Objective: To isolate the transforming growth factor-beta 1 (TGF-β1) phage model peptides from phage 12-mer display peptide library to inhibit the proliferation of keloid fibroblasts.
Methods: The phage display 12-mer peptide library was screened for 4 rounds with monoclonal anti-human TGF-β1 as the target to yield the specific phage model peptides. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used for the quantitative determination of cellular proliferation. Apoptosis was detected by the Annexin V-FITC/PI apoptosis detection kit and the cells were analyzed with flow cytometry. Immunofluorescent assay was employed to show the binding affinity of model peptides for keloid fibroblasts. Quantitative real-time polymerase chain reaction (PCR) was performed to detect the expressions of nuclear factor kappa B (NF-κB) and connective tissue growth factor (CTGF).
Results: Ten phage model peptides were obtained and they were similar to TGF-β1, TGF-β2, TGF-β receptor II (TβRII), TGF-β-induced factor, NF-κB or mitogen-activated protein kinase (MAPK). The results of MTT showed that four phage model peptides (No. 7 - 10) could inhibit the proliferation of keloid fibroblasts (P < 0.05). The results of apoptotic assessment showed that phage model peptides (No. 7 - 10) could slightly trigger the late apoptotic stage of keloid fibroblasts. The data of immunofluorescence assay revealed that the model peptides on phages rather than phages could bind to keloid fibroblasts. The findings of quantitative real-time PCR analysis suggested that the relative expression of NF-κB decreased in phage model peptides groups (No. 7 - 10). The quantitative expression was 0.28, 0.26, 0.46 and 0.30 respectively versus the negative control group. The relative expression of CTGF decreased in phage model peptides groups (No. 7 - 10). The quantitative expression was 0.26, 0.60, 0.34 and 0.17 respectively versus the negative control group.
Conclusion: Four phage model peptides (No. 7 - 10) isolated from phage display 12-mer peptide library can inhibit the proliferation of keloid fibroblasts via regulating the expressions of NF-κB and CTGF.