Title Optimizacija metode za izolaciju mikrobiote iz fecesa štakora u svrhu proteomskih istraživanja Title (english) Optimisation of rat faecal microbiota isolation method for proteomic analysis purposes Author Filip Sokol Mentor Sandra Kraljević Pavelić (mentor)Committee member Mirela Sedić (predsjednik povjerenstva)Committee member Elitza Petkova Markova - Car (član povjerenstva)Committee member Sandra Kraljević Pavelić (član povjerenstva)Granter University of Rijeka Defense date and country 2018-09-24, Croatia Scientific / art field, BIOTECHNICAL SCIENCES Abstract Proteomska istraživanja mikrobiote fecesa postaju sve značajnija zbog rastućih spoznaja o povezanosti različitih bolesti i disbalansa mikrobiote crijeva. Radi jednostavnosti, ali i potrebe kontrole prehrane te sastava mikrobiote, u istraživanjima se najčešće koriste modelni organizmi. Štakor u usporedbi s laboratorijskim mišem, ima sličnija farmakokinetička i farmakodinamička svojstva ljudima, te se stoga koristi kao pogodan modelni organizam u istraživanjima crijevne mikrobiote. Crijevna mikrobiota spada u najkompleksnije zajednice mikroorganizama koju čine bakterije, kvasci, virusi i ostali mikrobiote. U proteomskim istraživanjima crijevne mikrobiote kompleksnosti uzorka pridodaju dijelovi neprobavljene hrane i proteini domaćina koji u visoko osjetljivim proteomskim analizama, poput one koja se provodi spektrometrijom masa, prekrivaju prisutnost proteina izoliranih iz mikrobiote. Iz tog razloga važno je dalje razvijati i optimizirati već postojeće metode za izolaciju crijevne mikrobiote. Metode izolacije koje su se pokazale djelotvornima u dosadašnjim istraživanjima uključuju diferencijalna centrifugiranja i centrifugiranja u gradijentu. U ovom radu je provedena optimizacija postojećih metoda za izolaciju proteina mikrobiote iz fecesa, a početni uzorak je bio duboko zamrznuti feces štakora. U prvom se koraku optimizirao protokol za homogenizaciju fecesa, a zatim kod diferencijalnih metoda centrifugiranja i centrifugiranja u gradijentu mijenjani su parametri broja, jačine i vrste centrifugiranja, a kod centrifugiranja u gradijentu, mijenjana je koncentracija sredstva za stvaranje gradijenta, Histodenza. Kontrola efikasnosti izolacije vršena je bojanjem po Gramu i metilenskim modrilom uz promatranje mikroskopskih preparata svjetlosnim mikroskopom. Također je provedeno kemijsko i mehaničko liziranje stanica mikrobiote s ciljem izolacije proteina. Proteini su zatim kvantificirani i razdvojeni uz pomoć jednodimenzionalne (1D) gel elektroforeze s ciljem provjere uspješnosti njihove izolacije. Metoda centrifugiranja u gradijentu pokazala se kao najefikasnija metoda za izolaciju mikrobiote iz duboko zamrznutog fecesa pri čemu su bili zadovoljeni parametri razvnovrsnosti izolirane mikrobiote, a mikroskopski vidljivo onečišćenje, osobito ono uzrokovano neprobavljenim dijelovima hrane, bilo je minimalno.
Abstract (english) Proteome studies of feces microbiome are becoming more relevant due to the increasing number of evidences linking various diseases and disbalance of gut microbiota. Because of the possibility to control food intake and microbiome flora, as well as simplicity, model organisms are often used in microbiome studies. In comparison to laboratory mice, rats have pharmacokinetic and pharmacodynamic properties more similar to the human and therefore are used as a model organism in studies of gut microbiota. Intestinal microbiota is a complex community consisting of bacteria, yeasts, viruses and other. Undigested food components and host proteins obscure presence of microbial proteins in sensitive proteome analyses, such as mass spectrometry. For that reason, it is important to optimize existing methods for isolation of gut microbiota. Differential centrifugation and density gradient centrifugation have shown to be effective methods of isolation in previous studies. In this study, we have conducted optimization of existing methods used in isolation of fecal microbiota. Microbiota has been isolated from frozen rat feces. The first step was to optimize a protocol for feces homogenization, followed by optimization of centrifugation parameters. Parameters that were optimized included centrifugal force and the number of centrifugation steps, as well as previously mentioned centrifugation types. Histodenz, which is used as a density gradient media for density gradient centrifugation, was also tested in different concentrations. Efficacy of isolation was tested using Gram and methylene blue staining, detected with a light microscope. Chemical and mechanical disruption of the microbiota was performed to isolate proteins, followed by isolation and quantification using one-dimensional (1D) gel electrophoresis. Gradient centrifugation has shown to be the most efficient method in isolation of microbiota from frozen feces due to the preservation of microbiota composition and success rate of isolation, whereas contamination caused by undigested food fragments was minimal.
Keywords
crijevna mikrobiota
proteomika
diferencijalno centrifugiranje
centrifugiranje u gradijentu
Keywords (english)
gut microbiota
proteomics
differential centrifugation
density gradient centrifugation
Language croatian URN:NBN urn:nbn:hr:193:720926 Study programme Title: Biotechnology in medicine Type of resource Text File origin Born digital Access conditions Access restricted to students and staff of home institution Terms of use Created on 2018-10-31 09:46:34
