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

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.

Relaxin Inhibit Cardiac Fibrosis Induced by Phorbol 12-myristate 13-acetate

Relaxin is known to inhibit cardiac fibrosis. However, it is unclear whether relaxin could regulate the effects of Phorbol 12-myristate 13-acetate （PMA, PKC activator） on cardiac fibrosis. So the influence of relaxin on the cell proliferation and collagen expression induced by PMA in cultured cardiac fibroblasts was studied. It showed that PMA significantly increased cardiac fibroblasts proliferation, Type I pro-collagen protein expression, Type I pro-collagen mRNA expression, and rhRLX absolutely significantly decreased PMA induced effects on cardiac fibroblasts proliferation and Type I pro-collagen expressions, indicating that relaxin could inhibit cardiac fibrosis induced by PMA.

Regulation of activin receptor-interacting protein 2 expression in mouse hepatoma Hepa1-6 cells and its relationship with collagen type Ⅳ

AIM： To investigate the regulation of activin receptor-interacting protein 2 （ARIP2） expression and its possible relationships with collagen type Ⅳ （collagen Ⅳ） in mouse hepatoma cell line Hepal-6 cells. METHODS： The ARIP2 mRNA expression kinetics in Hepal-6 cells was detected by RT-PCR, and its regulation factors were analyzed by treatment with signal transduction activators such as phorbol 12-myristate 13-acetate （PMA）, forskolin and A23187. After pcDNA3- ARIP2 was transfected into Hepal-6 cells, the effects of ARIP2 overexpression on activin type Ⅱ receptor （ActRⅡ） and collagen Ⅳ expression were evaluated. RESULTS： The expression levels of ARIP2 mRNA in Hapel-6 cells were elevated in time-dependent manner 12 h after treatment with activin A and endotoxin LPS, but not changed evidently in the early stage of stimulation （2 or 4 h）. The ARIP2 mRNA expression was increased after stimulated with signal transduction activators such as PMA and forskolin in Hepal-6 cells, whereas decreased after treatment with A23187 （25.3% ± 5.7% vs 48.1% ± 3.6%, P 〈 0.01）. ARIP2 overexpression could remarkably suppress the expression of ActRⅡA mRNA in dose-dependent manner, but has no effect on ActRⅡB in Hepal-6 cells induced by activin A. Furthermore, we have found that overexpression of ARIP2 could inhibit collagen Ⅳ mRNA and protein expressions induced by activin A in Hapel-6 cells. CONCLUSION： These findings suggest that ARIP2 expression can be influenced by various factors. ARIP2 may participate in the negative feedback regulation of signal transduction in the late stage by affecting the expression of ActRIIA and play an important role in regulation of development of liver fibrosis induced by activin.

PMA及IL4序贯诱导人单核细胞系成为M2型巨噬细胞

目的将人单核细胞系U937及THP-1体外诱导得到M2型巨噬细胞。方法将U937及THP-1细胞以phorbol 12-Myristate 13-Acetate(PMA)及白介素-4(interleukin,IL-4)序贯诱导,采用q RT-PCR法及ELISA法检测M1/M2标志物m RNA及蛋白表达。结果 PMA使两种细胞转化为巨噬细胞,IL-4序贯诱导使巨噬细胞伸出伪足,序贯诱导后巨噬细胞M1表型标志物(IL-1β、IL-6、IL-12及i NOS)m RNA表达水平下调,M2表型标志物(IL-10、CD163)m RNA表达水平上调。同时,CCL18蛋白和m RNA表达水平均上调,而雷帕霉素可下调升高的CCL18表达水平。结论及IL-4序贯诱导人单核细胞系成为M2型巨噬细胞,雷帕霉素可逆转这一过程。