| Mastitis is the most common and influential disease in dairy cows,which can lead to a decline in milk yield and quality,and even the elimination of dairy cows,causing significant economic losses to the dairy industry.Exosomes(Exos)carry a large amount of important biological information such as proteins,RNA and lipids.Studies have shown that they have important functions in the body ’s immune function,growth and development,disease prevention and targeted drug delivery.However,the functions of miRNAs and proteins in milk exosomes of dairy cows and their changes in mastitis are still unclear.The purpose of this study was to explore the changes of protein and miRNA in milk exosomes of dairy cow mammary gland under different functional conditions such as health,recessive mastitis and clinical mastitis,and to screen out proteins and miRNAs related to inflammatory expression,so as to provide new ideas and scientific basis for the diagnosis and prevention of the disease.The specific results are as follows :1.The exosomes in the milk of healthy group,subclinical mastitis group and clinical mastitis group were isolated by ultracentrifugation,and verified by NTA(Nanoparticle Tracking Analysis,NTA),TEM(Transmission Electron Microscope,TEM)and WB(Western blot,WB).The results showed that the diameter of exosome particles in cow milk was 30-200 nm,and the shape was round or cup-shaped vesicle structure.The content of exosome protein in the milk of the subclinical mastitis group was the highest,followed by the clinical mastitis group,and the two groups were significantly higher than the healthy group(P < 0.05).From the perspective of particle concentration,the exosome particles in the recessive mastitis group were the most,followed by the clinical mastitis group,which were significantly higher than those in the healthy group(P < 0.05).Its performance trend was consistent with its protein concentration,but not consistent with the number of somatic cells.The detection results of exosome molecular marker proteins CD9,CD81 and TSG101 in each group were positive,indicating that exosomes were successfully isolated from milk under different functional states,which provided biological materials for subsequent experiments.2.A total of 104 728 799 original sequences were obtained by high-throughput sequencing.The length of miRNA was concentrated between 16-30 nt,with peaks at 22 nt and 30 nt,which was consistent with miRNA characteristics.Through comparative analysis,306 known miRNAs and 223 new miRNAs were identified.The predicted target genes of differentially expressed miRNAs were mainly enriched in inflammatory and immune pathways.In this study,bta-miR-378 / 378 c,miR-223-y and miR-200-z may be important factors related to mastitis.3.A total of 483 differentially expressed proteins were identified,of which 340 proteins were up-regulated and 184 were down-regulated.GO and KEGG functional enrichment analysis showed that these proteins were mainly involved in immune response,defense response,complement activation and coagulation cascade,cell transport and other metabolic pathways.Histones,fibrinogen,serine protease inhibitors and complement components screened from the protein interaction network were highly correlated with mastitis.In summary,this paper found that there were differences in the number,miRNA and protein of exosomes in the milk of dairy cows under different functional states.Among them,the expression of bta-miR-378 / 378 c,miR-223-y,miR-200-z,histone,fibrinogen and complement proteins were significantly different,mainly involving biological processes such as complement and coagulation cascade,Staphylococcus aureus infection,systemic lupus erythematosus and leukocyte transport,and metabolic pathways such as PI3K-AKt signaling pathway,chemokine signaling pathway and Toll-like receptor signaling pathway.The differentially expressed miRNAs and proteins screened in this study can be used as early biomarkers and provide new ideas and directions for the prevention and control of dairy cow mastitis. |
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