Vēža šūnu ekstracelulāro vezikulu frakcionēšana un raksturošana
Date
2014
Authors
Cīrulis, Aivars
Journal Title
Journal ISSN
Volume Title
Publisher
Latvijas Universitāte
Abstract
Ekstracelulārās vezikulas (EV) piedalās šūnai nevajadzīgo proteīnu aizvākšanā, starpšūnu komunikācijā, piemēram, pārnesot RNS, kā arī vīrusu daļiņu izplatīšanā. Pētījumi ir parādījuši, ka audzēju šūnu izdalītās vezikulas spēlē nozīmīgu lomu vēža attīstībā un tās var kalpot par biomarķieriem. Darba mērķis bija raksturot un salīdzināt plaušu vēža šūnu līniju COR-L23 izdalīto eksosomu un mikrovezikulu bagātinātas frakcijas, kā arī RNS saturu tajās. Pielietojot diferenciālcentrifugāciju un gēlfiltrāciju, tika iegūtas tīras eksosomu un mikrovezikulu frakcijas, kas tika apstiprinātas ar TEM un Western blotu. Pētījumā tika noskaidrots, ka rRNS daudzums vezikulās, salīdzinot ar šūnām, ir niecīgs. Tika izveidotas un nosekvenētas šūnu, mikrovezikulu un eksosomu pilna transkriptoma bibliotēkas ar Ion Proton™ sistēmu, kas sniedz iespēju veikt tālākas analīzes un rast atbildi uz dažādiem jautājumiem, piemēram, vai RNS profili būtiski atšķiras starp dažādiem EV veidiem.
Atslēgvārdi: Mikrovezikulas, Eksosomas, Jaunās paaudzes sekvenēšana, Vēzis
Extracellular vesicles (EVs) participate in unnecessary protein removal from the cell, intercellular communication, e.g., transfer RNA, and also in virus particle distribution. Studies have shown that tumour cell-derived vesicles play a role in cancer progression and can be used as biomarkers. Aim of this work was to isolate, characterize and compare secreted exosome and microvesicle enriched EV fractions from lung cancer cell line COR-L23. Using differential centrifugation and size exclusion chromatography we gained exosome and microvesicle fractions free of protein complexes and apoptotic bodies, which were confirmed by TEM and Western blot. In this study we confirm that the amount of rRNA in vesicles is insignificant, comparing to cells. Next, full transcriptome libraries were generated from cells, microvesicles and exosomes and sequenced using Ion Proton™ System, thus providing an opportunity to perform further analyses and answer to many questions, for example, do RNA profiles significantly differ between different types of EVs. Keywords: Microvesicles, Exosomes, Next generation sequencing, Cancer
Extracellular vesicles (EVs) participate in unnecessary protein removal from the cell, intercellular communication, e.g., transfer RNA, and also in virus particle distribution. Studies have shown that tumour cell-derived vesicles play a role in cancer progression and can be used as biomarkers. Aim of this work was to isolate, characterize and compare secreted exosome and microvesicle enriched EV fractions from lung cancer cell line COR-L23. Using differential centrifugation and size exclusion chromatography we gained exosome and microvesicle fractions free of protein complexes and apoptotic bodies, which were confirmed by TEM and Western blot. In this study we confirm that the amount of rRNA in vesicles is insignificant, comparing to cells. Next, full transcriptome libraries were generated from cells, microvesicles and exosomes and sequenced using Ion Proton™ System, thus providing an opportunity to perform further analyses and answer to many questions, for example, do RNA profiles significantly differ between different types of EVs. Keywords: Microvesicles, Exosomes, Next generation sequencing, Cancer
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Bioloģija