Screening And Identification Of Differential Proteins In Exosomes Of Bovine Mammary Epithelial Cells Treated With LPS

Mastitis is one of three major diseases of bovines recognized in the world and the economic loss to bovine breeding industry has been ranked first among all kinds of diseases for a long time.Escherichia coli is one of the most common pathogens causing mastitis.Lipopolysaccharide(LPS)is the main pathogenic factor,which can induce a variety of cell inflammation,apoptosis and autophagy.Mammary epithelial cells are the main functional cells of bovine mammary tissues,which can resist the infection of exogenous pathogens by secreting various immune regulatory factors.Exosomes are small vesicles that cells actively secrete extracellularly,which can carry proteins,lipids or nucleic acids.They play important functions in many physiological and pathological processes such as immune surveillance,inflammation,and cancer development.Among them,protein has certain tissue specificity as the bearer of life functions.Differentially expressed proteins are not only the key to distinguishing exosomes from different sources,but also an important way to study the origin of exosomes.Therefore,the purpose of this experiment is to screen the differentially expressed proteins in the exosomes of bovine mammary epithelial cells after LPS treatment by proteomics technology.To predict the potential functions of proteins in the exosomes of bovine mammary epithelial cells.It will lay an experimental foundation for further study on the regulatory role of exosome proteins in bovine mastitis.Bovine mammary epithelial cells were cultured in vitro and stimulated with 20 μg/m L LPS.The supernatant of cell culture was collected and exosomes were extracted by ultracentrifugation.The exosome markers CD9 and CD63 were detected by Western Blot.The exosomes were identified by transmission electron microscopy,protein concentration determination.The control and LPS stimulated histone samples were analyzed by unlabeled quantitative proteomics with three replicates in each group.Then,maxquant database was used for mass spectrometry data analysis.The differentially expressed proteins were screened and which were analyzed by functional clustering,GO and KEGG pathways.The test results are as follows:(1)Extraction and identification of exosomes: The protein concentration of the control group and LPS stimulation group were 0.578 μg/μL and 0.588 μg/μL.Under electron microscope,the size of the sample was similar to that of vesicle disk,with the diameter of 40-120 nm.It is in line with the expected characteristics of exosomes.Western Blot results showed that both CD63 and CD9 were positive.In conclusion,the exosomes of bovine mammary epithelial cells were successfully extracted.(2)Proteomics analysis: A total of 20 significantly different proteins were screened out of the control and LPS stimulated two sets of samples.15 of these proteins were up-regulated.These proteins include calcium homeostasis endoplasmic reticulum protein,inhibin-2,and peptidylproline Acyl cis-trans isomerase,DNA topoisomerase 2,histone H4,annexin,actinrelated protein 2/3 complex subunit 3,tumor susceptibility gene 101 protein,protein tyrosine kinase 7,transmembrane 4 L six family member 1,tubulin β-3 chain,RAS related,testin,myelin protein zero-like protein 1,G protein A inhibitory activity peptide 3.5 of these proteins were down-regulated,including 12 members of the solute carrier family 2,low molecular weight cytosolic acid phosphatase,fibrinogen beta chain,UDP-glucose 6-dehydrogenase,apolipoprotein C-II.GO and KEGG pathway enrichment analysis of the differentially expressed proteins that have been screened.The results show that these differences proteins mainly perform functions such as protein and RNA binding outside the cell,participate in processes such as single organisms and cellular protein metabolism,and involve multiple pathways such as the Rap1 signaling pathway,endocytosis,and regulation of the actin cytoskeleton.Screening significantly upregulated TSG101 and significantly down-regulated UGDH for verification,and found to be consistent with the proteomics identification results.In conclusion,the exosomes was isolated and extracted from bovine epithelial cells;The expression spectrum of exocrine protein was defined.The total proteins quantity of control group and LPS stimulation group was 1 520.A total of 20 significantly different proteins were screened,including 15 up-regulated proteins and 5 down-regulated proteins.Which were mainly involved in the process of Rap1 signaling pathway,endocytosis and actin cytoskeleton regulation.It is confirmed that after LPS treatment of bovine,the protein concentration and protein types of the exoderm increased.Which provided a basis for further study on the immune regulation function of the exocrine of the mammary epithelium.