Mechanism Analysis Of Vimentin And Periodic Growth Of Hair Follicle In Inner Mongolia Cashmere Goat

Cashmere which is produced from Inner Mongolia cashmere goat has great economic value and also is an important animal product in China.But related studies on the genetic breeding of cashmere goats,it is not only lagging behind other species in theory,but also in the field of genetics and breeding.The development and apoptosis process of cashmere are closely related to the skin follicles and the microenvironment in the skin.Therefore,the study of the proteomics of the skin at different stages of growth cycle is of great significance for understanding how the functional proteins related to the hair follicle growth cycle affect the character,yield and quality of the cashmere.The executor of physiological function in the organism is protein,and proteomics is an indispensable new science used to study and reveal the structure and function of the proteome.It is the scientific basis for revealing the life phenomena such as the essence and rule of life.Therefore,the study on the periodic growth of hair follicle of inner Mongolian cashmere goat from the aspect of proteomics is an effective method to explore its mechanism.In this study,the skin sample of Inner Mongolia cashmere goats was selected as the research object,and the skin of the hair follicles in the period of telogen and anagen was tested by proteomics,and the differentially expressed protein during the growth process and the specific expression protein in anagen of the skin follicles of Inner Mongolia cashmere goats were screened.We also predicted the protein-protein interaction network between the anagen-specific proteins and the differentially expressed proteins,and selected the key proteins.Than the results were verified by Western blot(WB).Co-immunoprecipitation(CO-IP)technique was used to screen the proteins interacting with vimentin,and by analyzing their structures and functions,we could study the roles of them in the process of hair follicle growth.Finally the expression trend and location of some vimentin interacting proteins were studied by Western blot(WB)and immunohistochemistry.The main results are as follows:(1)2199 proteins were identified in the anagen of Inner Mongolia cashmere goat skin sample,1647 proteins in the period of telogen,and 2974 total proteins in two periods.The total protein of the two periods of telogen and anagen was analyzed by GO component enrichment.The result showed that those proteins enriched in 205 Biological Process,151 Cellular Componant and 82 Mollecular Function.Through the analysis of KEGG pathway,678 proteins from 2974 total proteins were covered in 72 pathways.(2)through the analysis of SWATH data obtained by liquid chromatography mass spectrometry,832 special expression proteins of anagen and 311 differentially expressed protein of telogen and anagen were screened,of which 116 were down-regulated in growth period,and 195 proteins were up regulated in growth period,and were consistent with the results of WB.(3)GO component enrichment analysis was performed on 832 proteins which were specifically expressed in the growth period,The result showed that those proteins enriched in 89 Biological Process,74 Cellular Component,and 40 Mollecular Function.Through KEGG pathway analysis,339 proteins of growth specific proteins were covered in 40 pathways.The enrichment analysis of GO component of differential proteins was found to be enriched in 70 Biological Process,45 Cellular Componant and 31 Mollecular Function.Through the KEGG pathway analysis,131 differential proteins covered 32 pathways.(4)Prediction of PPIs of anagen-specific proteins and differential proteins revealed that TOP2 B,RPS27A and ALB were the three key proteins.ALB not only interacts with structural proteins such as VIM,but also interacts with CTNNB1.Transmembrane prediction results suggest that there may be transmembrane phenomenon in ALB..(5)The protein obtained by the Co-immunoprecipitation(CO-IP)of vimentin in the skin of Inner Mongolia cashmere goat was identified,and three biological repetitions were repeated to 16,19 and 22 proteins,401,732 and 684 peptides,1790,5315 and 7919 spectrum(1% FDR conditions).The protein obtained by Co-IP was enriched by GO component enrichment and enriched in 41 Biological Process,27 Cellular Componant and 24 Mollecular Function.(6)Protein-protein interaction network(PPI)prediction map of vimentin and its interaction protein was constructed by using STRING and Cytoscape software.And PSSpred online website and SWISS-MODEL database was used to predict the secondary structure and three-dimensional structure of vimentin and its interaction protein,and it is found that the proportion of vimentin and its interaction protein in the coil(C)and helix(H)is much higher than the strand(E),so it should all belong to fibrous protein.(7)The difference in the expression of keratin 5,keratin 10 and keratin 71 in the stages of telogen and anagen of Inner Mongolia cashmere goats was detected by Western blot technique.The expression of keratin 5,keratin 10 and keratin 71 in anagen was higher than in telogen,which was consistent with the expression trend of vimentin,indicating that there was a positive correlation between vimentin and keratin 5,keratin 10 and keratin 71.(8)The immunohistochemical results showed that keratin 5 and keratin 10 were expressed in the outer root sheath of the hair follicle.In the primary follicle,the expression of keratin 5 was the outer root sheath which was separated from the dermal papilla until the coarse hair extended to the outside of the skin;KRT0 was expressed only in the outer root sheath near the epidermis.In the secondary hair follicle,the expression of keratin 5 was the outer root sheath which was from the middle of the hair follicle until the cashmere extended to the outside of the skin;and the expression of KRT10 was the outer root sheath which was from the upper part of the hair follicle until the cashmere extended to the outside of the skin.