Background Transforming growth factor-β1 （TGF-β1） is known to have a role in keloid formation through the activation of fibroblasts and the acceleration of collagen deposition. The objective of this current study ...Background Transforming growth factor-β1 （TGF-β1） is known to have a role in keloid formation through the activation of fibroblasts and the acceleration of collagen deposition. The objective of this current study was to isolate TGF-β1 phage model peptides from a phage display 7-mer peptide library to evaluate their therapeutic effect on inhibiting the activity of keloid fibroblasts.Methods A phage display 7-mer peptide library was screened using monoclonal anti-human TGF-β1 as the target to obtain specific phages containing ectogenous model peptides similar to TGF-β1. Enzyme-linked immunosorbent assay （ELISA） was performed to select monoclonal phages with good binding activity, which underwent DNA sequencing. MTT assay and apoptosis assessment were used to evaluate the biological effects of the phage model peptides on keloid fibroblasts. Immunofluorescence assay was employed to show the binding affinity of the model peptides on phages causing keloid fibroblasts. Quantitative real-time PCR analysis was carried out to detect the expressions of nuclear factor κB （NF-κB） mRNA, connective tissue growth factor （CTGF） mRNA and TGF-β receptor Ⅱ （TβRII） mRNA in keloid fibroblasts.Results Specific phages with good results of ELISA were beneficiated. Four phage model peptides were obtained. The data of MTT showed that TGF-β1 and one phage model peptide （No. 4） could promote keloid fibroblasts proliferation,however, three phage model peptides （No. 1-3） could inhibit keloid fibroblasts proliferation. The results of apoptosis assessment showed that the three phage model peptides could slightly induce the apoptosis in 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 expressions of NF-κB mRNA and CTGF mRNA in the three phage model peptide groups decreased, while the expression of TβRII mRNA slightly increased.Conclusions Three phage model peptides isolated from a phage display 7-mer peptide library can inhibit keloid fibroblasts proliferation and induce the apoptosis in keloid fibroblasts. They can inhibit the activity of keloid fibroblasts by blocking TGF-β1 binding to its receptor and then regulating the expressions of NF-κB, CTGF and TβRII.展开更多
Background Keratinocyte growth factor （KGF） significantly influences epithelial wound healing. The aim of this study was to isolate KGF phage model peptides from a phage display 7-mer peptide library to evaluate the...Background Keratinocyte growth factor （KGF） significantly influences epithelial wound healing. The aim of this study was to isolate KGF phage model peptides from a phage display 7-mer peptide library to evaluate their effect on promoting epidermal cell proliferation. Methods A phage display 7-mer peptide library was screened using monoclonal anti-human KGF antibody as the target. Enzyme linked immunosorbent assay （ELISA） was performed to select monoclonal phages with good binding activity. DNA sequencing was done to find the similarities of model peptides. Three-（4,5-dimethylthiazol-2-yl） -2,5-diphenyl tetrazolium bromide （MTT） assay, immunofluorescence assay and quantitative real-time PCR analysis were employed to evaluate the effect of the phage model peptides on epidermal cells. Results Thirty-three out of fifty-eight （56.9%） of the isolated monoclonal phages exhibited high binding activity by ELISA. Ten of fifteen obtained phage model peptides were similar to KGF or epidermal growth factor （EGF）. MTT assay data showed that four （No. 1-4） of the ten phage model peptides could promote epidermal cell proliferation. The expression of keratinocyte growth factor receptor （KGFR） mRNA in the KGF control group and the two phage model peptide groups （No. 1 and No. 2） increased. Expression of c-Fos mRNA and c-Jun mRNA in the KGF control group increased, but did not increase in the four phage model peptide groups （No.1-4）. Conclusion Four phage model peptides isolated from the phage display 7-mer peptide library can safely promote epidermal cell proliferation without tumorigenic effect.展开更多
基金This study was supported by grants from the Nationa1 Natura1 Science Foundation of China （No. 30670571, 30772258 and 81071560）, Science and Technology Research Program of Shandong Province （No. 2009GG10002078）, Scientific Research Development Plan of the Department of Education of Shandong Province （No. J07WD03） and National Basic Research Program of China （973 Program） （No. 2005CB522603）.
文摘Background Transforming growth factor-β1 （TGF-β1） is known to have a role in keloid formation through the activation of fibroblasts and the acceleration of collagen deposition. The objective of this current study was to isolate TGF-β1 phage model peptides from a phage display 7-mer peptide library to evaluate their therapeutic effect on inhibiting the activity of keloid fibroblasts.Methods A phage display 7-mer peptide library was screened using monoclonal anti-human TGF-β1 as the target to obtain specific phages containing ectogenous model peptides similar to TGF-β1. Enzyme-linked immunosorbent assay （ELISA） was performed to select monoclonal phages with good binding activity, which underwent DNA sequencing. MTT assay and apoptosis assessment were used to evaluate the biological effects of the phage model peptides on keloid fibroblasts. Immunofluorescence assay was employed to show the binding affinity of the model peptides on phages causing keloid fibroblasts. Quantitative real-time PCR analysis was carried out to detect the expressions of nuclear factor κB （NF-κB） mRNA, connective tissue growth factor （CTGF） mRNA and TGF-β receptor Ⅱ （TβRII） mRNA in keloid fibroblasts.Results Specific phages with good results of ELISA were beneficiated. Four phage model peptides were obtained. The data of MTT showed that TGF-β1 and one phage model peptide （No. 4） could promote keloid fibroblasts proliferation,however, three phage model peptides （No. 1-3） could inhibit keloid fibroblasts proliferation. The results of apoptosis assessment showed that the three phage model peptides could slightly induce the apoptosis in 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 expressions of NF-κB mRNA and CTGF mRNA in the three phage model peptide groups decreased, while the expression of TβRII mRNA slightly increased.Conclusions Three phage model peptides isolated from a phage display 7-mer peptide library can inhibit keloid fibroblasts proliferation and induce the apoptosis in keloid fibroblasts. They can inhibit the activity of keloid fibroblasts by blocking TGF-β1 binding to its receptor and then regulating the expressions of NF-κB, CTGF and TβRII.
基金This study was supported by grants from the National Natural Science Foundation of China （No. 30670571 and No. 30772258）, Science and Technology Research Program of Shandong Province （No. 2009GG10002078）, Scientific Research Development Plan of the Department of Education of Shandong Province （No. J07WD03） and National Basic Research Program of China （973 Program, No. 2005CB522603）.
文摘Background Keratinocyte growth factor （KGF） significantly influences epithelial wound healing. The aim of this study was to isolate KGF phage model peptides from a phage display 7-mer peptide library to evaluate their effect on promoting epidermal cell proliferation. Methods A phage display 7-mer peptide library was screened using monoclonal anti-human KGF antibody as the target. Enzyme linked immunosorbent assay （ELISA） was performed to select monoclonal phages with good binding activity. DNA sequencing was done to find the similarities of model peptides. Three-（4,5-dimethylthiazol-2-yl） -2,5-diphenyl tetrazolium bromide （MTT） assay, immunofluorescence assay and quantitative real-time PCR analysis were employed to evaluate the effect of the phage model peptides on epidermal cells. Results Thirty-three out of fifty-eight （56.9%） of the isolated monoclonal phages exhibited high binding activity by ELISA. Ten of fifteen obtained phage model peptides were similar to KGF or epidermal growth factor （EGF）. MTT assay data showed that four （No. 1-4） of the ten phage model peptides could promote epidermal cell proliferation. The expression of keratinocyte growth factor receptor （KGFR） mRNA in the KGF control group and the two phage model peptide groups （No. 1 and No. 2） increased. Expression of c-Fos mRNA and c-Jun mRNA in the KGF control group increased, but did not increase in the four phage model peptide groups （No.1-4）. Conclusion Four phage model peptides isolated from the phage display 7-mer peptide library can safely promote epidermal cell proliferation without tumorigenic effect.
