Sustained small and intermediate size proteins expression in phorbol 12-myristate 13-acetate/ionomycine prolonged stimulated human fibroblasts

• Published in: Asian Pacific journal of tropical biomedicine Vol. 7; no. 5; pp. 432 - 436
• Main Authors: Abedian, Zeinab, Fattahi, Sadegh, Pourbagher, Roghayeh, Edrisi, Sahar, Mostafazadeh, Amrollah
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.05.2017; Dental Material Research Center, Faculty of Dentistry, Babol University of Medical Sciences, Babol, Iran%Cellular and Molecular Biology Research Center, Health Research Institute, Babol University of Medical Sciences, Babol,Iran; Cellular and Molecular Biology Research Center, Health Research Institute, Babol University of Medical Sciences, Babol,Iran; Wolters Kluwer Medknow Publications
• Subjects: Apoptosis; Biomarker; Fibroblast activation; Fibrosis; Ionomycine; Protein electrophoresis phorbol 12-myristate 13-acetate; Fibroblast activation; Biomarker; Ionomycine; Fibrosis; Apoptosis; Protein electrophoresis phorbol 12-myristate 13-acetate
• ISSN: 2221-1691; 2588-9222
• DOI: 10.1016/j.apjtb.2017.01.015

Objective:To compare the protein profile of culture supernatants in stimulated and unstimulated human fibroblasts to find some proteins indicating the presence of fibroblasts and their activation status.Methods:Dermal fibroblasts were stimulated with phorbol 12-myristate 13-acetate(PMA)/ionomycine for 72 h.MTT assay was done to determine cell viability and A/E fluorescent staining was used to evaluate the cell death pattern.Protein analysis was performed by gradient SDS polyacrylamide gel electrophoresis 8%–16%.Results:The supernatant of 24 h cultured both stimulated and unstimulated fibroblasts showed two bands in SDS-PAGE analysis with relative molecular weights of 8.59 and78.8 k Da.These bands density was decreased during the next 48 h in unstimulated cells while their expression was continued in PMA or PMA/ionomycine stimulated cells and a new 85.3 k Da band was appeared in unstimulated and 72 h PMA stimulated cells.Moreover,we found another seven small size(10–19.5 k Da) proteins in supernatants of48 h and 72 h unstimulated but not in PMA or PMA/Ionomycine stimulated fibroblasts.Most of these proteins expression were down regulated following fibroblast activation.This down-regulation is consistent with our finding that PMA or PMA/ionomycine stimulated cells exhibited a significant level of apoptosis cell death.Conclusions:Human fibroblasts produce some small to intermediate sized proteins with specific SDS-PAGE profile upon cell activation.Most of these proteins can be excreted in urine and can be immunogen theoretically so this data provided a reliable clue for fibrosis biomarker screening based on designation of an appropriated immunoassay.