Journal: Scientific reports

Article Title: Treatment of refractory cutaneous ulcers with mixed sheets consisting of peripheral blood mononuclear cells and fibroblasts.

doi: 10.1038/srep28538

Figure Lengend Snippet: Figure 1. Synergetic effect of mixed sheets consisting of PBMNCs and fibroblasts. (a) Comparison of VEGF secretion. The VEGF concentration in the culture medium was measured in PBMNCs, fibroblast sheets, and mixed cell sheets for 3 days. Normoxic condition: 37 °C, 20% O2 for 3 days. Hypoxic condition: 37 °C, 20% O2 for 2 days followed by 33 °C, 2% O2 for 1 day. (b) Secretion from PBMNCs increased VEGF production by fibroblasts. Fibroblasts were cultured for 48 h with or without the PBMNC-conditioned medium, and the VEGF concentration in the supernatant was analyzed by ELISA. (c) TGF-β1 concentration in fibroblasts and PBMNC culture medium at 48 h. (d) PDGF-BB concentration in fibroblasts and PBMNC culture medium at 48 h. (e) Neutralizing antibody against TGF-β1 and PDGF-BB inhibited VEGF production in fibroblasts. The PBMNC-conditioned medium was co-cultured with a neutralizing antibody against TGF-β1 or PDGF-BB, and the PBMNC-conditioned medium was added to fibroblasts. After 48 h, the VEGF concentration was measured by ELISA. (f) TGF-β1 and PDGF-BB recombinant proteins elevated VEGF production by fibroblasts. (g) The PBMNC-conditioned medium increased the expression levels of VEGF, collagen I, collagen III, α-SMA, and Axin2 mRNA. Fibroblasts were cultured with the PBMNC-conditioned or control medium for 48 h. The mRNA expression levels were determined using real-time PCR. ACTB was used as an endogenous control. The expression levels were compared with that in control medium, which is presented as 1.

Article Snippet: In the neutralizing antibody experiment, the conditioned medium was incubated for 90 min at 4 °C with anti-ATM antibody (38 μ g/mL, ab78, Abcam, Cambridge, UK) as a control antibody, anti-TGF-β 1 antibody (38 μ g/mL, ab64715, Abcam), or anti-PDGF-BB antibody (33 μ g/mL, AF-220-NA, R&D Systems, Inc.) and then added to fibroblasts.

Techniques: Comparison, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Recombinant, Expressing, Control, Real-time Polymerase Chain Reaction