II 3. Histologische und immunhistochemische Untersuchungen
II.5. Zellzyklusanalyse mittels FACS
Die FACS-Analyse (Fluorescence-Absorbance-Cell-Sorting) ermöglicht es, eine große Anzahl von Zellen in sehr kurzer Zeit einzeln auf ihre biochemischen oder biophysikalischen Eigenschaften zu untersuchen.
In einem sehr feinen Wasserstrahl wandern die zu untersuchenden Zellen hintereinander und werden dabei von einem Laser mit relativ kurzer Wellenlänge (!=488 nm) angeregt. Hierbei werden nun verschiedene Parameter bestimmt. Der Forward-Scatter (FSC) ist ein Maß für das Licht, welches im 180°-Winkel abgeschwächt aus der angeregten Zelle austritt und ist proportional zur Zellgröße. Der side-scatter (SSC) bezeichnet das im 90° Winkel abgegebene Streulicht und gilt als Maß für die Granularität. Aus diesen zwei Parametern können sehr gezielt Zellpopulationen bestimmt und definiert werden. Werden die Zellen zusätzlich mit einem Farbstoff oder einem fluoreszenzgekoppelten Antikörper inkubiert, so können neben den schon erwähnten Parametern weitere zur Fluoreszenz der Zellen erhoben werden, beispielsweise Rezeptorstatus, Differenzierungsmarker, etc.
Propidiumiodid interkaliert wie Ethydiumbromid mit DNA. Wird der Komplex aus DNA und Propidiumiodid mit kurzwelligem, blauen Licht angeregt, so emittiert dieser im roten Bereich. Diese Fluoreszenz wird über einen entsprechenden Filter detektiert. Die hierbei gemessene Lichtmenge ist direkt proportional zum DNA-Gehalt der Zelle. Visualisiert werden die erhobenen Werte in einem Histogramm, dessen x-Achse die Fluoreszenzintensität und dessen y-Achse die absolute Zellzahl repräsentieren. Über weitere statistische Analysen ist es nun möglich, die gemessenen Daten hinsichtlich Genauigkeit, Validität u.ä. zu untersuchen und die Anzahl der Zellen in den verschieden Abschnitten des Zyklus anzugeben.
p53cbs Promotor Luciferase-Gen
p53!
Luciferase Luciferin+ATP+H2O2+CoA
Licht+ADP+P+H2O
5.1. Zellzyklusanalysen von MCF-7-Zellen
Um ein möglichst große Anzahl von Zellen einer Population in der S- oder G2/M-Phase untersuchen zu können, wurden MCF-7 Zellen mit 0,5% FCS-supplementierten RPMI-Medium ohne den Zusatz von Insulin für 32 Stunden inkubiert. Hierdurch werden die Zellen in der G0/G1-Phase des Teilungszyklus arretiert. Nach der Zugabe von 10%igem FCS RPMI-Medium und Insulin wurde durch die im Medium enthaltenen Wachstumsfaktoren ein Teilungsstimulus induziert und die Zellen weitere 27 Stunden inkubiert. Nach dieser Zeit befinden sich etwa 65% der Zellen entweder in der S- oder G2/M-Phase. Bei einer weiteren Probe wurde direkt nach Zugabe des Wachstumsmediums mit unterschiedlichen Energiedosen bestrahlt, um einen Teilungsarrest zu induzieren und anschließend in An- oder Abwesenheit der modulatorischen Peptide MCF-7-Zellen auf ihre Teilungsaktivität mittels FACS untersuchen zu können. Andere Versuche, Zellen mit Hydroxyharnstoff (RNA-Polymerase-Inhibitor)oder Mimosin (Dihydrofolatreduktase-Inhibitor) synchronisieren zu können, erwiesen sich wegen der Toxizität der verwendeten Stoffe als nicht durchführbar.
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