| BackgroundChronic wound is a significant public health problem worldwide and imposes a heavy burden on individual patients and global society as a whole.With the acceleration of population aging,diabetes mellitus is currently the major cause of chronic wound.Wound healing is a dynamic and complex process involving a variety of cells,extracellular matrix and growth factors.Growth factors play crucial roles in initiating and fascinating the different stages of wound healing.However,in diabetic individuals,the type and amount of growth factors are defected and the signal between the factors and cells is also disrupted.In light of this,administration of exogenous growth factors may be a new scheme to accelerate diabetic tissue repair.Transforming growth factor β1(TGF-β1)and Keratinocyte growth factor(KGF)are closely implicated in the process of wound healing.Though many efforts have been made to develop growth factors as therapeutic agents,to date,their characteristics such as large molecular weight,complex structure,complicated synthesis process,functional diversification,poor stability,short biological half-life and strong immunogenicity have always been obstacles in further application.At present,the research and application of peptides are boosting.Peptides have many advantages over biomolecules and protein drugs due to their specificity,increased diffusion and tissue penetration,rapid blood clearance,chemical stability,and ease of synthesis in large scale and low cost.Hence,the preparation of TGF-β1 and KGF active peptides with biological activities of natural growth factor of TGF-β1 and KGF,will open up a prospective way for the treatment of diabetic wounds.Objective1.To isolate key sequences of TGF-β1 from the phage display 12-mer peptide library,to synthesize the bioactive peptides,and to evaluate their biological effects on fibroblasts in vitro and to evaluate their biological effects on cutaneous wound healing in streptozotocin induced diabetic rats.2.To obtain KGF phage model peptides from the phage display 7-mer peptide library,to detect their biological effects on epidermal cells in vitro and to evaluate the in-vivo effect on promoting diabetic wound healing.MethodPart Ⅰ:Using monoclonal anti-human TGF-β1 antibody as the target,a phage display 12-mer peptide library was screened to isolate target sequences.The key sequences of TGF-β1 were chosen through alignment analysis.Based on these sequences,bioactive peptides were produced.After being purified,structurally modified and fluorescence labeled,the peptides were added to the cultured fibroblasts,The effects of the biosynthesized peptides on fibroblasts were evaluated.Part Ⅱ:Full-thickness excisional wounds were made on the dorsums of 50 rats which were then randomly divided into five groups:negative control group(normal saline(NS)),two TGF-β1 control groups which were respectively treated with low-dose TGF-β1(5ng/ml)and high-dose TGF-β1(50ng/ml),and two model-peptide-treated groups which were respectively treated with low-dose model peptide(5 ng/ml,)and high-dose model peptide(50 ng/ml).At day 14 post-injury,rats were euthanised and wounds were assessed by gross,histopathology,immunohistochemistry,immunofluorescence and quantifcational real-time polymerase chain reaction.Part Ⅲ:Using monoclonal anti-human keratinocyte growth factor(KGF)antibody as the target in this study,we screened the phage display 7-mer peptide library to isolate key sequences of KGF.Through alignment analysis,the key sequences of KGF were selected and used to synthesize bio-active peptides.After purification,structural modification and fluorescence label,the phage model peptides of KGF were added to epidermal cells.CCK-8 assay,quantitative real-time PCR analysis and western blot analysis were employed to evaluate the effect of the phage model peptides on epidermal cells.Part Ⅳ:Full-thickness excisional dorsal wounds were created in the diabetic rats,after which the rats were randomly divided into five groups:negative control group[normal saline(NS)],two KGF control groups which were respectively treated with low-dose KGF(5 ng/mL,K-LD)and high-dose KGF(50 ng/mL,K-HD),and two KGF model-peptide-treated groups which were respectively treated with low-dose model peptide(5 ng/mL,M-LD)and high-dose model peptide(50 ng/mL,M-HD).On day 14 post-injury,wound closure was followed by digital planimetry and wound tissues were harvested for histological assay and real-time polymerase chain reaction.ResultPart Ⅰ:Ten specific sequences similar to that of TGF-β1 were isolated from a phage display 12-mer peptide library.Seven key sequences were chosen to generate bioactive peptides of TGF-β1.Only one model peptide could bind to fibroblast and promote its proliferation.The result of quantitative real-time PCR analysis and western blot analysis indicated the model peptide could promote both the mRNA expression and protein expression of TGF-β RⅡ in fibroblasts.Part Ⅱ:A significant increase in rate of wound closure was observed in TGF-β1 model peptide groups in comparison to negative control group.The results of histopathological staining revealed that re-epithelization and collagen deposition in model peptide-treated groups were significantly higher than those in negative control group.The results of immunohistochemistry and immunofluorescence tests showed that Ki67-positive,VEGFA-positive,CDS 1-positive,a-SMA-positive,CD206-positive cells in model peptide and TGF-β1 control groups were more than those in negative control group.Furthermore,comparing with the mRNA expression profile in negative control groups,mRNA expression profile in model peptide group showed a decrease in pro-inflammatory cytokines and an increase in anti-inflammatory cytokines and collagen.Part Ⅲ:Among the 26 sequences isolated from a phage display 7-mer peptide library,only one sequence was chosen to generate bioactive peptide of KGF.The synthesized peptide could bind to epidermal cells,facilitate cellular proliferation,and promote both the mRNA expression and protein expression of KGF receptor.Part Ⅳ:Wounds treated with KGF model peptide closed markedly faster than negative control wounds and were comparable to KGF treated wounds.Histology and immunohistology results demonstrated significantly higher levels of re-epithelization,granulation tissue formation and vascularization in the model peptide groups.Furthermore,real-time polymerase chain reaction expression of KGFR,collagen I and transforming growth factor-β1 in model peptide groups,were also generally higher than that in negative control group.ConclusionIt is an efficient way to harvest phage model peptides from phage display peptide library.Phage-displayed TGF-β1 peptide can bind to the receptor of fibroblasts,play a positive biological role in vitro and facilitates diabetic wound healing through accelerating re-epithelialization,enhancing collagen deposition,promoting neo-vascularization,and inhibiting inflammatory response in vivo.Phage-displayed KGF model peptide can bind to epidermal cells,facilitate cellular proliferation in vitro and promote wound healing through accelerating re-epithelialization,enhancing dermal regeneration,and inducing angiogenesis.TGF-β1 and KGF phage model peptides may be of therapeutic interest in diabetic wound healing process and lead to a new opportunity for promoting diabetic wound healing. |
PDF Full Text Request