| Exosomes are a special class of nanoscale vesicles that its main biological functions are:immune function,intercellular information communication,promotion of angiogenesis,as one of biological indicators for the diagnosis and treatment of the occurrence and development of digestive diseases.Exosomes,also known as intraluminal vesicles(ILVs),are special vesicles that are released by fusion of multivesicular bodies(MVBs)with the plasma membrane in living cells.Exosomes have been widely reported in mammalian gut systems,but studies in vivo have been limited.The zebrafish digestive system is different from mammals,and no reports of exosomes have been reported in the zebrafish gut.This study used the zebrafish gut as the research object to explore and interpret the exosomes and MVBs derived from zebrafish gut mucosa at the molecular and cellular levels.It is important to reveal the gut function such as teleost fish,and the research results provide the theoretical basis for the study of zebrafish gut diseases.Experiment Ⅰ The Development,secretion and dynamic distribution of exosomes and MVBs in zebrafish gut absorptive cells.The anatomical and histological structure of the zebrafish gut was observed by stereomicroscope and hematoxylin-eosin staining(HE)technique;real-time quantitative PCR(RT-qPCR)was used to analyze the expression of exosomes-specific markers CD63 and TSG101 mRNA in different gut segments;Western blotting(WB)was used to detect the expression of CD63 and TSG101 protein in different gut segments;The distribution characteristics of CD63 and TSG101 protein immunopositive cells in different gut segments of zebrafish were further observed by immunohistochemical method(IHC);the exosomes positive particles were analyzed in different gut by immunofluorescence labeling(IF).On this basis,the morphological characteristics and transient dynamic secretion process of exosomes and their MVBs in different intestinal segments were elucidated by transmission electron microscopy(TEM).The results showed that the zebrafish gut showed a typical "Z" appearance,which was divided into the anterior gut,mid gut and posterior gut.The gut wall consisted of three layers:mucosal layer,muscular layer and serosal layer.The mucosal layer is divided into mucosal epithelium and lamina propria,and the mucosal epithelium is clearly visible with well-arranged Absorptive cells(ACs).The expression of CD63 and TSG101 mRNA in different gut segments of zebrafish was different,especially in the mid gut,and the expression in the posterior was the lowest.The results of CD63 and TSG101 protein levels were consistent with the previous,both showing the most significant in the mid gut.It was further found that compared with the negative control group,CD63 and TSG101 protein immunopositive cells were mainly concentrated in mucosal epithelial cells of different gut segments,while the lamina propria was weakly positive.CD63 and TSG101 immunopositive particles are mainly distributed in the apical cytoplasm of different gut mucosal epithelial cells,especially in the mid gut.On this basis,the ultrastructure indicates that exosomes and their MVBs are mainly distributed in the mid gut mucosal epithelial ACs compared with the anterior and posterior.Further observation revealed that the early endosomes(ee)in the exosomes pathway existed in the apical cytoplasm and basal cytoplasm of ACs,and there was a phenomenon of mutual fusion.At the same time,it is observed that late endosomes(le)are located above the nucleus of such cells,which have the characteristics of low electron density,inward buding,and less intracellular ILVs.Importantly,there are two kinds of MVBs with different electron densities in the cytoplasm of ACs,one with smaller diameter,higher electron density,less intracellular volume containing ILVs,and the other with relatively larger diameter,the electron density is low,and there are a large number of ILVs in the cavity.In addition,there is a typical structure in which exosomes are secreted into the gut lumen by exocytosis at the apical cell membrane of the ACs.Importantly,a rich distribution of exosomes was observed in the mucus layer above the microvilli,and occasionally a complete MVBs-like structure was present in the gut lumen.Based on the above results,the morphological characteristics and distribution dynamics of ACs exosomes and their MVBs in the mid gut of zebrafish were preformed.These results reveal that exosomes and their MVBs are mainly formed and secreted by the mid gut absorptive cells of the zebrafish,and is mainly secreted by the gut lumen.Experiment Ⅱ Cytogenetic characteristics of zebrafish gut goblet-derived exosomes and their MVBs.The morphological structure and distribution characteristics of exosomes and MVBs in zebrafish goblet cells(Goblet cells,GCs)were systematically analyzed using glycogen Schiff’s reaction technique(PAS),IHC and TEM techniques.The results showed that the exosomes-specific marker protein CD63 immunoreactive cells were mainly distributed in the gut mucosa epithelium,and the lamina propria was weakly positive.On this basis,it was found that GCs were mainly distributed between the epithelium of the mid gut mucosa through ultrastructural observation,with scattered and clustering distribution.The cytoplasm of the top of the GCs is enlarged,containing a large amount of mucous particles,and the cytoplasm of the basal part is narrow,so the appearance is a typical "cup" shape.Importantly,in addition to the developed endoplasmic reticulum,MVBs with smaller diameters and lower electron densities were observed in the basal cytoplasm of GCs,and MVBs contained less ILVs in the lumen.In the cytoplasm at the top of this type of cells,MVBs are also found in the center and sides of the mucus particles,mostly in the shape of a flat ellipse,and the cavity contains ILVs.Therefore,cell morphological characteristics suggest that the secretory process of exosomes and its MVBs may be closely related to the secretory process of mucosal particles in GCs.These results revealed that zebrafish gut goblet cells can secrete exosomes in addition to synthesizing and secreting mucin.Experiment Ⅲ Ultrastructural identification of exosomes and MVBs in zebrafish gut mucosa immune-related cells The morphological structure and distribution characteristics of exosomes and their MVBs in zebrafish gut mucosa immune-related cells were identified by TEM.The results showed that there were typical MVBs in the cytoplasm of Lamina propia lymphocytes(LPLs),which were round and low in electron density,the cavity contained ILVs within different sizes,and the MVBs were mostly located in the caldera of the nucleus.In addition to lymphocytes,the lamina propria also contains high post-endothelial venules.The venule is composed of high columnar high endothelial cells(HECs).Under electron microscope,there are a large number of membrane vesicles,mitochondria and MVBs in the cytoplasm.The latter contains many ILVs and the shape is round with low electron density characteristics.At the same time,HECs and adjacent cells were found around the high endothelium venules,and the information was exchanged by releasing the exosomes through the budding of basal cytoplasmic membrane.The first ultrastructure showed that mitochondria-rich cells(MRCs)existed between zebrafish gut mucosal epithelial cells,and their cytoplasm contained a large number of mitochondria and tubular systems.According to whether the cytoplasmic membrane at the top of the cell is connected to the gut lumen,it is classified into "open" MRCs and "closed" MRCs,and typical MVBs are found in their cytoplasm.In summary,a pattern of exosomes and their MVBs in gut mucosal immune-related cells was mapped.These results revealed that zebrafish gut exosomes and their MVBs are derived not only from absorptive cells,but also from goblet cells,lamina propria lymphocytes,high endothelial cells,and mitochondria-rich cells. |
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