Auf der Grundlage der von uns ausgewählten chondrogenen Mutterzelllinie RCJ3.1(C5.18) sind im Rahmen dieser Arbeit zwei unterschiedliche Ansätze zur Beobachtung und Quantifizierung der chondrogenen Differenzierung erarbeitet worden. So wurde im ersten Teil eine Rezeptorzelllinie hergestellt und charakterisiert, die im Rahmen zunehmender Differenzierung eine gut zu beobachtende Grünfluoreszenz aufweist und mit der wir somit einen Marker für die chondrogene Differenzierung besaßen. Mit deren Hilfe konnten wir anschließend beispielhaft die Einflüsse von Wnt-Proteinen auf die Entwicklung und Differenzierung dieser Reporterzellen untersuchen. Die hierbei von uns gemachten Beobachtungen bewiesen zum einen die aussagekräftige, stabile Reportereigenschaft unserer neuen Zelllinie und gleichzeitig konnten wir weitestgehend übereinstimmend mit bis dahin veröffentlichten (und retrospektiv auch mit danach erschienen) Arbeiten108 die Effekte der unterschiedlichen Wnts auf die chondrogene Differenzierung bestätigen und teilweise auch um zusätzliche Erkenntnisse erweitern. So konnten wir z. B. erste Beweise für die gegensätzlichen Effekte der einzelnen Mitglieder der Wnt-Familie auf die chondrogene Differenzierung in vitro liefern.109
Im zweiten Teil wurde ebenfalls auf Basis der oben genannten Mutterzelllinie ein anderes Modell zur Untersuchung der chondrogenen Differenzierung entwickelt, indem die von uns neu etablierte klonale Rattenzelllinie R-tTA-24 nach stabiler Transfektion eines Tetracyclin regulierten Genexpressionssystems110 aufgrund des darin enthaltenen Tetracyclintransaktiva-tors (=tTA) eine regulierte Gen-Expression111 ermöglicht. Zur weiteren Austestung wurde zunächst mittels einer zweiten Transfektion ein bidirektionales Plasmid112, welches gleichzeitig für EGFP und für Luziferase kodiert, stabil unter den Promotor eingebracht, um anschließend eine dosisabhängige Regulation beider Proteine nachweisen zu können. Damit war die Funktion des Tetracyclintransaktivator auch in einer klonalen Rattenzelllinie bewiesen und gleichzeitig eine neue Zelllinie mit der Möglichkeit zu weiterführenden Untersuchungen der chondrogenen Differenzierung von uns etabliert worden.113
Die chondrogene Differenzierung unterliegt einer großen Anzahl von Regulatoren und Einflüssen114, so seien hier nur die oben angesprochenen Wnts genannt, die bereits in ungezählten Untersuchungen näher beleuchtet wurden, oder die im letzten Jahr publizierten Einflüsse von Hypoxie auf die Chondrogenese.115 Dabei sind diese regulierenden Einflüsse sowohl in der fetalen/embryonalen und juvenilen Knochen- und Knorpelentwicklung116 als auch in der Frakturheilung117 sowie der Pathogenese unterschiedlicher (Knochen-/Knorpel-) Erkrankungen118 bis hin zur Entstehung von Knochentumoren119 von großer Bedeutung.
108 Vergl. u. a. Bergwitz et al, 2001; Church, 2002; Weidinger et Moon, 2003; Topol et al., 2003; McEven et al., 2001; Kuhl et al., 2000; Galceran et al., 1999; Kengaku et al., 1998
109 Vergleich hierzu insb. Kapitel 4.2.5
110 Baron, Gossen, Bujard, 1997, Gossen et Bujard, 1992
111 Baron et Bujard, 2000; Bujard, 1999; Freundlieb, Baron et al., 1997
112 Baron, Freundlieb et al., 1995
113 Vergl. Bergwitz et al., 2000
114 s. u. a. DeLise, Fischer et Tuan, 2000
115 Schipani, 2005; Robins, Akeno et al. 2005
116 u. a. Kobayashi et Kronenberg, 2005; Karsenty et Wagner, 2002; Wagner et Karsenty, 2001; Huelsken et Birchmeier, 2001; Tickle et Munsterberg, 2001; Hall et Miyake, 1995
117 u.a. Zhong, Gersch et Hadjiargyrou, 2006; Hadjiargyrou, Lombardo, Zhao et al., 2002 ; Gregory, Gunn et al., 2005 ; Otto, Rao, 2004
118 u. a. Hartmann, 2006; Urano, 2006; Johnson et Rajamannan, 2006; Rawadi, Roman-Roman, 2005; Sen, 2005;
Ishikawa, 2005; Westendorf, Kahler et Schröder, 2004; Shum et Nuckolls, 2002; Sen, Chamorro, 2001
119 Uren, Wolf et al., 2004; Hoang, Kubo et al., 2004
Ein ferneres Ziel ist sicherlich die Detektion und gezielte Therapie von Tumorerkrankungen auf molekularer Ebene.120 So sind bereits mehrere Arbeiten veröffentlicht, in denen Wnts/Wnt-Antagonisten bzw. Mitglieder der Wnt-Pathways als Biomarker zur Detektion unterschiedlicher Tumoren121 oder zur Einschätzung der Prognose122 gemessen wurden. Aus dem immer mehr zunehmenden Verständnis der überaus komplexen Regulations-mechanismen auf molekularer Ebene ergeben sich zudem bereits erste (gedankliche) Ansätze zur Prävention und Therapie.123
Die von uns im zweiten Teil etablierte Zelllinie R-tTA-24 ist trotz vieler denkbarer Einsatzmöglichkeiten (bisher) für keine zusätzlichen Untersuchungen verwandt worden, so wurde der Tetracyclintransaktivator zwar auch in vitro in anderen Zelllinien124 eingesetzt und in seiner Form weiter modifiziert125, seine überwiegende Bedeutung gewinnt der tTA jedoch im Einsatz in vivo in transgenen Mäusen.126
120 Smalley et Dale, 2001
121 Urakami, Shiina, Enokida et al., 2006; Marsit, Karagas et al., 2005; Wong, Lo et al., 2002
122 u. a. Veeck, Niederacher et al., 2006 ; Dejmek, Leandersson et al., 2005; Dejmek, Dejmek et al., 2005;
Jonsson, Dejmek et al., 2002
123 u. a. Buchanan et DuBois, 2006 ; You, Kim, He et al., 2006; Caldwell, Jones et al., 2006; You, Kim et al., 2006; van Es et Clevers, 2005; Gregory, Gunn, Reyes et al., 2005; Reguart, He et al., 2005; Leris, Roberts et al., 2005; Zhou, An, Jiang et al., 2002; Zhu, Zheng et al., 2002; Sen, Chamorro et al., 2001;
124 Gallagher, Schoning et al., 2003; Howe, Skryabin et al., 1995
125 Dugray, Geay et al., 2001; Kafri, van Praag et al., 2000; Freundlieb, Schirra-Muller, Bujard, 1999; Shockett, Difilippantonio et al., 1995
126 u. a. Tietge, Kozarsky et al., 2003; Radomska, Gonzalez et al., 2002; Zhu, Zheng et al., 2002; Christen et von Herrath, 2002; McCloskey, Turnbull et al., 2005; Gossen, Freundlieb et al., 1995; Furth, St. Onge et al., 1994
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