Dokument: Charakterisierung der p21-GFP-Expression in Genom-editierten humanen Zelllinien
| Titel: | Charakterisierung der p21-GFP-Expression in Genom-editierten humanen Zelllinien | |||||||
| Weiterer Titel: | Characterization of p21-GFP expression in genome-edited human cell lines | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=60612 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20220919-105715-9 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Deutsch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Büchel, Simon [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof.Dr Fritz Boege [Gutachter] PD Dr. rer. nat. Stork, Björn [Gutachter] | |||||||
| Stichwörter: | p21, DNA-Schädigung, Expressionsdynamik, Zellzyklus | |||||||
| Dewey Dezimal-Klassifikation: | 600 Technik, Medizin, angewandte Wissenschaften » 610 Medizin und Gesundheit | |||||||
| Beschreibungen: | Diese Arbeit beschäftigt sich mit den Antwortmechanismen humaner Zellen auf eine DNA-Schädigung, welche essentiell zur Tumorprävention ist. Untersucht wird das Tumorsupressorprotein p21, welches eine wesentliche Rolle in der Kontrolle des Zellzyklus einnimmt. Bei einer Schädigung der Körperzelle wird die Expression von p21 induziert, welches einen Zellzyklusarrest induziert, um die DNA-Reparatur zu ermöglichen.
Studien haben gezeigt, dass sich die Induktion von p21 auf Einzelzellniveau unterschiedlich darstellt. In manchen Zellen erfolgte eine sofortige Induktion der p21 Expression, in anderen eine verzögerte und in einigen gar keine. In dieser Arbeit sollte zum einen geprüft werden, ob diese individuelle p21-Antwort abhängig ist von der Zellzyklusphase, in der die Zellschädigung stattgefunden hat. Zum anderen sollte untersucht werden, ob sich die Dynamik der p21-Expression zwischen verschiedenen Arten der DNA-Schädigung unterscheidet. Für die Experimente wurden HT-1080- Fibrosarkomzellen verwendet, in denen p21 durch CRISPR/Cas9-Genomeditierung mit dem grün fluoreszierenden Protein GFP fusioniert wurde. Bei einer Zellschädigung kann somit die Expression des p21-GFP-Fusionsproteins unter dem Fluoreszenzmikroskop beobachtet werden. Durch die Expression eines Fusionsproteins aus dem Zellzyklusmarker PCNA und dem roten Fluoreszenzprotein RFP kann anhand der Verteilung der roten Fluoreszenz der Zellzyklus bestimmt werden, da sich diese in G1-, S- und G2-Phase unterscheidet. Zusätzlich stand ein durch CRISPR/Cas9-generierter p21-Knockout-Klon zur Verfügung. Um zu prüfen, ob die p21 Antwort vom Zellzyklus zum Zeitpunkt der Zellschädigung abhängt, wurden die Zellen mit Camptothecin geschädigt und die p21-GFP-Induktion 48 Stunden lang nachverfolgt. Dabei hat sich zwar bestätigt, dass einzelne Zellen stärker nach der Schädigung fluoreszieren, aber eine Zuordnung zum Zellzyklus war durch technische Probleme des Mikroskops nicht möglich. Zusätzlich konnte durch einen Vergleich des p21-Knockout-Klons mit dem Wildtyp gezeigt werden, dass Zellen ohne p21 trotz Schädigung häufiger in die S-Phase gehen, die Zellzyklus-Kontrolle also behindert ist. Um die zweite Frage zur p21-Expression bei verschiedenen Arten der DNA-Schädigung zu bearbeiten, wurden die Zellen mit unterschiedlichen Dosen an UVA Strahlen geschädigt und die p21-GFP-Expression quantifiziert. Es hat sich ein sigmoidaler Anstieg der mittleren Fluoreszenz gezeigt. Da bei Röntgenstrahlen ein linearer Anstieg stattfindet, verursacht die Art der DNA-Schädigung also tatsächlich unterschiedliche p21-GFP-Expressionskinetiken. Außerdem zeigte sich, dass p21-GFP-exprimierende Zellen in G1 und G2 verharren, in welcher Phase vermehrt war jedoch abhängig von der Schädigungsart. Zusammenfassend konnte gezeigt werden, dass die p21-GFP-Induktion schadensspezifische Kinetiken aufweist, sodass dieses Zellsystem als biologisches System zur Toxizitätsbestimmung verschiedenster Substanzen geeignet erscheint.This work deals with the response mechanisms of human cells to DNA damage, which is essential for tumor prevention. The tumor suppressor protein p21 is investigated, which plays an essential role in cell cycle control. Upon damage to the somatic cell, the expression of p21 is induced, which induces cell cycle arrest to allow DNA repair. Studies have shown that the induction of p21 varies at the single-cell level. In some cells there was an immediate induction of p21 expression, in others a delayed induction and in some none at all. In this work, it was the aim to determine whether this individual p21 response is dependent on the cell cycle phase in which cell damage occurred. The second aim was to investigate whether the dynamics of p21 expression differ between different types of DNA damage. For the experiments, HT-1080- fibrosarcoma cells were used in which p21 was fused to the green fluorescent protein GFP by CRISPR/Cas9 genome editing. Thus, upon cell injury, expression of the p21-GFP fusion protein can be observed under the fluorescence microscope. By expressing a fusion protein of the cell cycle marker PCNA and the red fluorescent protein RFP, the cell cycle can be determined based on the distribution of red fluorescence as it differs into G1, S, and G2 phases. In addition, a p21 knockout clone generated by CRISPR/Cas9 was available. To test whether the p21 response depends on the cell cycle at the time of cell damage, cells were damaged with camptothecin and p21-GFP induction was followed up for 48 hours. This confirmed that individual cells fluoresced more strongly after damage, but assignment to the cell cycle was not possible due to technical problems with the microscope. In addition, by comparing the p21 knockout clone with the wild type, it was shown that cells without p21 enter S phase more frequently despite damage, thus cell cycle control is impaired. To address the second question about p21 expression in different types of DNA damage, cells were damaged with different doses of UVA radiation and p21-GFP expression was quantified. A sigmoidal increase in mean fluorescence was shown. Thus, since a linear increase occurs with X-rays, the type of DNA damage actually causes different p21-GFP expression kinetics. Moreover, p21-GFP-expressing cells were shown to persist in G1 and G2, but in which phase increased was dependent on the type of damage. In summary, it was shown that p21-GFP induction exhibits damage-specific kinetics, so that this cell system appears to be suitable as a biological system for toxicity determination of a wide variety of substances. | |||||||
| Quelle: | Literaturverzeichnis
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(2019): Effects of different doses of X-ray irradiation on cell apoptosis, cell cycle, DNA damage repair and glycolysis in HeLa cells. In: Oncology letters 17 (1), S. 42–54. DOI: 10.3892/ol.2018.9566. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Medizinische Fakultät » Institute » Zentralinstitut für Klinische Chemie und Laboratoriumsdiagnostik | |||||||
| Dokument erstellt am: | 19.09.2022 | |||||||
| Dateien geändert am: | 19.09.2022 | |||||||
| Promotionsantrag am: | 28.04.2022 | |||||||
| Datum der Promotion: | 08.09.2022 |
