ABSTRACT
Objective To study interaction proteins with secreted apoptosis-related protein 1 (SARP1) in fibroblasts of hypertrophic scar and to analyze its molecular mechanisms.Methods The recombinant adenovirus Ad-SARP1 was successfully constructed and transfected into the fibroblasts of hypertrophic scar in culture.The proteins were precipitated by immunoprecipitation and separated by SDS-PAGE,then it was stained with Coomassie blue and proteins from SDS-PAGE gel electrophoresis strip were analyzed with enzymolysis and mass spectrometric in turn.The peptide sequences were obtained according to mass spectrometry and the database were searched automatically.Results The results showed that in control cells,Ad-SARP1 and Ad-EGFP infected cells,were precipitated 7 protein bands,their molecular weights were about 93 × 103,43 × 103,40 × 103,37 × 103,31 × 103,26 × 103 and 12× 103,respectively; without SARP1 antibody the protein bands did not precipitate.Analysis of the 6 protein bands showed that proteins that might interact with SARP1 included periostin precursor (OSF-2),asporin precursor (PLAP1),phosphoglycerate kinase 1,rCG50690,apolipoprotein A-I precursor (Apo-AI),and thioredoxin 1 (TRX1).Conclusions The interaction proteins of SARP1 can be obtained by immunoprecipitation combined with liquid chromatography/mass spectrometry and ion trap detection technology.These results provide new clues for the mechanism of SARP1 regulates the apoptosis signal pathway of HSFb.
ABSTRACT
Objective To explore the effects of secreted apoptosis-related protein 1 (SARP1) on apoptosis of the hyperstrophic scar fibroblasts (HSFB) and its regulating mechnisms.Methods The recombinant vector was identified by enzyme digestion analysis.And the virus supernatant of the recombinant vector was extracted from packaged 293 cells,then it infected the skin fibroblasts from hypertrophic scar patients,which aimed to promote its expression of SARP1 protein.After adenovirus infection,the expression of SARP1 in the fibroblasts was confirmed by RT-PCR and Western blot.The effect of SARP1 on proliferation of HSFB was detected by MTT assay,and the effect of SARP1 on apoptosis of HSFB was detected and change of the cells functions were analyzed by FACS.Results Recombinants were confirmed.After adenovirus infection,both protein and mRNA of SARP1 were detected in HSFB.And the mRNA value of SARPlwas detected to increase significantly by RT-PCR and the protein expression was detected to increase significantly by Western blot (P＜0.05).The proliferation in the groups of the adenovirus infection and HSFB was positively regulated by SARP1 (P＜0.01) and the apoptosis of them was inhibited by the expression of SARP1 as compared to the control groups of HSFB and Ad-EGFP.It showed that the apoptosis index decreased as compared the group of infected fibroblasts to the control group by flow cytometry.Conclusions SARP1 could be highly expressed in HSFB by adenovirus infection,exhibiting the proliferation-enhancing and apoptosis-inhibiting effects on HSFB.