Isolation and identification of hepatocellular exosomes and their effects on the expression of miR-122/33 and immune-related genes in grass carp(Ctenopharyngodon idella)

Exosomes are nano-scale vesicles with a phospholipid bimolecular membrane, which can participate in many physiological processes. The aim of this study was to investigate the isolation and identification methods of extracellular exosomes from grass carp hepatocyte and to conduct preliminary study on the effects of exosomes secreted from fatty hepatocyte on the expression of miRNAs and immune-related genes in grass carp hepatocytes. In this study, The exosomes of grass carp hepatocytes L8824 were extracted by ultracentrifugation. The morphology of exosomes was observed by Electron Microscopy, the parcicle size and quantity of exosomes were determined by Nano-particle Tracking Analysis. Meanwhile, we analyzed the expression of the specific protein CD63 using Western blot. Then, we used exosomes,which wwew secreted from normal hepatocytes and fatty hepatocyte induced by oleic acid to incubate grass carp hepatocytes, and the effects of two different exosomes on the transcription levels of miR-122/33 and immune-related genes, which are TNF-α, NF-κB, IL-1β, IL-6 and IL-10, were detected by real-time qPCR. Our results indicated that the exosomes secretion of grass carp hepatocytes culture medium were uneven vesicles of 30-150 nm, round or oval, and had a complete membrane structure. The expression of exosomes marker protein CD63 was positive, and the NTA results showed that the exosome vesicles accounted for more than 50 % of all the vesicles. Additionally, the exosomes of fatty liver cells significantly increased the expression of miR-122 and the immunogenic genes (TNF-α, IL-1β and IL-6) in hepatocytes (P<0.05). These results suggest that the exosomes of grass carp hepatocytes can be isolated successfully by ultracentrifugation. Moreover, exosomes may play a key role in the regulation of immunity of grass carp hepatocytes.