primers for PCR amplification,and the rat Kruppel-like factor-4 open reading frame is inserted into the carrier pShuttle-CMV refactoring pShuttle CMV-factor-4 plasmid Kruppel samples,chloramphenicol acetyltransferase gene as a comparison,by pShuttle CMV-Kruppel-like factor-4 and common transfection pAdeasy-1 sample to build Ad BJ518 virus cells-Kruppel factor-4 adenovirus Ad-Con virus and the control group,the concentration of 90%of infections of LA-4 cells respectively the concentration of 1 and 5 moi Ad-factor-4 adenovirus Kruppel sample and the control group Ad-Con virus,measured Kruppel-like factor-4 mRNA and protein expression level.In beta actin,for internal,calculation and relative value of the proteins under test.Ad-Kruppel factor-4 adenovirus infections LA-4 lung epithelial cells in mice in F-12 k culture medium adding 10%fetal bovine serum,at 37 0 C,the quantitative analysis using western blot method Ad-Kruppel-like factor-4 adenovirus protein,is made with beta actin,calculation and relative value of the proteins under test.3.3 Transforming growth factor beta induced Ad-Kruppel like factor-4 adenovirus infections LA-4 lung epithelial cells in mice,detection Plasminogen activator inhibitor type-1,transformation of epithelial mesenchymal markers cells adhesion protein and the change of Collagen I contentUsing 0,5 ng/ml transforming growth factor beta interference 0,5 MOI Ad-Kr uppel factor-4 adenovirus infections mice lung epithelial LA-4 cells,using western blot method to measure the Plasminogen activator inhibitor type-1,transformation of epithelial mesenchymal markers cells E-adhesion protein and the change of Collagen I content,is made with beta actin,calculation and relative value of the proteins under test.ResultsPart one1.1 Transforming growth factor-beta interference in mouse lung epithelial LA-4 cells.Kruppel-like factor-4 mRNA and protein expression changesKruppel-like factor-4 mRNA level significantly lowered,and the dose of relevance,2 and 5 ng/ml and 10 ng/ml between the group of Kruppel-like factor-4 lists of mRNA level difference was statistically significant(P<0.05,or P<0.01,between 2 and 10 ng/ml group,P<0.05).Kruppel-like factor-4 mRNA level with the time extension significantly cut,with 5 ng/ml of transforming growth factor beta,over 12 hours post-treatment(H.P.T.)mRNA cut its Kruppel-like factor-4(P<0.05,or P<0.01),and 3 hours,6 hours difference between interference group was statistically significant(P<0.05),western blot experimental analysis showed that the transforming growth factor-beta induced LA-4 cell Kriippel-like factor-4 protein expression significantly lowered a dose dependent variation(P<0.01 or P<0.05)and time dependence(P<0.01 or P<0.05).1.2Transforming growth factor-beta promote mice lung epithelial LA-4 cells Plasminogen activator inhibitor-1 rise,promoting the transformation of epithelial mesenchymalUsing western blot reaction,1,2,5 or 10 ng/mL transforming growth factor beta interference in mice lung epithelial LA-4 cell,the plasminogen activator inhibitor type-1 protein significantly increase,epithelial markers epithelial cell calcium mucin,and measuring dependence,at the same time we are using western blot method to detect the transforming growth factor beta induced mice lung epithelial LA-4c cells fibrosis markers of alpha SMA and Collagen I,2,5 or 10 ng/mL transforming growth factor beta can promote the level of the alpha-SMA and Collagen I rise.Part twoMice lung epithelial LA-4 cell transfection the siRNA-PAI-1,inhibition of transforming growth factor beta induced mice lung epithelial LA-4 cell interstitial transformationPlasminogen activator inhibitor type-1 specific siRNA transfection lung epithelial LA-4 cells in mice after 24 hours,compared with the control siRNA group,Plasminogen activator inhibitor-1 mRNA and protein levels significantly reduced,transforming growth factor-beta transfection induced the siRNA-PAI-1 of lung epithelial LA-4 cells in mice.the plasminogen activator inhibitor type-1 rise was not significant(p<0.01),and transforming growth factor beta induced dye siRNA-PAI-1 of mice lung epithelial LA-4 cells,epithelial calcium adhesion proteins rise,fibrosis markers of alpha SMA and Collagen I rise is not obvious.Part three3.1 Kruppel-like factor-4 overexpression of adenovirus infection in mice lung epithelial LA-4 cells establishment and validation of the modelKruppel-like factor-4 coding sequence amplification and cloning to shuttle plasmid,and then in LA-4 cells with adenovirus genome plasmid and plasmid adenovirus recombinant,infection Kruppel-like factor-4.Ad-Kruppel-like factor-4 virus infection in mice lung epithelial cell,LA-4 sample tested the Kruppel factor-4 expression of infection in the plural for 1 or 5 moi environment three hours after infection,Kruppel-like factor-4 mRNA level,(P<0.001 or P<0.0001).At its peak in 12 hours after infection(P<0.0001).Kruppel-like factor-4 expression increased protein content.(P<0.01,P<0.001 or P<0.001)3.2Kruppel-like factor-4 overexpression of adenovirus infection in mice lung epithelial LA-4 cells reduce transforming growth factor-beta for plasminogen activator inhibitor type-1,and inhibition of transforming growth factor beta induced LA-4 interstitial cell transformationKruppel-like factor-4 overexpression of mice lung epithelial LA-4 cell,compared with control group,the plasminogen activator inhibitor type-1 mRNA level dropped significantly,(p<0.05 or p<0.01 infection measurement for 1 and 5 MOI),western blot detection protein,according to the results of plasminogen activator inhibitor type-1 protein expression is significantly decreased,Kruppel-like factor-4 overexpression of mice lung epithelial LA-4 cells,transforming growth factor beta promote epithelial calcium adhesion protein secretion to increase,Collagen Ⅰ secretion decreased,therefore,above all,Kruppel-like factor-4 expression,can reduce of transforming growth factor-beta plasminogen activator inhibitor type-1 raised effect,inhibition of transforming growth factor beta induced LA-4 cell interstitial cell transformation.Conclusion1.Transforming growth factor-beta interfer mice lung epithelial cell,Kruppel-like factor-4 mRNA and protein expression level were significantly lower,and as dose of correlation and time correlation.2.Transforming growth factor beta promote mice lung epithelial LA-4 cells plasminogen activator inhibitor-1,and the transformation of epithelial mesenchymal,and a measurement correlation.3.Mice lung epithelial LA-4 cell transfected siRNA-PAI-1,after plasminogen activator inhibitor type-1 mRNA and protein levels significantly reduced,transforming growth factor-beta transfection induced siRNA-PAI-1 of lung epithelial LA-4 cells in mice after plasminogen activator inhibitor type-1 don’t rise,transforming growth factor beta induced dye siRNA-PAI-1 of mice lung epithelial LA-4 a significant rise in cell calcium epithelial cell adhesion protein,fibrosis markers alpha SMA and Collagen I rise is not obvious.4.Kriippel-like factor-4 overexpression of adenovirus infected in mice lung epithelial LA-4 cells,plasminogen activator inhibitor type-1 mRNA level dropped significantly,plasminogen activator inhibitor type-1 protein expression was significantly decreased;Kruppel-like factor-4 over expression of mice lung epithelial LA-4 cells,transforming growth factor-beta lead to the plasminogen activator inhibitor type-1 protein expression levels;Kruppel-like factor-4 overexpress in mice lung epithelial LA-4 cells,transforming growth factor beta can promote epithelial calcium adhesion protein levels rise,lower the level of Collagen I;Kru ppel-like factor-4 expression,can reduce of transforming growth factor-beta plasminogen activator inhibitor type-1 raised effect,inhibition of transforming growth factor beta induced LA-4 cell interstitial cell transformation. |
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