| In recent years,the research on the promotion of wound healing by medical biomaterials has received increasing attention from clinical medicine.As a natural biological material,human hair keratin has good biocompatibility,biodegradability and certain wound repairing effects.It has been reported in the literature and preliminary clinical verification.How keratin biomaterials regulate the rapid healing of wounds is a new demand and goal for clinical precision treatment.However,human hair extraction keratin is a complex proteins that composed of many different molecular weight.Different molecular weight proteins have different biological functions on dermal wound healing.How does the molecular weight affect the biological activity and function of the protein has not been studied.Therefore,based on the molecular weight and dermal wound repair,this article studied the influence of molecular weight on the dermal wound healing effect of human hair keratin,and explains the regulation and synergistic effect of different molecular weight human hair keratin in the whole process.From the view of molecular weight,the role of human hair keratin in the healing process of skin wounds was analyzed,and the active fragments promoting skin healing were screened out,which provide new methods and ideas for the development of new protein wound repair materials.The main contents and results of present study are follows:A low molecular weight keratin(5-30 kDa),a medium molecular weight keratin(30-50 kDa)and a high molecular weight keratin(more than 50 kDa)were prepared by ultrafiltration separation.Three kinds of keratin hydrogels with molecular weight range were prepared,and their physicochemical properties were studied.The results showed that the glycine content in LMWK was the highest,and the elastic modulus(G')of each molecular weight hydrogel group increased with the molecular weight.However,the low molecular weight group with the lowest mechanical strength has the fastest degradation rate in vivo and in vitro,and it can be completely degraded within 2 weeks.The application of HaCaT cell scratch model showed that human hair keratin had an increasing effect on the proliferation and migration of HaCaT cells.Compared with the HMWK group,LMWK had the most significant effect.In the evaluation of the wound tissue of the SD rat whole skin slice model,the LMWK and MMWK groups had a 78.78% reduction in wounds after 7 days of treatment compared with the HMWK group,showing a stronger wound healing effect.After 28 days of treatment,the wound treated by LMWK and MMWK returned to normal.In contrast,the wound healing process of the blank control group and the HMWK group were still in the repair stage,forming an incomplete epidermis.The above studies indicate that the physicochemical properties of human hair keratin,the ability to promote cell proliferation,migration,and the ability to promote wound healing are affected by molecular weight and promote wound healing by improving epithelialization,angiogenesis,collagen deposition,and remodeling.The application of proteomics technology to study the effects of keratin in different molecular weight ranges on the expression of endogenous proteins during skin damage repair.Compared with the blank control group,415 proteins were differentially expressed on the 1st day after LMWK keratin repair and 101 proteins were differentially expressed after 7 days.After the 1st day of HMWK keratin repair,298 proteins were differentially expressed;after 7 days of repair,267 proteins were differentially expressed.Bioinformatics analysis of all differential proteins,functional enrichment of GO and KEGG pathways showed that LMWK regulates cell signaling during wound healing by regulating the intermediate filament protein of the cytoskeleton on the first day after wound repair.Migration,and on the 7th day after repair,HMWK began to regulate leukocyte activation and cell activation,significantly increasing cell growth and ROS clearance.The WB technique was used to verify the most obvious activity-repairing markers of protein manganese superoxide dismutase(Sod2),aminopeptidase N(Anpep),allograft inflammatory factor-1(Aif1),vimentin(Vim).The above studies showed that the LMWK group had initiated the expression of active repair markers on day 1 and showed a stronger ability to regulate wound healing to promote wound healing.To investigate the effect of recombinant human hair keratin with different molecular weights on the healing effects of wounds.Application of genetic engineering technology,expression of type I keratin K37 and type II keratin K81.The characteristic peaks of UV,FT-IR and CD are consistent with the data of human hair extract data,showing good biocompatibility and biodegradation.The study found that after 7 days of treatment,the wound size of RK37 and RK81 decreased to 49.66% and 56.4%,respectively.RK81 with better wound healing effect,and intercept all ?-helices according to its secondary structure to construct low molecular weight recombinant keratin(RK81-1A?,RK81-1B? and RK81-2?),UV and FT-IR characteristic peak of low molecular weight proteins are consistent with the full sequence recombinant protein data,and the CD shows that its secondary structure consists of ?-helical structure.RK81-1A? is a soluble protein,and the others are in the form of inclusion bodies and are partially soluble.On the 7th day,the wound healing rates of RK81-1A?,RK81-1B?and RK81-2? were 82.77%,59.33% and 61.63%,respectively,which could initiate the expression of active repair markers.The above studies showed that the pro-healing ability of the protein fragment RK81-1A? with wound repair activity was most significant compared with other proteins(P<0.001).In order to improve the solubility of low molecular weight recombinant protein,RK81-1B? and RK81-2? were obtained after gene sequence modification by using site-directed mutagenesis technology.And named them as SRK81-1B? and SRK81-2?,keeping their structure similar but hydrophilicity improved.RK81-1A? was not modified due to the absence of Cys.Bioinformatics analysis of the modified SRK81-1B? and SRK81-2? main structure is also ?-helix,but its solubility and ability to promote wound healing have been enhanced.The study found that after 7 days of treatment,the wound healing rates were 72.97% and 77.83%,respectively.RK81-1A?exhibits better wound repair activity compared to the two low molecular weight recombinant proteins after modification.The above research shows that solubility can be improved by mutating the Cys of keratin and affecting the wound healing properties of the protein.In the present study,ultrafiltration technology was used to separate human hair keratin with different molecular weights.The effects of different molecular weight on the proliferation and migration of HaCaT cells and the healing of wounds in SD rats were investigated.The regulation pathway of human hair keratin with different molecular weights throughout the process were analyzed based on proteomics techniques.Selected the best effect of keratin in the molecular weight range of 5-30 kDa,obtaining a single human recombinant keratin,molecular weight interception and site-directed mutagenesis according to its sequence structure,screening the better water-soluble and active fragments,for the development of clinical wounds.The new keratin material needed for repair provide the research basis and transformation ideas. |
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