Vpliv z gastrinom spodbujenih zunajceličnih veziklov na migracijo naivnih celic raka želodca

Bricman, Vesna (author), Trebušak Podkrajšek, Katarina (mentor), Hudler, Petra (comentor)

Najpogostejši dejavniki za razvoj raka želodca so okužba s patogeno bakterijo Helicobacter pylori, spremembe v izražanju genov in gastro-ezofagealni refluks. Povečano izločanje peptidnega hormona gastrina, zaradi refluksa ali okužbe s H. pylori, stimulira proliferacijo želodčnih celic, kar poveča pojavnost karcinoidov v želodcu. Gastrin v patoloških pogojih zavira apoptozo, spodbuja celično proliferacijo in migracijo, kar vodi v povečano tveganje za nastanek raka želodca. Študije so pokazale, da na procese nastanka in napredovanja rakov med drugim vplivajo tudi zunajcelični vezikli, ki jih izločajo predrakave in rakave celice. Zunajcelični vezikli sodelujejo pri zapleteni medcelični komunikaciji, saj vsebujejo različne molekule, ki delujejo na tarčne celice. Zunajcelični vezikli so zanimivi za preučevanje, saj njihova sestava odraža sestavo celice izvora in je odvisna od stanja v celici. Glede na nastanek, velikost in izvor, razdelimo zunajcelične vezikle na tri podskupine: eksosomi, mikrovezikli in apoptotska telesca. Večji vezikli, ki jih izločajo rakave celice, se imenujejo onkosomi. V naši raziskavi nas je zanimalo, ali zunajcelični vezikli, izolirani iz celične linije raka želodca MKN45, spodbujene z gastrinom, pospešijo migracijo naivnih celic raka želodca MKN45 ter ali pospešijo migracijo naivnih normalnih epitelijskih celic MCF-10A. Z diferencialnim ultracentrifugiranjem smo ločili izolirane vezikle na eksosome, mikrovezikle in onkosome. Nato smo njihove lastnosti ovrednotili s pretočno citometrijo in optičnim sledenjem posameznemu nanodelcu. Preverili smo njihov vpliv na migriranje naivnih celic raka želodca MKN45 in naivnih zdravih celic MCF-10A, s testom celjenja ran (angl. wound healing assay). Primerjali smo migracijo celic, ki smo jim dodali vezikle, izolirane iz celic, spodbujenih z gastrinom, in migracijo celic, ki smo jim dodali vezikle, izolirane iz celic, ki niso bile spodbujene z gastrinom. Ugotovili smo, da onkosomi in mikrovezikli, izolirani iz celic MKN45, spodbujenih z gastrinom, ne vplivajo na migracijo naivnih celic MKN45. Eksosomi pa njihovo migracijo zavirajo. Na migracijo naivnih normalnih epitelnih celic MCF-10A nobena podskupina zunajceličnih veziklov, izoliranih iz celic MKN45, spodbujenih z gastrinom, ni imela vpliva.

• Language: Slovenian
• Keywords: rak želodca, celična linija MKN45, gastrin, zunajcelični vezikli, migracija
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2022
• PID: 20.500.12556/RUL-135290
• Publication date in RUL: 05.03.2022

Secondary language

• Language: English
• Title: Effect of gastrin-induced extracellular vesicles on naive gastric cancer cell migration

Abstract

The most common factors for developing stomach cancer are infection with pathogenic bacteria Helicobacter pylori, changes in gene expression and acid reflux. Over-expression of peptide hormone gastrin due to acid reflux or H. pylori infection stimulates proliferation of gastric cells, which increases the formation of carcinoids in the stomach. In pathological conditions gastrin inhibits apoptosis, increases cell proliferation and migration, which leads to increased stomach cancer risk. Studies have shown that extracellular vesicles, secreted from pre-cancerous and cancerous cells, influence both the development and the progression of cancer. Extracellular vesicles are involved in complex cell-to-cell communication, because they carry different biomolecules, which have an effect on target cells. The composition of vesicles reflects the composition of cells of origin and it depends on the state the secreting cell is in. This makes extracellular vesicles interesting for studying. Based on their biogenesis, size and origin we divide extracellular vesicles into three subpopulations: exosomes, microvesicles and apoptotic bodies. Larger vesicles secreted form cancer cells are known as oncosomes. We were interested if extracellular vesicles isolated from gastric cancer cell line MKN45, induced with gastrin, increase the migration of naïve gastric cancer cell line MKN45 and normal epithelial cells MCF-10A. Using differential centrifugation, we separated isolated extracellular vesicles into three populations: exosomes, microvesicles and oncosomes. They were evaluated by flow cytometry and nanoparticle tracking analysis. We tested their ability to affect cell migration on MKN45 cells and on MCF-10A cells using a “wound healing assay”. We compared the migration of cells exposed to extracellular vesicles isolated from MKN45 cells induced with gastrin, and migration of cells exposed to extracellular vesicles isolated from MKN45 cells that were not induced with gastrin. Our results showed that oncosomes and microvesicles isolated from MKN45 cells induced with gastrin did not affect the migration of naïve MKN45 cells, while exsosomes reduced their migration. Oncosomes, microvesicles and exosomes isolated from MKN45 cells induced with gastrin did not affect the migration of naïve MCF-10A cells.

• Keywords: gastric cancer, MKN45 cell line, gastrin, extracellular vesicles, migration