Identification And Proteomic Analysis Of Exosomes Released By White Blood Cell Of Lamprey

Lamprey,as an important link of evolution between invertebrate and vertebrate,has the oldest adaptive immune defense mechanism.Through the study of lamprey immune mechanism to reveal the origin and evolution of adaptive immunity has an important value.Exosomes are 40~130 nm vesicle-like structures,which contain a large number of proteins,nucleic acids,lipids and other components.It plays an important role in the process of intercellular communication,immune response and tumorigenesis.Now,the related research of exosomes is mainly concentrated in higher vertebrates,such as human and mouse.However,there is little report on exosomes in the lamprey.Due to the highly conservative in morphology and sretory process of exosomes,we take the Lampetra japonica as the experimental material.Based on the methods for purification and identification of the mammalian cells-derived exosomes,we optimized the experimental methods,isolated and purified the exosomes derived from white blood cell of lamprey,and the morphology of exosomes was further characterized.On the other hand,because the molecular markers of exosomes in lamprey is still unknown,we have cloned the homologous genes in lamprey of the exosomes marker Alix and the endoplasmic reticulum marker GRP78 respectively.Furthermore,Western Blot was used to detect a number of marker molecules including L-Alix in the exosomes released by white blood cell,which proved that the purified product was exosomes.Finally,through the high-throughput analysis of the protein components of the exosomes derived from white blood cell,it was found that the exosomal proteins can be divided into two types of proteins.The first type is marker molecules in exosomes,such as CD81,Annexin A7,Integrin beta,Alix etc.,another type is importent immune regulatory protein,such as TGF-?,CD45.We then cloned the TGF-? homologous genes in lamprey,and analyzed the domain composition the physical and chemical properties of the protein by bioinformatic tools.The regulation function of cytokine TGF-? carried by exosomes in adaptive immune system of lamprey is worthy of further research.In summary,we have successfully established purification and identification methods of white blood cell-derived exosomes in lamprey.Through the characterization of diameter and protein contents,we found that the morphological and molecular markers of exosomes are relatively conservative between lamprey and other species,and there are several importent immune regulatory proteins in exosomes.Our results will lay the foundation for clarifying the important function of exosomes in the immune cell communication of the lamprey.Futher study is of great significance for revealing the regulation mechanism of the adaptive immune system of the lamprey.