| Background:Keloid is a kind of pathological scar.It is characterized by excessive accumulation of extracellular matrix and active fibroblast proliferation.Its pathogenesis remains unclear,with no effective methods for diagnose,treatment and relapse monitoring.Keloid has familial aggregation and heredity.Researches about familial keloid showed that the lesions are often multiple.Keloid pedigrees have different clinical manifestations.It is presumed that different protein expression spectrum and expression characteristics may exist between familial keloids and sporadic keloids.Compared with hypertrophic scar,keloid is charactered with long-term sustained growth beyond the original lesion,high recurrent rate,rare to regression,tumor heterogeneity and invasiveness.While the identification of keloid and hypertrophic scar relies on clinical features.The identification for atypical keloids or hypertrophic scar is difficult.As the clinical diagnosis will affect the follow-up treatment plan directly,it is of great significance to search for a more scientific and effective identification method.Protein is the direct supporter of genetic material.It plays an important role in the progress of disease,the reaction of the body and the prognosis of disease.Currently,the research about specific biomarkers of keloid and hypertrophic scar is still imperfection.To study the protein expression profile and protein interactions of keloid is of great significance for disease diagnose,distinguish and recurrence detection.Objective:To explore the protein expression profiles of keloid,hypertrophic scars,and normal skin.To analyze the function and biological processes of differential proteins,and protein-protein interaction between different groups.To find out specific protein markers for the diagnose and distinguish of pathological scar.To provide theoretical and methodological basis for pathgenesis and development of pathological scar.Methods:In this study,the tissue and serum samples of familial keloid(FK),sporadic keloid(SK),hypertrophic scar(HS),normal skin(NS)were collected.The proteins were extracted and identified using the isobaric tags for relative and absolute quantitation(iTRAQ)technique and tandem mass spectrometry.The differences of tissue and serum protein expression among 4 groups were analysed.Bioinformatics analysis of different expression proteins were conducted,including gene ontology(GO)analysis,Kyoto Encyclopedia of Genes and Genomics(KEGG),GO and KEGG cluster of orthologous groups of proteins,clustering and protein-protein interaction(PPI)analysis.A total of 8 proteins were selected for technique verification of tissue sample proteomic profiling.Western blot and immunohistochemistry were used to verify the expression of proteins.Compare the different expression proteins between tissue and serum proteomic results.Select different expression proteins existed in both tissue and serum samples.ELISA was used for verification.Try to find specific protein biomarkers for pathological scar diagnose,and the identification of keloids and hypertrophic scars.Results:1.The proteomic profiling and protein verification of tissue samplesA total of 2450 differentially expressed proteins were identified.P<0.05 and a difference of more than 1.2 folds(up-regulation and down regulation)were selection criteria.Compared with NS,250 up-regulated and 281 down-regulated proteins were identified in FK;281 up-regulated and 232 down-regulated proteins were identified in SK;199 up-regulated and 233 down-regulated proteins were identified in HS.Compared with SK,64 up-regulated and 164 down-regulated proteins were identified in FK;79 up-regulated and 169 down-regulated proteins were identified in HS.Compared with HS,124 up-regulated and 115 down-regulated proteins were identified in FK.The proteins were identified by GO,KEGG pathway enrichment and cluster analysis.Western blot and immunohistochemistry were used to verify the expression of several proteins to verify the reliability of iTRAQ technique.2.The proteomic profiling and protein verification of serum samplesA total of 860 differentially expressed proteins in serum were identified.A difference greater than 1.2 folds(up-regulation and down regulation)and p<0.05 were selection criteria.Compared with NS,13 up-regulated and 22 down-regulated proteins were identified in FK;28 up-regulated and 25 down-regulated proteins were identified in SK;8 up-regulated and 14 down-regulated proteins were identified in HS.Compared with SK,16 up-regulated and 27 down-regulated proteins were identified in FK.Compared with HS,17 up-regulated and 5 down-regulated proteins were identified in FK;24 up-regulated and 11 down-regulated proteins were identified in SK.The bioinformatics analysis of the differential proteins was carried out and compared with the tissue differential proteins to find the consistent expression proteins.The basic information of these proteins was obtained on NCBI website.Classified the proteins according to their function and cell location.Identify 5 candidate biomarkers for ELISA verification based on the existing knowledge,database notes and researches.The results showed that fibronection were significantly up-regulated in SK and HS compared with NS.ConclusionIn this study,the tissue and serum samples' protein expression profile of familial keloid,sporadic keloid,hypertrophic scar and normal skin was obtained using iTRAQ technique.The different expression proteins among these groups were clarified.It provides a solid foundation for specific biomarker identification,pathogenesis revelation,clinical diagnosis,treatment and relapse detection.In this study,iTRAQ technique were used to detect tissue and serum samples of keloid,hypertrophic scar and normal skin.The protein expression profiles of four groups were obtained,and the differences in protein expression between each two groups were analyzed.The protein function,pathways and protein-protein interactions were investigated.It provided a solid foundation for the search of specific protein markers which can be used in analysing the pathogenesis,clinical diagnosis,treatment,relapse detection of keloids. |
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