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Quantifying extracellular vesicle secretion from single neuro-endocrine cells to understand how they affect hormonal secretion
Högskolan i Skövde, Institutionen för biovetenskap.
2020 (engelsk)Independent thesis Advanced level (degree of Master (One Year)), 20 poäng / 30 hpOppgave
Abstract [en]

Extracellular vesicles (EV’s) are small lipid bilayer vesicles that are generated by almost all kind of cells in a body. EV’s are considered as one of the key intercellular messengers regulating cell signalling mechanisms. Earlier studies have shown that, in metabolic diseases like diabetes and obesity, as well as during hypertension and neurodegenerative disease there is increased production and secretion of EV’s. Secretion mechanism of EV’s is yet unknown. The aim of this study was to investigate the EV production and secretion mechanism in type-2-diabetes and in parallel to EV studies, measurement of SST secretion, to elucidate how it is influenced by EV’s. Tetraspanin was used to label EV’s and the efficiency was evaluated by using TIRFM and considering how many exosomes they label per cell and how well they express. Further, the EV marker were exploited in studying trafficking events of EV’s at the plasma membrane. This included EV approach to PM through docking/visiting, and EV loss from PM through undocking. EV labelling showed that CD63 and CD151 were two efficient markers for live-cell imaging by TIRF microscopy (TIRFM). Trafficking analysis of EV’s showed that number of visiting events were significantly higher compared to docking and undocking events. To know how many of total EV’s in a cell are ready to fuse with plasma membrane, rate of displacement of EV’s was monitored. This showed, small fraction of EV’s were slow-moving, probably docked at the PM while rest EV’s were fast-moving, either visiting or undocking EV’s. Docked EV’s fuse with plasma membrane. SST secretion from δ-cells was studied using pancreatic islets. There are no currently reliable means to measure δ-cells SST secretion. Commercially available antibodies against SST were evaluated compared to antibodies developed in the lab. Efficiency of the antibodies was studied by analyzing number of δ-cells and their distribution in an islet. The results showed that the antibodies against SST that were developed in the lab have a higher efficiency compared to the commercially available antibodies in δ-cells in an islets and tissue. These antibodies were used for staining δ-cells in type-2 diabetic vs healthy islets. Decrease in number of δ-cells in diabetic islets was observed. Therefore, these developed antibodies can be used for future hormone secretion studies.

sted, utgiver, år, opplag, sider
2020. , s. 32
Emneord [en]
EV’s, markers, fusion, trafficking, metabolic mechanism, TIRFM, δ cells, antibodies, SST (somatostatin)-SST14 and SST28, distribution
HSV kategori
Identifikatorer
URN: urn:nbn:se:his:diva-19336OAI: oai:DiVA.org:his-19336DiVA, id: diva2:1511004
Eksternt samarbeid
Mercodia AB
Fag / kurs
Bioscience
Veileder
Examiner
Tilgjengelig fra: 2020-12-17 Laget: 2020-12-17 Sist oppdatert: 2020-12-17bibliografisk kontrollert

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