The Experimental Study Of KGF And TGF-β1 Phage Model Peptides On Improving The Quality Of Wound Healing

Wound healing is a complex process that restores tissue integrity. Abnormal wound healing will result in chronic wound and scar hyperplasia. Diabetes patients and long-term bedridden patients often appear chronic wound, which is a common clinical problem and need to be resolved. Keloid is is a special type of pathological scar, which influences the physical functions and causes aesthetic problems. Keloid is frequently recur after treatment, making it hard to achieve successful prevention and treatment of keloid.Keratinocyte growth factor (KGF) is a multifunctional growth factor with epithelial cell specificity, produced by interstitial cells and secreted via a paracrine pathway. KGF is closely related to epithelial wound healing, embryonic development, tumor formation and development and immune reconstitution. When trauma occurs, keratinocyte proliferation, migration and cytoskeletal rearrangement are largely controlled by KGF, released by the underlying fibroblasts, significantly influencing the epithelial wound healing process. However, the tumorigenicity of KGF limits its use.Transforming growth factor-p1 (TGF-p1) is known to be closely associated with scar formation. TGF-β1 is the most representative cell division inducing agent and the strongest fibrosis-promoting factor. TGF-p1 can promote vascularization and induce granulation tissue formation, and stimulate extracellular matrix protein synthesis and deposition, inhibit the production of collagenase and promote wound fibrosis. The expression of TGF-β1 in keloid is higher than that in normal skin. TGF-β1 signaling pathway is associated with scar formation. TβRII, Smad, MAPK, NF-kappa B and CTGF are very important intracellular mediums of TGF-β1 signaling pathway. The current studies focus on the prevention and treatment of scar hyperplasia by adjusting TGF-p1 signing pathway, which has good prospect of research and application.Phage display technology is a method that monoclonal antibodies or cells are used as the target to harvest analog epitope and ligand, including receptor blocking pharmacon and excitomotor, polypeptide vaccine and drugs. Phage M13 is a good vector for growth factors, which has the advantages of low immunogenicity, and being readily diffusible. It can express fusion protein, keep the conformation of ectogenous peptides, and help ectogenous peptides against the effect of proleases in the wounds.The objectives of the study were to isolate KGF and TGF-p1 phage model peptides from phage display peptide library. The influence of KGF phage model peptides on promoting epidermal cell proliferation was detected. Based on sequence analysis and in vitro cell test results, the specific sequences of KGF were chosen. pComb3-KGF active peptides were constructed and expressed, which would be used to promote wound healing. The influence of TGF-β1 phage model peptides on keloid fibroblasts were detected to select phage model peptides with competitive inhibition to be used to prevent and treat scar hyperplasia. Research on these aspects has not yet been reported.Part one:The study of KGF phage model peptides'screening and promoting epidermal cell proliferationObjective The objective of the 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 for four rounds using monoclonal anti-human KGF antibody as the target. Phage titer was done to evaluate the recovery of phages. ELISA was performed to select monoclonal phages of the fourth round with good binding activity. DNA sequencing was done to find the similarities of model peptides. MTT assay was used to evaluate the effect of phage model peptides on promoting epidermal cell proliferation. Immunofluorescence assay was employed to evaluate the cell affinity of phage model peptides. Quantitative real-time PCR analysis was employed to evaluate the expressions of KGFR, humanβ-defensin 3, c-Fos and c-Jun in epidermal cells.Results After the four rounds of screening, abundant of specific phage modeling peptides were harvested. Twenty-five phage modeling peptides were isolated from the phage display 7-mer peptide library. Some of the isolated phage modeling peptides exhibited high binding activity by ELISA. MTT data showed that four phage modeling peptides could promote epidermal cell proliferation. The results of immunofluorescence assay showed that the phage modeling peptides had good cell affinity. The expression of keratinocyte growth factor receptor (KGFR) and humanβ-defensin 3 in the KGF control group and the two phage model peptide groups increased. The expression of c-Fos and c-Jun in the KGF control group increased, but did not increase in the phage model peptide groups.Conclusions KGF phage model peptides can be isolated from the phage display 7-mer peptide library, which can safely promote epidermal cell proliferation. Two phage model peptides can promote the expression of humanβ-defensin 3 in epidermal cells to control the wound infection. Phage model peptides can not promote the expression of c-Fos and c-Jun. Part two:The study of pComb3-KGF active peptides' construction and promoting epidermal cell proliferationObjective Based on sequence analysis and in vitro test results, the specific sequences of KGF were chosen. pComb3-KGF active peptides were constructed and expressed to be used to detect the effect of promoting epidermal cell proliferation.Methods pComb3 was used as the vector, and its heavy chain was chosen as the location to insert exogenous gene. Spel and XhoI were chosen as the restriction endonuclease. Based on sequence analysis and in vitro test results, four sequences were chosen. Primers were designed. The normal skin fibroblasts were gathered with adherent tissue culture method from human skin. The total RNA was extracted from the cells. The selected genes were obtained using RT-PCR method. The genes were subcloned into pComb3 vector after SpeⅠand XhoⅠdouble-digest. The technology of phage display was used to display the inserted genes on the phage surface. Phage DNA was extracted and purified followed with restriction enzyme digestion, PCR and gene sequencing. MTT assay was used to evaluate the effect of pComb3-KGF active peptides on promoting epidermal cell proliferation. Immunofluorescence assay was employed to evaluate the cell affinity of pComb3-KGF active peptides. Quantitative real-time PCR analysis was employed to evaluate the expression of KGFR, humanβ-defensin 3, c-Fos and c-Jun in epidermal cells.Results The genes were obtained by RT-PCR, and were subcloned into pComb3 vector. The results of restriction enzyme digestion, PCR and gene sequencing proved that the genes had been inserted into the pComb3 vector. The proteins of the genes were expressed on the surface of the vector. MTT data showed that all of the four pComb3-KGF active peptides could promote epidermal cell proliferation. The results of immunofluorescence assay showed that they had good cell affinity. Moreover, the expression of KGFR in the KGF control group and two pComb3-KGF active peptide groups increased, and the expression of human β-defensin 3 in the KGF control group and three pComb3-KGF active peptide groups increased. The expression of c-Fos and c-Jun in the four pComb3-KGF active peptides groups was weaker than that in the KGF control group.Conclusions pComb3-KGF active peptides are successfully constructed and expressed, which can promote epidermal cell proliferation. They can promote the expression of human P-defensin 3 in epidermal cells to control the wound infection. The intensity of them promoting c-Fos and c-Jun expression was weaker than the wild-type KGF.Part three:The study of TGF-β1 phage model peptides' screening and inhibiting the activity of keloid fibroblastObjective The objective of the study was to isolate TGF-p1 phage model peptides from phage display 7/12-mer peptide library to evaluate their therapeutic effect on inhibiting the activity of keloid fibroblasts.Methods Phage display 7/12-mer peptide library were screened for four rounds using monoclonal anti-human TGF-β1 as the target to obtain specific phages containing ectogenous model peptides which is similar to TGF-β1.ELISA was performed to select monoclonal phages with good binding activity, which underwent DNA sequencing. MTT assay was used to evaluate the effect of phage model peptides on normal fibroblasts and keloid fibroblasts. Apoptosis of keloid fibroblasts induced by phage model peptides was detected using the annexin V-FITC/PI apoptosis detection kit, and cells were analyzed using a flow cytometer. Immunofluorescence assay was employed to show the binding affinity of the modeling peptides for TGF-β1 causing keloid fibroblasts. Quantitative real-time PCR analysis was carried out to detect the expression of NF-Kappa B, CTGF and TβRII in keloid fibroblasts. Results After the four rounds of screening, abundant of specific phage modeling peptides were harvested. Four phage modeling peptides were isolated from the phage display 7-mer peptide library. Ten phage modeling peptides were isolated from the phage display 12-mer peptide library. The isolated phage modeling peptides exhibited high binding activity by ELISA. MTT data showed that seven phage model peptides could inhibit keloid fibroblasts proliferation. The results of apoptosis assessment showed that the seven phage model peptides could slightly induce the apoptosis in keloid fibroblasts. The data of immunofluorescence assay revealed that the phage model peptides had good cell affinity. The findings of quantitative real-time PCR analysis suggested that the expression of NF-Kappa B and CTGF in the seven phage model peptides groups decreased, while the expression of TβRII slightly increased.Conclusions TGF-β1 phage model peptides can be isolated from the phage display 7/12-mer peptide library. Seven phage model peptides can inhibit keloid fibroblasts proliferation and induce the apoptosis in keloid fibroblast.