Isolation And Identification, Immune Activity And Anti-virus Effection Of Biliary Exosomes Of Chicken

Exosomes is protein vesicles with biologic activity derived from living cells late endosomes. Exosomes can be isolated from a variety of culture supernatants in vitro and body fluids. Characteristics of exosomes from different sources of cells vary wildly from contained ingredients. However, in generally, exosomes from different types of cells are common in size (20to170nm), buoyant density(1.12-1.19g/mL), and contain the following substances: major histocompatibility complex class I and/or class II molecules, heat shock protein, integrin, four-transmembrane proteins, cytoskeletal proteins, as well as some biological enzymes.In recent years, domestic and foreign researchers concentrated on different cells and body fluids in mice or human fluid, while in poultry it’s still blank. In2010, Masyuk A I successfully extract exosomes in mice bile. We speculated that chicken bile also contains exosomes, and which are important for local immune homeostasis.Exosomes were isolated from bile of specific pathogen free (SPF) chickens at age of42-43days by ultracentrifugations and filtration. Sucrose gradient fractionation showed the density is between1.13and1.19g/ml. Electron microscopy observation showed that exosomes with the diameter of30-150nm, were present in the space of Disse and bile canaliculus of liver. Chicken biliary exosomes displayed typical saucer-shaped, rounded vesicles morphology. Proteomic analysis by Liquid Chromatography-Mass Spectrum (LC-MS) revealed that chicken biliary exosome contained196proteins, including exosomal markers and several unique proteins, such as HSP70, C-reactive protein, CCCH type antiviral protein, mucin and vitronectin. HSP70protein family often identified as a sign of exosomes. C-reactive protein, produced by liver cells, has an important sign of inflammation as acute phase proteins; mucins of epithelial cells play a lubrication and cell signal transduction function, and they can form a chemical barrier; a great mount of immunoglobulin and zinc finger protein CCCH-type anti-viral proteins indicated that biliary exosomes play an important role in the immune response. In this study, we isolated white blood cells of thymus, spleen, liver of42-43day-old SPF chicken, and pulsed a certain dose of bile exosomes co-culture for a period of time. Then measured population of CD4/CD8, La (B cell marker)/MHC II, MHC I/Monocyte (Macrophage) by flow cytometry. The results show that compared with control, pulsed with chicken biliary exosomes, the population of CD4+T cells and MHC-I from liver was significantly increased, but there was little effect on MHC-II. Strikingly, while there were no obviously changes in neither spleen nor thymus lymphocytes. At the same time point, however, we observed naive CD4+T, CD8+T cells of spleen co-cultured with biliary exosomes cells survival the numbers of survival cells increased without any growth factors.This means that the chicken bile exosomes enhance the ability of liver lymphocytes in immune response, and play an significance role in immune and targeted on the cells of origin.We first found that biliary exosomes have zinc finger CCCH-type antiviral protein. This protein can inhibit the replication of retroviruses. Therefore, we designed experiments to study the antiviral activity of chicken bile exosomes. In many poultry retrovirus, we chose the J subtype avian leukosis virus (ALV-J) as a model virus. ALV-J induce tumor diseases and immune suppression in chicken. At present, no effective vaccines or drugs can prevent or cure these diseases. The zinc finger protein CCCH-type antiviral protein discovered in the biliary exosomes may be the hope for the treatment of avian leukosis. In this study, we found that:a certain dose of exosomes can inhibit the replication of viruses. In addition, Gould’s Trojan horse hypothesis is that ALV-J can rob the endocytosis pathway of exosomes, proposed that high concentrations of exosomes may become virus invasion accomplices. Therefore, to decide the appropriate dose and time of exosomes is particularly important to our future research in antiviral activity.