Extraction Mechanism Of Human Hair Keratin And The Effect On Cell Proliferation

Human hair keratin has been widely used in tissue repair,hemostasis,cell culture and other fields due to its good biological activity and biocompatibility.Human hair is extracted to obtain water-soluble human hair keratin,which is the basis of biomedical applications.However,the chemical reactions of different kinds of human hair keratin in the extraction process and the changes of human hair keratin in different extraction processes are not clear yet.Since there are 17 kinds of keratin in human hair,it is difficult to isolate a single keratin from the extract of human hair,which limits the research on the variation of keratin during the extraction process.The recombinant keratin has the characteristics of single component and high purity.It is an important way to analyze the extraction rule of human hair keratin by exploring the changes of different recombinant human hair keratin in the extraction process.Based on this,three kinds of human hair keratin(K31,K35,K38)and three kinds of human hair keratin(K81,K84,K86)were selected as research objects by analyzing the contents of various kinds of human hair keratin and the homology of human hair keratin in human hair extract.By simulating the reduction reaction with β-mercaptoethanol as reducing agent,the changes of different kinds of human hair keratin in reduction extraction were investigated.Recombinant human hair keratin was treated by the oxidation method and the enzyme method,and the effects of different extraction methods on its change reaction were analyzed.The cell proliferation activity of different kinds of recombinant human hair keratin after reduction treatment was further investigated,and the effect of different treatments on cell proliferation was explored.The main research contents and conclusions of this paper are as follows:(1)Recombinant human hair keratin with single component was obtained.Three kinds of human hair keratin(K31,K35,K38)and three kinds of human hair keratin(K81,K84,K86)were screened out by the content and homology differences of human hair keratin,and single component recombinant human hair keratin was obtained by prokaryotic expression.SDS-PAGE results showed that the molecular weight of recombinant keratin was consistent with that of literature,and the target band was single.FT-IR and CD studies showed that the chemical and secondary structures of recombinant human hair keratin were similar to those of extracted keratin.(2)The changes of different kinds of recombinant human hair keratin after reduction treatment were clarified.FT-IR and SDS-PAGE results showed that the chemical structure of keratin after reduction treatment was similar to that before reduction treatment,and its molecular weight was close to that of the corresponding recombinant human hair keratin.RK31 has the highest yield and RK35 has the lowest yield,which may depend on the hydrophilicity of the head and tail non-helical domains.Contact Angle measurement showed that the hydrophilicity of type I keratin was higher than that of type II keratin before reduction treatment.After the reduction treatment,the hydrophilicity of keratin increased,and there was no significant difference between type I and type II keratin.The contents of total sulfhydryl group and disulfide bond decreased after the reduction treatment,and the contents of type II were lower than that of type I.The CD study found that the reduction treatment could reduce the proportion of irregular crimp of type I keratin and increase the proportion of irregular crimp of type II keratin,but RK86 was an exception.(3)The changes of recombinant human hair keratin treated by different extraction methods were described.FT-IR showed that sulfonic acid group characteristic peaks appeared after oxidation treatment.Yield analysis showed that the reduction method was mild and the extraction rate was the highest.The yield of alkaline keratin was higher by the oxidation method.The enzymatic hydrolysis method has low yield and minimum molecular weight.The hydrophilicity of Contact Angle was increased to some extent and the enzymatic hydrolysis was the least.The contents of total sulfhydryl group and disulfide bond decreased significantly,and the reduction degree was greater than that of oxidation method and enzymatic hydrolysis method.The CD study found that all three methods could transform part of the α-helix into random coils.(4)The cell proliferation effects of different kinds of human hair keratin and different extraction methods were analyzed.The results of cell proliferation showed that different kinds of keratin promoted cell proliferation more than the blank control group.RK81 and RK84 had the best ability to promote cell proliferation before reduction treatment.There was no significant difference between type I keratin and type II keratin after reduction treatment.When the concentration of keratin was 25μg/m L,the increased proportion of type I keratin hydrophilic or "regular curl" and type II keratin "regular curl" may promote cell proliferation.When keratin concentration was 1mg/m L,RK35 was significantly better than RK31 and RK38,which might be related to the adhesion sequence.Among the three treatments of RK31,ERK31 had no effect on cell proliferation,while ORK31 had the best effect on cell proliferation.Among the three treatments of RK81,ERK81 had the best result,while RRK81 was relatively poor.q PCR results showed that RK81 and ORK81 significantly reduced the m RNA expression level of PI3 K,and significantly increased the expression level of Braf,MEK1,ERK2 and Cyclin D1,which may promote cell proliferation through MAPK signaling pathway.It may also be involved in the regulation of CDK4/6-Cyclin D elevation and the inhibition of PI3 K related pathways.In conclusion,based on the single component characteristics of recombinant human hair keratin,this paper explored the change and reaction rules of different kinds of recombinant human hair keratin in the reduction extraction process,and the influence of different extraction methods on the chemical changes of recombinant human hair keratin,and then studied the effect of different human hair keratin on its cell proliferation.The completion of this paper not only deepens the understanding of the extraction mechanism of human hair keratin,but also has a certain guiding role in the selection of extraction method of human hair keratin.