| In the wool industry,poultry market and hairdressing industry,millions of tons of biomaterials rich in keratin can be produced every year.In order to avoid waste of keratin biomaterial resources,chemical methods are usually used to extract keratin from natural biological materials such as human hair,feathers and wool for recycling.Human hair keratin has no heterogeneity and rejection reaction because it is derived from humans,and the human hair keratin extracted by reduction method has good water solubility,and has functions of hemostasis and wound healing.Therefore,the further study on the extraction method of human hair keratin and the biological function of keratin has important significance and value.The complexity of the extracted components of human hair keratin makes it difficult to study the reaction mechanism during the extraction of keratin.Except for the disulfide bond breaking during the extraction process,little is known about the chemical changes of keratin,which hinders the development of keratin extraction technology.In view of this,this topic proposes for the first time to obtain a single human hair recombinant keratin by DNA recombination technology,and to reduce the recombinant keratin in accordance with the reduction extraction method.By comparing the physical and chemical properties of the recombinant keratin before and after reduction,clarify the keratin extraction process Chemical changes,preliminary exploration of the reduction and extraction mechanism of human hair keratin.In addition,this topic also compared and evaluated the degradability,biocompatibility and tissue repair effect of keratin extract and recombinant keratin before and after reduction.The main research contents and conclusions are as follows:1.Based on the classification and sequence conservation of human hair keratin,this subject selected a type I keratin 37 and a type II keratin 81 for expression to obtain recombinant keratin 37(RK37)and Recombinant Keratin 81(RK81),Which was reduced according to the reduction extraction method to obtain water-soluble recombinant keratin(SRK37)and Soluble Recombinant Keratin 81(SRK81),and obtained the keratin extract from human hair by the reduction extraction method.The physical and chemical detection and analysis results of the above five kinds of keratin are as follows:(1)The measurement results of contact angle showed that the contact angles of recombinant keratin RK37 and RK81 were reduced from 38.2°and 42.9°to24.6°and 30.6°respectively,and the hydrophilicity was significantly improved;(2)The disulfide bond and thiol measurement results showed that after reduction,the disulfide bond content of RK37 and RK81 decreased from 3.30 and 1.09 mol/10~5 g to 1.23 and0.66 mol/10~5 g,respectively,and the thiol content increased from 0.04 and 0.02 mg/m L to 0.09 and 0.04 mg/m L.It indicates that the disulfide bond of keratin is broken by about 50%during the reduction process;(3)FT-IR results show that the chemical structure of keratin before and after reduction has not changed significantly;(4)TGA results show that the thermal stability of SRK37 and SRK81 is reduced;(5)The results of SDS-PAGE and MALDI-TOF-MS indicate that the molecular weight of RK37 and RK81 decreases after reduction;(6)Amino acid analysis results showed that the total amino acid content of recombinant keratin decreased after reduction,from 93.3 and 91.9g/100.0 g of RK37 and RK81 to 54.1 and 50.0 g/100.0 g,respectively.Among them,the reduction of amino acid content such as cystine and arginine indicates that in addition to the disulfide bond breakage,an amino acid reaction occurred during the extraction of keratin,resulting in a decrease in the molecular weight of recombinant keratin;(7)CD spectrum shows that the secondary structure of recombinant keratin is mainly composed ofα-helix;Be St Sel analysis shows that the content ofα-helix after the reduction of recombinant keratin increases,and the content of random coil decreases,indicating that the amino acid reaction mainly occurs in the random coil region.2.The degradability and biocompatibility of five kinds of keratin were evaluated.The experimental results show that all five keratin materials can be degraded.Each keratin is non-toxic to cells,can promote cell proliferation,and has good cell compatibility.There is no damage to animal organs at the implantation site of keratin materials,and no abnormal expression of pro-inflammatory factors in animals.The keratin has good biocompatibility.3.In order to meet the clinical requirements for the moisture retention,breathability and safety of repair materials,the keratin composite carboxymethyl cellulose(CMC)hydrogel material was prepared by freeze-thaw method,and the physical and chemical properties of the hydrogel material Analyzed.The tissue repair effect of each hydrogel material was evaluated by the SD rat skin full-cut wound model.The results show that each hydrogel material has a uniform microporous structure,and the SRK37/CMC,SRK81/CMC and Keratin extract/CMC groups have higher porosity,swelling and water holding capacity.Haematoxylin Eosin(HE)staining and Masson’s Trichrome(MTC)staining results show that the water-soluble keratin composite hydrogel can promote angiogenesis,epidermal formation and collagen synthesis at the wound site faster.In addition,the results of tissue repair of water-soluble keratin(SRK37,SRK81 and Keratin extract)and CMC in different ratios(1:3,1:5,1:7)were shown to be complex.Increased sex keratin content accelerates wound healing.In summary,in addition to the disulfide bond breakage during the keratin reduction and extraction process,an amino acid reaction also occurs in the random coil region of the protein structure,which reduces the molecular weight of keratin.We also found that after reduction,the recombinant keratin has improved water solubility and hydrophilicity,and reduced thermal stability.During the reduction and extraction process,the disulfide bond of keratin is broken by about 50%.The above findings have laid a theoretical foundation for the in-depth discussion of the keratin extraction mechanism.In addition,compared with recombinant keratin and keratin extracts,the highly purified water-soluble recombinant keratin after the reduction shows stronger wound healing ability in the body,and also has good biocompatibility,which can be used for new tissue repair Provide reference for material design and development. |
