diplomsko delo
Patrik Levačič (Author), Nataša Debeljak (Mentor)

Abstract

Endogeni kanabinoidni sistem (ECS) kontrolira številna fiziološka in patološka stanja. Sestavljata ga kanabinoidni receptor tipa 1 (CB1) ter tipa 2 (CB2), njuni endogeni ligandi (endokanabinoidi) ter proteini, ki regulirajo biosintezo in degradacijo endokanabinoidov. CB1 je visoko izražen v možganskem tkivu, CB2 je izražen izključno v celicah imunskega sistema. Z G proteini sklopljen receptor 55 (GPR55), je kanabinoidni receptor, kateri je glede na aminokislinsko zaporedje, manj kot 15 % podoben CB1 in CB2. Ima sposobnost vezave raznolikih endogenih ter eksogenih kanabinoidnih ligandov, zato je GPR55 glavni kandidat za kategorizacijo kot kanabinoidni receptor tipa 3. Izraža se v možganskem tkivu, endotelijskih celicah ter prebavnem traktu. V sklopu diplomskega dela smo želeli z uporabo metode točkovnega nanosa določiti ustrezne delovne koncentracije primarnih in sekundarnih protiteles proti CB1, CB2 in GPR55. Poleg tega smo za nadaljnjo kvantitativno določitev CB želeli določiti ustreznost protiteles dveh proizvajalcev, Santa Cruz Biotechnology in Abcam, in izbrali protitelesa tistega proizvajalca, s katerimi smo dobili boljše rezultate pri zaznavi. V ta namen smo iz izbrane celične linije pripravili celične lizate in topni frakciji celičnega lizata izmerili koncentracijo proteinov s kompletom standardov govejega serumskega albumina (BSA). Vzorce, nanešene na membrano za imunodetekcijo, smo inkubirali z izbranimi protitelesi in nato slikali s kamero LAS 4000, ki ima sposobnost zajemanja kemiluminiscenčne svetlobe. Naši rezultati nakazujejo, da so protitelesa proti CB1 in CB2 obeh proizvajalcev ustrezna. Odločili smo se, da so protitelesa proizvajalca Santa Cruz Biotechnology ustreznejša za imunodetekcijo CB1 in CB2 v primerjavi s protitelesi proizvajalca Abcam, in so kot taka primerna za nadaljnjo uporabo pri kvantitativnem določanju CB z metodo prenosa Western. Za izbrana protitelesa smo se odločili, ker je jakost signalov bolj zvezna in bolje posname gradient redčitev oz. koncentracij. Raziskave o izražanju GPR55 v celičnih linijah raka dojke so redke Na podlagi rezultatov našega dela smo potrdili izražanje GPR55 v nesmrtni celični liniji raka dojke T-47D.

Keywords

rak dojke;celična linija raka dojke;endogeni kanabinoidni sistem;kanabinoidni receptorji;protitelesa;točkovni nanos;diplomska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.11 - Undergraduate Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [P. Levačić]
UDC: 577.27:616-006.6(043.2)
COBISS: 32541699 Link will open in a new window
Views: 458
Downloads: 73
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Other data

Secondary language: English
Secondary title: Selection of antibodies for quantitative determination of cannabinoid receptors in breast cancer cell lines
Secondary abstract: The endogenous cannabinoid system (ECS) controls a number of physiological and pathological conditions. They consist of the cannabinoid receptor type 1 (CB1) and type 2 (CB2), their endogenous ligands (endocannabinoids), and proteins that regulate endocannabinoid biosynthesis and degradation. CB1 is highly expressed in the brain tissue and CB2 is expressed exclusively in the cells of the immune system. G protein-coupled receptor 55 (GPR55) is a cannabinoid receptor that is, based on the amino acid sequence, less than 15 % similar to CB1 and CB2. The ability to bind various endogenous and exogenous cannabinoid ligands is the reason why GPR55 is a prime candidate for categorization as a cannabinoid receptor type 3. It is expressed in the brain tissue, endothelial cells and the digestive tract. Within this diploma thesis, we wanted to determine the appropriate working concentrations of primary and secondary antibodies against CB1, CB2 and GPR55 using the dot blot method. In addition, for further quantitative determination of CB, we wanted to determine the suitability of antibodies from two manufacturers, Santa Cruz Biotechnology and Abcam, and selected antibodies from that manufacturer, with which we obtained better detection results. For this purpose, cell lysates were prepared from the selected cell line and the protein concentration of the soluble fraction of the cell lysate was measured with a set of bovine serum albumin (BSA) standards. Samples applied to the membrane for immunodetection were incubated in selected antibodies and then imaged with a LAS 4000 camera, which has the ability to capture chemiluminescent light. Our results suggest that antibodies against CB1 and CB2 from both manufacturers are appropriate. We decided that the antibodies manufactured by Santa Cruz Biotechnology were more suitable for the immunodetection of CB1 and CB2, in comparison with antibodies manufactured by Abcam, and as such were appropriate for further use in the quantitative determination of CB using the Western method. We opted for the selected antibodies because the signal strength is more continuous and better imitates the dilution or concentration gradient. Because little research has been done on the expression of GPR55 in breast cancer cell lines, we also confirmed the expression of GPR55 in the immortal T-47D breast cancer cell line based on the results.
Secondary keywords: breast cancer;endogenous cannabinoid system;breast cancer cell line;antibodies;dot-blot;
Type (COBISS): Bachelor thesis/paper
Study programme: 1000371
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, UNI Biokemija
Pages: 48 str.
ID: 12033041