| Objectives To screen the differential proteins related to the activation of fibroblasts treated with transforming growth factor(TGF)-?1 by using a isobaric tags for relative and absolute quantitation(iTRAQ)profile in order to provide new ideas for the study of the pathogenesis and prevention strategies of silicosis fibrosis.Methods Primary culture of Wistar rat lung fibroblasts by adherent method and transformed into myofibroblasts by TGF-?1 stimulation,which were divided into control and TGF-?1 stimulation group.iTRAQ was used to label protein samples,and liquid chromatography-tandem mass spectrometry(LC-MS/MS)was used to analyze the labeled proteins,and the differentially expressed proteins in the control group and TGF-?1stimulation group were screened.The silicotic rats were made by a HOPE-MED 8050 exposure control apparatus.The experimental group was divided into control group for 24weeks(control 24w)and silicotic model group for 24 weeks(silicosis 24w).The rat lung fibroblasts were cultured in vitro by enzyme digestion at 24 weeks in the control group and24 weeks in the silicotic model group.siRNA knockdown Very low-density lipoprotein receptor(VLDLR)and Syndecan-2(SDC2)were constructed and transfected into human embryonic lung fibroblast(MRC-5)cells.After screening the effective silencing sequences,they were divided into: negative control group(Negative Control,NC);VLDLR gene silencing group(VLDLRsiRNA#3);SDC2 gene silencing group(SDC2siRNA#3);negative control + TGF-?1 group(NC + TGF-?1);VLDLR gene silencing + TGF-?1 group(VLDLRsiRNA#3+TGF-?1);SDC2 gene silencing + TGF-?1 group(SDC2siRNA#3+TGF-?1).VG staining to observe the morphology of lung tissue.Detect the expression and localization of Collagen type V(pro COL V),Collagen type XI(pro COL XI),VLDLR and SDC2 use Immunohistochemical staining.Test the expression of Collagen type I(pro COL I),?-smooth muscle actin(?-SMA),pro COL V,pro COL XI,Vascular cell adhesion protein 1(VCAM1),Transmembrane protein 214(TM214),VLDLR and SDC2 in lung tissue and fibroblasts cells by Western blot.Results 1 A total of 1,648 proteins were identified by iTRAQ.Compared with the control group,196 differentially expressed proteins in the TGF-?1 group were 1.20 times,and the expression of 20 differentially expressed proteins was 1.50 times,including 13 upregulated proteins and 7 down-regulated proteins.Bioinformatics analysis showed that the differentially expressed proteins were mainly related to cell proliferation,apoptosis,inflammatory response,signal transduction pathway and other functions.2 VG staining results showed that the alveolar walls of the lung tissues in the control 24 weeks group were thin and clear without collagen deposition,while the alveolar walls of the lung tissues in the model 24 weeks group were widened with more cellular and fibrous silica nodules formation and red collagen deposition.3 Immunohistochemical staining results showed that compared with the control group,the positive expressions of pro COL V,pro COL XI,VLDLR and SDC2 in the model 24 weeks group were enhanced,mostly expression in silicotic nodules and interstitial fibrosis regions.4 Western blot results showed that the levels of pro COL I,?-SMA,pro COL V,pro COL XI,VCAM1,VLDLR and SDC2 in model 24 weeks group were up-regulated to 11.09,27.17,7.03,3.61,12.16,2.39 and 21.12 folds in control 24 weeks group,while TM214 showed no difference.The expressions of pro COL I,?-SMA,pro COL V,pro COL XI,VCAM1,VLDLR and SDC2 in primary cultured silicosis rat fibroblasts were significantly up-regulated,which were 6.16 times,5.63 times,29.34 times,13.74 times,4.09 times,80.58 times and 4.06 times of the controlgroup,while TM214 showed no difference.5 Cell transfection experiment:(1)Western blot results showed that VLDLR,SDC2 plasmid vector silencing sequence found that compared with the negative control group,VLDLRsiRNA#3 group had significant silencing effect,and the expression of VLDLR decreased to 6.65% in the negative control group;Similarly,compared with the negative control group,SDC2siRNA#3 group had significant silencing effect,and the expression of SDC2 decreased to 21.14% in the negative control group.(2)Western blot results showed that the expressions of pro COL I,?-SMA,pro COL V and pro COL XI in VLDLRsiRNA#3 group were decreased to32.57%,42.55%,60.85% and 16.65% in the NC group,similarly,the pro COL I,pro COL V,and pro COL XI expressions in SDC2siRNA#3 group were decreased to 5.43%,19.41%,and 8.13% in the NC group,while there was no difference in the expression of ?-SMA.(3)Western blot results showed that in the VLDLR transfection experiment,compared with the NC group,the expressions of pro COL I,?-SMA,pro COL V and pro COL XI in the NC+TGF-?1 group were 3.78 times,3.71 times,12.38 times and 10.78 times higher than that in the NC group.The expressions of pro COL I,?-SMA,pro COL V and pro COL XI in the VLDLRsiRNA#3+TGF-?1 group were decreased to 73.66%,55.70%,22.47% and 50.06% of the NC+TGF-?1 group.Similarly,in SDC2 transfection experiments,the expressions of pro COL I,?-SMA,pro COL V and pro COL XI were significantly up-regulated after TGF-?1 treatment,and the expressions of pro COL I,pro COL V,and pro COL XI were decreased after SDC2 gene silencing pretreatment to44.95%,32.04%,and 37.63% of the NC+TGF-?1 group,while there was no difference in the expression of ?-SMA.Conclusion 1 196 differentially expressed proteins related to TGF-?1 mediated fibroblast activation were identified by iTRAQ,in which pro COL V,pro COL XI,VCAM1,VLDLR and SDC2 may be involved in the occurrence and development of silicosis fibrosis.2 Kockdown VLDLR and SDC2 can inhibit collagen synthesis in MRC-5cells at the basic level and under the induction condition of TGF-?1.Figure14;Table20;Reference141... |
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