Isolation Of Milk-derived Exosomes And Identification Of MiRNAs Associated With Mastitis

With the rapid development of society and economy,the people’s demand for milk is increasing.In the actual production process,bovine mastitis is a difficult problem to solve.Mastitis brings ten thousands of economic losses every year.A large number of studies have found that micro RNAs(mi RNAs)are a type of endogenous non-coding small RNA molecules that play an important role in many important biological processes.Exosomes are small vesicles containing a small number of biomolecules including DNA,mi RNA,proteins and lipids,which function by participating in intercellular communication.The previous studies have found that mi RNAs in breast tissue have a regulatory effect on milk quality traits.At the same time,some mi RNAs are closely related to the occurrence of bovine mastitis.However,there are few studies on milk-derived exosomal immune-related mi RNAs and their functions.This experiment selected mastitis and healthy cattles as the research objects based on the analysis of the DHI data of the lactating herd.The milk-derived exosomes in the two groups of milk samples were separated,and the morphological structure and particle size of the separated exosomes were analyzed.The differences in the expression levels of marker proteins and immune-related mi RNAs in milk-derived exosomes in each group were detected.At the same time,the two groups of milk-derived exosomes were co-cultured with bovine mammary epithelial cells and the expression of exosomal mi RNA on immune-related factors in bovine mammary epithelial cells were tested.The results of the study are as follows.1.This experiment analyzed the DHI data of the milk production performance of the lactating herd in the fourth quarter of 2019 on the lactating dairy farm,and found that the somatic cell number and somatic score of the mastitis dairy cattle population and the healthy dairy cattle population are significantly different(P<0.01),and there are significant differences in milk production,milk protein rate and lactose rate between the mastitis group and the normal healthy group(P<0.01).2.In this experiment,the optimized ultracentrifugation method was used to successfully separate milk-derived exosomes from mastitis cattles and healthy lactating cattles.According to the characterization and analysis of exosomes,the milk-derived exosomes isolated in this experiment are round and partly concave.The size of the exosomes separated in each group is between 50 and 150 nm,mainly distributed in the 100 nm range.The expression levels of TSG101 and CD81,which are the surface markers of exosomes,were detected.Both milk-derived exosomes from mastitis cattles and healthy milk-derived cattles were expressed in large quantities,and there was no difference between the groups,Successfully separated milk-derived exosomes.3.The expression levels of immune-related mi RNAs in the milk-derived exosomes of the mastitis group and the healthy group were detected.The results showed that compared with the healthy group,the expression of immune-related mi RNAs in the mastitis group had significant changes,and the expressions of mi R-142-5p,mi R-223,mi R-103,mi R-146 a and mi R-29 a were significantly increased(P<0.01),the expression of mi R-200 b was significantly lower(P<0.01).4.After co-cultivation of milk-derived exosomes from mastitis dairy cattle and bovine mammary epithelial cells,the expression levels of differential mi RNA and immune-related genes in mammary epithelial cells also changed.Compared with the blank control group,the expression levels of mi R-146 a and mi R-29 a in the cells of the milk-derived exosomes treatment group of mastitis dairy cows and the healthy milkderived exosomes treatment group increased significantly(P<0.01).The milk-derived exosome treatment group of mastitis cattle and the milk-derived exosomes of healthy cattle The m RNA expression levels of immune-related genes TLR2 and TLR4 genes in the cells of the treatment group,LPS treatment group and blank control group showed a significant decrease(P<0.01).In summary,this experiment is based on the DHI milk production performance analysis of the lactating herd.Mastitis and healthy cattle are selected as research objects.The milk-derived exosomes of the two groups of cattle are extracted and identified,and the milk-derived exosomes of the two groups of milk-derived exosomes are detected.The expression level of immune-related mi RNA,and then through two sets of milkderived exosomes co-cultured with bovine mammary epithelial cells,preliminary screening confirmed that mi R-146 a and mi R-29 a in bovine milk-derived exosomes can be used as molecules predicting bovine mastitis diagnostic signs are expected to provide the theoretical and technical basis for milk production and diagnosis of cattle mastitis.