MiRNA Profiling Of Exosome Isolated From LPS-Challenged Dairy Cows Mammary Epithelial Cell

Dairy cow mastitis is a disease that is harmful,expensive,and difficult to control in dairy farms.For a long time,the disease has been one of the major diseases that have plagued the healthy development of the world dairy industry.Mammary epithelial cells are not only a natural barrier against the invasion of pathogenic microorganisms,but also initiate the body’s earliest immune recognition and immune response to pathogenic microorganisms,and play an important role in coordinating the follow-up immune molecules and immune cell responses.Exosomes are small vesicles secreted by cells.They are 40-120 nm membrane-encapsulated structures,which can specifically encapsulate some proteins,lipids or miRNA.They are biologically active and can be absorbed by recipient cells.Material transport and information transfer between cells.LPS stimulation of epithelial cells may affect the miRNA pattern in exosomes,which in turn affects the development of breast inflammation and milk quality.Therefore,this experiment intends to isolate and identify exosomes produced by LPS-stimulated mammary epithelial cells,and to screen for key miRNA that play a regulatory role in exosomes produced by epithelial cells during breast infection.In this experiment,the ultracentrifugation method was used to extract the mammary gland epithelial cells from normal cows and the exosomes derived from the mammary gland epithelial cells after 6 hours of LPS stimulation.The extracted exosomes were determined by protein concentration,transmission electron microscopy and Western blot.Techniques are identified and analyzed for high-throughput sequencing to screen for miRNA that may regulate breast inflammation.The results showed that the exosome particle diameter was between 40-120 nm under electron microscope,and the homogeneity was good,which accorded with the expected characteristics of exosomes.The results of Western blot showed that the exosome surface markers CD63,HSP70 and HSP90 were all positive.The exosome was obtained by successful extraction.The sequence obtained by sequencing was subjected to new miRNA prediction analysis,and a total of 400 new miRNA were predicted.The differential expression analysis showed that there were 44 differentially expressed miRNA,of which 23 were up-regulated and21 were down-regulated;GO and KEGG bioinformatics analysis of 43784 target genes corresponding to the expressed miRNAs showed that the differential miRNA selected were involved in biological processes such as protein binding,Th17 cell differentiation and regulation,metabolic processes,and cellular communication regulation,through high-throughput.In the sequencing results,miRNA that may affect the inflammation of the breast were screened,and the expressions of inflammation-related bta-miR-146 a,bta-miR-148 a,and bta-miR-224 were different.It is speculated that these miRNAs regulate the development of breast inflammation.In this study,normal and LPS-stimulated exosomes derived from mammary gland epithelial cells were isolated,and differential miRNAs in exosomes were screened to lay the foundation for further study on whether exosomes produced by mammary epithelial cells can regulate PMN function during breast infection.