Retseptori internalisatsioonianalüüs
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Date
2012
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Tartu Ülikool
Abstract
Cellomics HCS platvorm on paindlik high-content screening lahendus, mis koos oma lisamoodulitega võimaldab uurida mitmesuguseid erinevaid bioanalüüse alates valkude translokatsioonist kuni rakutsüklite jälgimiseni elus rakkudes. HCS on tõestanud ennast kui tõhus ja paindlik analüüsiviis ning on kasvava tähtsusega nii akadeemilistes uurimustes kui ka farmaatsias ravimiarenduseks.
Cellomics Colocalization V3 pildianalüüsiprotkolli kasutades piltidelt võetud mõõdud kinnitavad, et Endoteliin A retseptorvalgu (ETAR) internalisatsiooni aktiivsus Icosageni poolt arendatavates rakkudes käitub reeglipäraselt temale lisatud peptiidi Endoteliin-1 (ET-1) suhtes. Matemaatiliselt on võimalik reeglipära kirjeldada neljaparameetrilise sigmoidse kurviga. Kuna uuritavate andmete jaotused polnud mitte ühegi uuritava parameetri puhul normaaljaotusega, siis määrati matemaatilised mudelid üle andmete mediaanväärtuste. Mudelid valideeriti võrreldes neid one-way ANOVA mudeliga, ning testide tulemusena võib väita, et määratud kurvid iseloomustavad mõõdetud andmeid hästi.
Bioloogiline hüpotees, et bioanalüüs töötab, on võimalik kinnitada retseptori ning tema internalisatsiooni sihtpunktiks olevate endosoomide kattuvuse alusel. Efektiivsed peptiidi kontsentratsioonid on internalisatsiooni tööle hakkamiseks 0.29nM, poole aktiivsuse saavutamiseks 1.6nM ning maksimaalse efektiivsuse saavutamiseks 10nM.
Lisaks tuvastati töö käigus, et internalisatsiooni on võimalik iseloomustada ka endosoomimärget kasutamata, uurides Kolmogorov-Smirnov kahe valimi testist (K-S test) väljundina saadavaid andmete jaotuvuste kaugusi üksteisest. K-S testi tulemused näitasid ka testi potentsiaali erinevate katsete normaliseerimiseks hilisema võrdluse huvides.
High-content screening has proved itself to be a highly efficent method for analyzing bioassays in various different fields from oncology to drug screening. Cellomics HCS platform is a modular system which provides the means to analyze cellular and subcellular processes by using fluorescent dyes and automatic fluorescent microscopy. The aim of this thesis is to describe receptor internalization bioassay in biological system developed by Icosagen. Main focus of the thesis is to describe both image and data analysis methods and processes behind the final description of the assay. Studied assay is Endothelin A receptor internalization assay and the goal was to investigate the relation of peptide Endothelin-1 and internalization activity. By using Cellomics Colocalization V3 BioApplication for image analysis and fitting four parameter logistic curve on receptor and early-endosome overlap medians it was found that the Endothelin-1 effective doses are 0.29nM for the assay to show any activity, 1.6nM for half-maximal efficency and 10nM for maximum internalization. As an alternative data analysis method Kolmogorov-Smirnov two sample test for difference in variance was also tested. Results of this approach were fitted to four-parameter logistic model and the sigmoidal curves suggested that this method can be used as an alternative to more common study of mean or median values.
High-content screening has proved itself to be a highly efficent method for analyzing bioassays in various different fields from oncology to drug screening. Cellomics HCS platform is a modular system which provides the means to analyze cellular and subcellular processes by using fluorescent dyes and automatic fluorescent microscopy. The aim of this thesis is to describe receptor internalization bioassay in biological system developed by Icosagen. Main focus of the thesis is to describe both image and data analysis methods and processes behind the final description of the assay. Studied assay is Endothelin A receptor internalization assay and the goal was to investigate the relation of peptide Endothelin-1 and internalization activity. By using Cellomics Colocalization V3 BioApplication for image analysis and fitting four parameter logistic curve on receptor and early-endosome overlap medians it was found that the Endothelin-1 effective doses are 0.29nM for the assay to show any activity, 1.6nM for half-maximal efficency and 10nM for maximum internalization. As an alternative data analysis method Kolmogorov-Smirnov two sample test for difference in variance was also tested. Results of this approach were fitted to four-parameter logistic model and the sigmoidal curves suggested that this method can be used as an alternative to more common study of mean or median values.