Stereotaxie 2011 monograph Myc astrocytoma German glioblastoma Myc doctoralThesis Radke, Josefine Radke Josefine https://archiv.ub.uni-marburg.de/diss/z2011/0430/cover.png Pathologie Astrozytom https://doi.org/10.17192/z2011.0430 p53 Bei dem Glioblastoma multiforme handelt es sich um den häufigsten primären Hirntumor. Es handelt sich um einen sehr malignen Tumor, der Menschen in mittlerem Alter betrifft und sich durch eine infauste Prognose auszeichnet. Trotz einer Kombination aus Operation, Radiatio und verschiedenen Ansätzen der Chemotherapie, versterben die meisten Pateinten bereits im ersten Jahr nach Diagnosestellung. Es wird zwischen primären und sekundären Glioblastomen unterschieden. Das primäre Glioblastom entwickelt sich völlig neu (de novo). Es gibt weder radiologische, noch histologische Hinweise auf einen vorausgegangenen weniger malignen Tumor. Häufige Mutationen sind EGFR-Amplifikation, Deletion des p16INK4a und LOH (loss of heterozygosity) von 10q. Im Gegensatz dazu, entwickelt sich das sekundäre Gliobastom durch Progression eines niedriggradigen oder anaplastischen Astrozytoms. Bereits in den niedrigmalignen Vorläuferläsionen kommt es häufig zu einem Verlust des Tumorsuppressors p53. Das mittlere Erkrankungsalter liegt bei 45 Jahren. Das Glioblastom ist bevorzugt in den Großhirnhemisphären, insbesondere frontotemporal, lokalisiert. Makroskopisch zeichnen sich diese Tumoren durch eine bunte Schnittfläche aus. Charakteristisch sind gelbliche Nekrosen, Blutungen, grau-weiß aussehendes Tumorgewebe und diffuses infiltrierendes Wachstum. Rein histologisch können beide Tumorentitäten nicht voneinander unterschieden werden. Um den Ursprung des Tumors und dessen Tumorbiologie sowie Infiltration und Migration besser verstehen zu können und um neue therapeutische Strategien zu entwickeln, werden gute Mausmodelle benötigt. In der Vergangenheit gab es einige gute Ansätze zur Entwicklung eines solchen Modells. Leider gelang es keinem Modell das infitrative Wachstum im Gehirn nachzuempfinden, welches für die Läsion so charakterisch ist. Desweiteren gibt es Unstimmigkeiten in der wissenschaftlichen Gemeinschaft bezüglich der Ursprungszellen des Glioblastoms. Es konnte bisher nicht herausgefunden werden, ob es sich bei diesen Zellen um differenzierte Astroyzten oder aber neurale Progenitoren handelt. Holland et al. entwickelten ein vielversprechendes Glioblastom-Modell durch Gentransfer und eine daraus resultierende Überexpression von K-Ras uns Akt in neuralen Progenitoren. Des Weiteren publizierten Lassman et al. ein Modell mit differenzierten Astroyzten, die durch eine Überexpression von c-Myc einen undifferenzierten Phänotyp zeigten. Das Ziel der vorliegenden Arbeit war die Entwicklung eines Glioblastom-Modells mit differenzierten Astrozyten jedoch ohne eine Überexpression von K-Ras, da dieses eine für das Glioblastoma multiforme eher untypische Mutation darstellt. Diese Arbeit konzentrierte sich somit auf die Verwendung von p53-knockout Astrozyten. Diese wurden aus neugebornenen p53-knockout Mäusen gewonnen und kultiviert. Die kultivierten Astrozyten wurden mit einem Überstand von kultivierten und vorher transfizierten Phoenix-Zellen infiziert. Bei den Phoenix-Zellen handelt es sich um eine Zelllinie, die nach einer Transfektion mit bestimmten Plasmiden fähig ist, einen Retrovirus zu produzieren, der widerum für die Infektion anderer muriner Zellen zur Verfügung steht. Somit konnten in dieser Arbeit 3 verschiedene Zelllinien generiert werden: p53-knockout Astrozyten, die Akt, Myc oder Akt und Myc überexprimierten. Durch die Überexpression von Akt konnten die Zellen immortalisiert werden. Myc-überexprimierende Astrozyten zeigten ein rapides Zellwachstum und einen undifferenzierten Phänotyp. Verschiedene Passagen der Kulturen wurden immunhistochemisch gegen Ki67, GFAP und neurale Marker für Progenitoren wie Olig2, Nestin, Musashi-1 und CD133 gefärbt. Es kam zu einem Verlust des GFAP und einer positiven Immunhistochemie für die neuralen Marker der Progenitoren in späten Passagen. Mit Hilfe eines RNA Protection Assay (RPA) konnte auf RNA-Ebene bewiesen werden, dass es sich bei den Kulturen um reine Astrozytenkulturen handelt, die auch im Verlauf späterer Passagen keine neuronalen Marker, wie Synaptophysin, Calbindin oder Neurofilament exprimierten. Im Verlauf der Arbeit injizierten wir stereotaktisch Myc und Akt überxprimierende p53 knockout-Astrozyten (p53MA) in das rechte Striatum von Bl6- und RAG2-/- Mäusen. Nach 21 Tagen wurden die Hirne der Tiere entnommen und Kryostatschnitte angefertigt. Durch eine HE-Färbung konnte das Tumorwachstum nachgewiesen werden. Zusätzlich wurden die Schnitte immunhistochemisch gegen CD4, CD8 und Mac-1 gefärbt. 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Scientifically there are two different entities – the primary and secondary glioblastoma multiforme, which cannot be distinguished histologically. The tumor shows a rapid diffuse infiltrative growth, polymorphic nuclei, necrosis and angiogenesis. The primary glioblastoma develops de novo. Common mutations seen in primary glioblastomas are loss of heterozygosity of 10q, EGFR-ampilfication and deletion of p16INK4a. There is no evidence of a low-grade neoplasia in diagnostic imaging or histology. In contrast to that the secondary glioblastoma progresses from low-grade or anaplastic astrocytomas into a high-grade lesion. The mutation of the tumor suppressor p53 is often seen in those low-grade lesions. As already mentioned the glioblastoma multiforme is a very aggressive type of cancer which affects middle-aged people. In order to understand the cancers origin, its metastasis, infiltration and migration good tumor models are needed. Furthermore understanding the tumor biology is also necessary to develop new therapeutic strategies. There have been various approaches in the past to develop a mouse model for the glioblastoma mutiforme. Unfortunately they all were not able to show infiltrative growth in the brain, which is a strong characteristic of that lesion. Besides that there is a deep disagreement in the scientific community whether the glioblastoma multiforme originates from differentiated astrocytes or neural progenitors. Holland EC et al. were able to create a promising glioblastoma model through gene transfer resulting in an overexpression of K-Ras and Akt in neural progenitors. Furthermore Lassman et al. published a similar tumor model with differentiated astrocytes that showed an undifferentiated phenotype through overexpression of c-Myc. Following these tumor models this work wanted to create a glioblastoma model from differentiated astrocytes without the overexpression of K-Ras since the mutation of K-Ras is not a common mutation seen in glioblastoma multiforme. P53 knockout astrocytes from newborn p53 knockout mice were cultured. The cultured astrocytes were infected with a supernatant from cultured Phoenix cells. After transfection with certain plasmids this cell line is capable of producing a retrovirus that is able to infect murine cells. As a result this work was able to create 3 different cell lines: Astrocytes that over expressed Akt, c-Myc or Akt and c-Myc. Through the overexpression of Akt cells were immortalized. In contrast to that c-Myc seemed to be responsible for a rapid growth and an undifferentiated phenotype of the cultures astrocytes. The cultures astrocytes were stained against Ki67, GFAP and against neural progenitor marker like Olig2, Nestin, Musashi-1, CD133 showing a loss of GFAP and a positive immunostaining for the progenitor marker in late cell passages. Using RNA Protection Assay this work was able to proof that our cultures astrocytes did not overexpress neuronal markers like for example neurofilament, synptophysin or calbindin. Furthermore stereotactic injection was used to place p53 knockout astrocytes that overexpressed Akt and c-Myc in the striatum of Bl6- and RAG2-/- mice. After 21 days animals were sacrificed, cryosections of the brains were made and the resulting tumor was stained with hematoxylin and eosin. To finish tumor sections were stained against CD4, CD8 and Mac-1 showing no infiltration of the immune system in the animals´ brains. Akt application/pdf stereotaxis Publikationsserver der Universitätsbibliothek Marburg Universitätsbibliothek Marburg