ABSTRACT
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 and 78.8 kDa. 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 kDa band was appeared in unstimulated and 72 h PMA stimulated cells. Moreover, we found another seven small size (10–19.5 kDa) proteins in supernatants of 48 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.
ABSTRACT
Background: the human leukocyte antigen [HLA] matching between organ donor and recipient is an acceptable strategy in clinical transplantation since 1964. However, in bone marrow transplantation, finding matched donors is often problematic. Thus new method for down regulation of HLA can be an alternative strategy to solve this problem
Objective: to examine the effect of serum starvation on HLA class I expression in human peripheral blood mononuclear cells [PBMCs]
Methods: PBMCs were cultured in RPMI-1640 supplemented with 10% FBS [non-starved cells] as well as in medium only [starved cells] for 16, 24, 48, 72, 96h under standard cell culture conditions. The pattern of cell death and HLA class I expression was determined by flowcytometry. Antigenicity of the starved PBMCs was evaluated in a one-way mixed lymphocyte culture by MTT assay
Results: mean fluorescence intensity [MFI] of different indicated starved PBMCs gradually decreased and this reduction was stable after 96h of re-feeding with medium containing FBS. Under serum starvation condition, PBMCs showed apoptotic cell death pattern. There was a linear correlation between percentages of cells, which exhibited the late apoptosis death pattern and serum starvation period [r=0.88, p<0.01]. Surprisingly, the starved PBMCs lost their stimulatory property in mixed culture with allo-reactive lymphocyte
Conclusions: membrane HLA class I expression could be stably reduced in 96h starved human PBMCs culture condition, decreasing their allo-reactivity while their viability rate is enough for possible clinical application