Study On The Promoting Effect Of Human Hair Keratin Hydrogel On The Repair Of Skin Radiation Combined Injury In Rats

Objective:With the wide application of nuclear technology and radiation in medicine and other fields,the incidence of skin radiation combined injury is increasing.The radiation complex injury is difficult to recover in clinical practice.Therefore,this thesis is to prepare human hair keratin hydrogel and study its promoting role in the repair of combined radiation injury of rat skin.Methods:1.Preparation and characterization of human hair keratin.Washing of uncolored human hair with sodium dodecyl sulfonate?SDS?,stirring and cleaning,degreasing and drying;reduction filtration with thioglycolic acid?TGA?,filtrate 6000 rounds per munite centrifugation,low-molecular-weight ultrafiltration membrane dialysis purification,freeze-drying,polyacrylamide gel electrophoresis analysis of human hair keratin amino acid molecular weight distribution;and the amino acid content of human hair keratin was determined by precolumn derivative chromatography of ninhydrin.2.Preparation and characterization of human hair keratin hydrogel.Human hair keratin was incubated with H₂O₂ to form cross-linked human hair keratin hydrogel.The morphology and structure of human hair keratin hydrogels were observed by scanning electron microscopy.FT-IR spectroscopy was used to analyze human hair keratin extract and human hair keratin hydrogel structure;Elastic modulus and viscosity modulus were measured by rotational rheometer.3.Cytology test.Human keratinocytes?HaCaT cells?were divided into control group,irradiation group and hydrogel treatment group.After incubation for 48 h under normal cell culture condition,irradiation group and hydrogel treatment group were irradiated 5 Gy gamma rays.Hydrogel treatment group were treated with human hair keratin hydrogels in the medium,and the activity of cells was measured by MTT test.4.Animal experiments.Eighteen male SD rats were randomly divided into simple skin injury control group,combined radiation injury group and hydrogels treatment group.The control group was treated with 1.0 cm×1.0 cm excision of the back skin.The rats in combined radiation injury group were irradiated with gamma rays 5 Gy,and were excised at 1.0 cm×1.0 cm in the irradiation site.The rats in the hydrogels group were irradiated with gamma rays 5 Gy.After excised at 1.0 cm×1.0 cm in the irradiation site,the wound was completely covered with human hair keratin hydrogel.Wound healing was observed and recorded at 1 d,7 d,14 d,21 d.Wound closure rate was calculated,and skin of wound edge was stained with H&E.Results:the molecular weight distribution of human hair keratin was concentrated at 45 kDa and 55 kDa,mainly containing 17 kinds of amino acids.The contents of Glu,Arg and Leu were the highest,accounting for19.54%±0.21%,10.46%±0.11%and 9.82%±0.19%respectively.The human hair keratin hydrogel has obvious interconnected porous structure under scanning electron microscope.Human hair keratin extract and hydrogel have several characteristic absorption peaks.Viscosity and elastic modulus increase with the raise of oscillation frequency.The viscous modulus in the same oscillation frequency was always higher than elastic modulus,which showed a good viscosity.The activity of HaCaT cells in the hydrogel group was significantly higher than that in the control group and the irradiation group,were 120.50%,100%and 65.33%respectively.All SD rats had the same wound size on day 1.The wounds of rats in the hydrogel treatment group were completely covered by the hydrogel.The wounds of rats in simple skin injury control group and compound skin injury control group were exposed.All of the wounds significantly narrowed at 7 d and 14 d.The wounds of rats in hydrogel treatment group were recovered obviously than others two groups.The wounds of rats in hydrogel treatment group were basically covered with new skin at 21 d.A small amount of the wounds of rats in simple skin injury control group were not covered by newborn skin.In the compound skin injury control group,the area of skin were not covered by newborn skin was the largest.Compared with the simple skin injury control group and the compound skin injury control group,the closure rate of the wounds of rats in the hydrogel treatment group was significantly higher,which were 98.60%,76.48%and60.17%at the 21st day,respectively.H&E staining showed that the wounds of rats in compound skin injury control group showed severe surface erosion on day 1,and only epidermal hyperplasia was observed from day 7 to day 14.The wounds of rats in hydrogels treatment group had formed a thin epidermis on day 14,and the wound healing process entered the remodeling stage on day 21 after treatment,and the epidermis was close to maturity.Conclusion:In this study,biomaterials of human hair keratin hydrogel with supporting function,viscosity,permeability and biocompatibility,which can improve the activity of HaCaT cells after irradiation and can promote the repair of complex skin radiation injury,are successfully prepared.