Isolation,identification And Proteomic Analysis Of Milk-derived Exosomes Which From Different Animals

Act as a carrier,exosomes has lots of important physiological function.Milk is rich in exosomes,however,differences in the morphological,kinds of protein and functions in milk exosomes existed among different sources of milk,especially from different species of animals.The aim of this study was to isolate and identify the milk-derived exosomes from different species of animals and further to perform the proteomic detection to analysis the protein function in milk-derived exosomes.The details are as follows:Experiment 1: Isolate of milk-derived exosomes in holstein cow,yak,and camel milk through differential ultracentrifugation,and then using transmission electron microscopy(TEM),protein quantification,western blot(WB)and nanoparticle tracking analysis(NTA)to identify the milk-derived exosomes from holstein cow,yak and camel milk.Cytotoxicity tests were performed to determine the toxicity of the milk-derived exosomes from holstein cow,yak and camel milk.(1)The results of this research indicated that morphological of milk-derived exosomes from holstein cow,yak,and camel milk were similar to the other biological samples;(2)Protein quantification There was no significant difference in exosome protein concentration between Holstein cows and yak milk(P = 0.82),while there was a significant difference in the concentration of exosome protein between Holstein cows and camel milk(P < 0.05),yak milk There was a significant difference between the protein concentration of Chinese and foreign urinary protein and that of camel milk(P < 0.05);(3)the marker proteins of exosomes such as ALIX,CD63,CD81,TSG101 and HSP70 were identified in the milk-derived exosomes from holstein cow,yak,and camel milk;(4)the peaks detected through NTA of milk-derived exosomes from holstein cow,yak,and camel milk are relatively simple,which are all within 150 nm,however,the size of the exosomes of camel milk is larger than holstein cow and yak milk;(5)and the cytotoxicity tests results of the milk-derived exosomes from holstein cow,yak,and camel milk on the small intestinal epithelial cells of rat proved that the activity of the cells added with milk-derived exosomes from holstein cow,yak,and camel milk was much higher than the ones in control group,thus,milk-derived exosomes isolated from holstein cow,yak,and camel milk through differential ultracentrifugation could be used for further research.Experiment 2: The aim of this study was to indentify different proteins of milk-derived exosomes from holstein cow,yak and camel milk using labele-free proteomic quantification and bioinformatics analysis.(1)A total of 1,746 proteins were identified(holstein cow 759,yak 980,camel 1284).(2)Furthermore,GO analysis was performed to analysis the cellular component,molecular function and biological process of milk-derived exosomes from holstein cow,yak and camel milk,and the results showed that milk-derived exosomes are mainly involved in ATP synthesis and toll-like receptor signaling pathways.(3)Quantitative results showed that there were 373 different proteins found in milk-derived exosomes from holstein cow,yak and camel milk,including 22 differential proteins are up-regulated in holstein cow milk,115 in yak milk and 236 in camel milk.These differential proteins were involved in the pathways of angiogenesis,viral bacterial infections and cancer,however,the up-regulated proteins of milk-derived exosomes from camel milk mostly were related to the role of anti-inflammatory and anti-cancer.All the results above could provide a reference for further study on milk-derived exosomes.Experiment 3: The aim of this study was to preliminary explore the advantages and disadvantages between two different methodologies used for extracting exosomes from jersey milk.Exosomes was firstly obtained through differential ultracentrifugation and kit extraction,and then was compared by transmission electron microscopy(TEM),protein quantitative,western blot(WB)and nanoparticle tracking analysis(NTA).Cytotoxicity test was performed for exosomes got from kit extraction.(1)The results of TEM showed that the two methods had typical saucer-like structure vesicles,but under the TEM field of vision,exosomes was more by kit extraction than differential ultracentrifugation;(2)the concentration of exosomes protein extracted by the kit was significantly higher than that of differential ultracentrifugation(P < 0.05);(3)the exosomes marker protein ALIX and CD81 expressed by two methods,but the abundance of CD81 extracted by differential ultracentrifugation was particularly low,TSG101 was not identified,but also identified by kit extraction,(4)the particle size distribution of the exosomes extracted by the two methods was in line with 30-150 nm.(5)Further cytotoxicity test proved that the exosomes extracted through kits has no disadvantage on the proliferation of cell,which could be used in further research,especially for cell culture.Kit extraction is a suitable strategy for exosomes extracted from jersey milk,which is better than the differential centrifugations method in this study.