The Study Of Construction Of KGF Phage Model Active Peptides And Prevention Of Radiotherapy-Induced Oral Mucositis

Oral cancer is a detrimental and malignant disease. After a longtime exploration, it is widely accepted that radiotherapy (RT) is one of the most effective treatments for oral cancer. However, radiotherapy-induced oral mucositis (RTOM) often occurs after RT, and to date, effective treatment for RTOM is still unavailable. This situation significantly impairs the living quality of the patients and inevitably disturbs the systemic therapy for oral cancer. To prevent RTOM, it is of particular importance to stimulate the proliferation of the oral mucosal cells and repair the damaged oral mucosa, which has been the priority in the research field of tumor supportive care.Keratinocyte growth factor (KGF) is a growth factor specifically expressed in epithelial cells and plays an important role in epithelial wound healing. KGF is highly expressed during the wound healing processes and induces a series of intracellular signaling cascades via interaction with its receptor on the epithelial cells. KGF stimulates the cell growth, promotes the epithelial cells to migrate from wound edge to the deeper matrix, and accelerates reepithelialization. Several studies have suggested that KGF can be used to treat RTOM. KGF could reduce apoptosis and DNA damage of the oral epithelial cells, and stimulate the proliferation and migration of these cells, which makes it a promising, novel therapy to protect oral mucosa and accelerate tissue regeneration. Indeed, it has been reported that local application of an RGF spray or an oral injection of RGF is capable of promoting the healing of oral mucositis through enhancing the proliferation and migration of the epithelial cells and increasing the biological activity of these cells.However, many studies confirmed that KGF is closely correlated with the development, progression and metastasis of tumors. Therefore, KGF cannot be used to treat oral mucositis in cancer patients because of its potential capability of promoting tumor development. Moreover, the clinical application of FGF is significantly limited by other problems such as a short half-time, poor stability, easy degradation and inactivation.Phage random peptide library (PRPL), encompassing a great range, has been widely used in imitating antigenic epitopes and protein engineering, especially in producing molecular polypeptide. It has been used to develop new medications of accelerating wound healing and receptor agonists of the growth factors. Using this technique, the researchers have successfully developed various mimic peptides for growth factors such as such as FGF, CTGF and VEGF. Compared with the natural protein, synthetic peptide has a smaller molecular weight, more specific biological effects, greater stability, lower immunogenicity, higher solubility, and easier to be largely manufactureed.In our previous studies, we obtained multiple pivotal gene segments which encode biologically active KGF fragments (see attachment for the detailed results). In this study, we selected the pivotal gene segments of KGF using the solid phase peptide synthesis (SPPS) technology, and then synthetized and optimized the obtained biologically active peptide of KGF. In this way, we provide a new medicine and a novel biomaterial for preventing and treating complications of the oral cancer patients who are receiving RT. As an auxiliary therapy for oral cancer, our results considerably improve the living quality of the patients and make it easier for the patients to receive standard treatment for oral cancer. Part I:The harvest of KGF phage model peptide and in vitro experimentObjective The objective of the study was to isolate KGF phage model peptides from a phage display7-mer peptide library to evaluate their effect on stimulating the proliferation of oral mucosal epithelial cells.Methods KGF monoclonal antibodies were used to select the biologically active KGF fragments from the phage library for four times. Phage titer was determined to evaluate the recovery of the phages. ELISA was performed to select monoclonal phages of the fourth round with good binding activity. DNA sequencing was used to evaluate the genetic similarities of model peptides. MTT assay was used to evaluate the effect of phage model peptides on the proliferation of oral mucosal epidermal cells. Immunofluorescence assay was employed to evaluate the cell affinity of phage model peptides. Quantitative RT-PCR analysis was employed to evaluate the expressions of KGFR, human β-defensin3, c-Fos and c-Jun in the epidermal cells.Results After the four rounds of screening, specific phage model peptides were harvested. Twenty-five phage modeling peptides were isolated from the phage display7-mer peptide library. Some of the isolated phage modeling peptides exhibited high binding activity as shown by ELISA. MTT assay showed that four phage modeling peptides could promote the proliferation of oral mucosal epidermal cells. Immunofluorescence assay displayed that the phage modeling peptides had a high cell affinity. The expression levels of keratinocyte growth factor receptor (KGFR) and human defensin β3were increased in the KGF group and the two phage model peptide groups when compared with the negative control group. The expression levels of c-Fos and c-Jun were increased in the KGF group when compared with the negative control group, while showed no difference between the phage model peptide groups and the negative control groups. Conclusions KGF phage model peptides can be isolated from the phage display7-mer peptide library, which can safely promote the proliferation of the oral mucosal epidermal cells. Two phage model peptides can promote the expression of human defensinP3in the epidermal cells to control the wound infection. Phage model peptides can not promote the expression of c-Fos and c-Jun. Part II:The construction of KGF phage model active peptide and in vitro experimentObjective Based on the sequence analysis and the in vitro experiments, the specific sequences encoding the biologically active KGF fragments were selected. pComb3-KGF active peptides were constructed and expressed to be used to evaluate the effect of these peptides on promoting the proliferation of oral mucosal epidermal cells.Methods The heavy chain of pComb3was selected to be the location to insert the exogenous gene. SpeⅠ and Xhol were chosen as the restriction endonuclease. Based on the sequence analysis and the in vitro experiments, four sequences were chosen. Primers were designed. The normal oral mucosal epidermal cells were gathered using adherent tissue culture method. The total RNA was extracted from the cells. The selected genes were obtained using RT-PCR method. The genes were subcloned into the pComb3vector after double-digested with Spel and Xhol. The technology of phage display was used to display the inserted genes on the phage surface. Phage DNA was extracted, purified, digested with restriction endonucleases, and evaluated with PCR and gene sequencing. MTT assay was used to evaluate the effect of pComb3-KGF active peptides on promoting the proliferation of oral mucosal epidermal cells. Immunofluorescence assay was employed to evaluate the cell affinity of pComb3-KGF active peptides. Quantitative RT-PCR analysis was employed to evaluate the expression of KGFR, human β-defensin3, c-Fos and c-Jun in epidermal cells.Results The genes were obtained by RT-PCR, and subcloned into pComb3vector. The results of restriction enzyme digestion, PCR and gene sequencing proved that the genes were inserted into the pComb3vector. The proteins of the genes were expressed on the surface of the vector. MTT assay showed that all of the four pComb3-KGF active peptides could promote the proliferation of oral mucosal epidermal cells. The results of immunofluorescence assay showed that these active peptides had high cell affinity. Moreover, the expression level of KGFR was significantly increased in the KGF group and two of the pComb3-KGF active peptide groups, and the expression level of human β-defensin3was elevated in the KGF group and three of the pComb3-KGF active peptide groups. The expression levels of c-Fos and c-Jun were significantly lower in the four pComb3-KGF active peptides groups when compared with those in the KGF group.Conclusions pComb3-KGF active peptides are successfully constructed and expressed, which can promote the proliferation of oral mucosal epidermal cells. These active peptides can also promote the expression of human defensin β3in oral mucosal epidermal cells to control the wound infection. However, these active peptides displayed a weaker effect on enhancing the expression levels of c-Fos and c-Jun when compared with the wild-type KGF. Part three:The effect of KGF phage model active peptide used in the treatment of radiotherapy-induced oral mucositisObjective To establish a rat model of radiation-induced oral mucositis, and to investigate the in vivo effects of KGF phage model active peptides on treating radiation-induced oral mucositis and promoting the healing of oral ulcers.Methods A Varian23EX linear accelerator was used for the irradiation process. The distance between the irradiation source and the skin was100cm, with a radiation area of10×10cm2and a rack angle of180d°. Twenty-fire male Wistar rats were used in this study,20of which received the irradiation and the other non-irradiated rats served as controls. Tongue and buccal mucosa of the20rats was irradiated for6minutes to receive a radiation dose of20Gy with a dose rate of40CGy/min. The general situation, body weight and the oral mucosa of the irradiated and the non-irradiated rats were examined on a daily basis. The animals were sacrificed on3,6and9days after the irradiation. Histological examination of the oral mucosa was performed to evaluate the optimal conditions to establish a rat RTOM model. Twenty Wistar rats were then used to establish the RTOM model using the aforementioned method. Fifteen of the RTOM rats were randomly divided into3groups to receive subcutaneous injections of the KGF phage active peptides at a dose of lmg/kg/d for5days on3(Group A).6(Group B), or9(Group C) days after the irradiation. The general situation, body weight and the oral mucosa of the rats were examined daily until the animals were sacrificed and subjected to histological analysis. The other five RTOM rats received only vehicle injections served as controls.Results One day after the irradiation, the rats showed no significant changes in food or water intake, which was decreased on2days after the irradiation. The body weight of the irradiated rats was decreased slowly, reached its lowest point on9～12days after the irradiation, and then showed a slow recovery. Erythema was observed in the tongue and buccal mucosa on3days after the irradiation. Histological examination showed vacuolar degeneration in the tongue squamous cells, and a small amount of infiltrated inflammatory cells were observed in the lamina propria. Ulcers were observed on the6th day after irradiation. Histological examination showed that the filiform papillae of the tongue were missing, the spinous cells were degenerated and exfoliated, and a small amount of inflammatory cells infiltrated into the lamina propria. The epithelial cells of the buccal mucosa also exfoliated.Larger ulcers were observed on the9th day after the irradiation. Histological examination showed that the filiform papillae of the tongue exfoliated, the basal cells and the squamous cells were degenerated, and a massive amount of inflammatory cells infiltrated into the lamina propria. The epithelium cells of the buccal mucosa exfoliated. The radiation-induced ulcer in the buccal mucosa could be self-repaired3weeks after a single20Gy irradiation.After subcutaneous injections of KGF phage active peptides, Group A showed no ulcers. Moreover, the ulcers of Group B showed no progression after the application of KGF active peptides, and the ulcers were healed3days after the KGF peptide injections. The ulcers of Group C progressed slightly after the irradiation, and the ulcers were healed6days after the KGF peptide injections. Histological analysis showed a normal structure of the tongue epithelium and a few infiltrated inflammatory cells in the lamina propria. In contrast, the ulcers of the control group were not healed until3weeks after the irradiation.Conclusion A rat model of radiation-induced oral mucositis (RTOM) was successfully established. Using this rat model, the in vivo effect of the KGF phage active peptides on the prevention and treatment of RTOM was investigated. The optimal conditions of applying the KGF phage active peptides were also evaluated. We found that the KGF active peptides can effectively reduce the severity of RTOM. promote epithelialization of the wound area, and accelerate the healing of the ulcers, indicating that KGF active peptides are a promising therapy for RTOM.