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Titolo/Abstract/Parole chiave

MECCANISMO TRASFORMANTE DEI VIRUS ONCOGENI HPV IN CELLULE EPITELIALI UMANE DELLA CERVICE UTERINA

Bosi, Silvia (2012) MECCANISMO TRASFORMANTE DEI VIRUS ONCOGENI HPV IN CELLULE EPITELIALI UMANE DELLA CERVICE UTERINA. Tesi di Dottorato , Università degli studi di Ferrara.

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    Abstract

    The aims of this study are: (i) to set up a culture protocol to derive pathological and normal keratinocytes from small human cervical intraneoplasia (CIN) biopsies and normal uterine cervix (NUC) tissues; (ii) to investigate the expression prfile induced by transforming mechanisms due to human papillomavirus (HPV) in CIN keratinocytes clones from different CIN grades, CIN1-3/CIS, using the microarray technique. In this study a new approach to establishing CIN and NUC keratinocyte cultures from small tissue fragments was developed. CIN specimens and corresponding normal tissues, which were used as controls, were digested with collagenase. Tissue-derived fibroblasts and keratinocytes were co-cultured in calcium serum medium conditions. Primary co-cultures were subsequently sub-cultured. Single keratinocyte clones from primary co-cultures were expanded using a culture medium which was optimized in our laboratory. Primary clones from CIN and normal tissues, as well as expanded clones, were tested by immunofluorescence for epithelial and cervical markers such as 5-, 14-, 17- and 19-keratins, and p63. Our results indicate that primary CIN, as well as normal keratinocyte clones, can be obtained in a co-culture system with live human fibroblasts in calcium and serum medium conditions. The clone number varied depending on the grade of CIN lesions from which clones originated. CIN and normal keratinocytes from single clones, when cultured with our new medium, grew at a high rate with uniform morphology. The second objective of our study was to identify genes that are related to progression and transformation inducted by HPV in CIN lesions using the microarray technique. One clone of CIN1; two clones of CIN2 and corresponding normal clones; one clone of CIN3 and corresponding normal; one clone of in situ cervical cancer (CIS), were investigated with microarray technique for analysis of differential gene expression profile. The expression of ~ 41,000 genes were compared and 598 genes resulted differentially expressed between CIN and NUC. Among 598 genes, 152 genes were upregulated and 262 were down-regulated. We selected some genes with a greater than 2 fold difference between CIN and NUC for validation of microarray results with RTqPCR. Some genes were selected as possible candidates involved in progression from CIN1 to CIN3/CIS. Among up-regulated we identified PFKFB3, FOXD2, HOXB3, HOXB4, HOXA3, HOXA4, HOXA5, EMX2, WNT4 and among down-regulated APOBEC3B, APOBEC3F, PTPN3, CLDN11, S1PR5, IL1B. These results will need to be validated in a larger series of dysplasia to identify whether our selected genes could play a role in cervical tumorigenesis. In conclusion, our study reports, for the first time, a co-culture system of keratinocytes from minute CIN and normal cervix biopsies and offers a potential of developing newer diagnostic markers and therapeutic targets for the prevention and treatment of the cervical carcinoma.

    Tipologia del documento:Tesi di Dottorato (Tesi di Dottorato)
    Data:8 Marzo 2012
    Relatore:Martini, Fernanda
    Coordinatore ciclo:Cuneo, Antonio
    Istituzione:Università degli studi di Ferrara
    Dottorato:XXIV Anno 2009 > FARMACOLOGIA E ONCOLOGIA MOLECOLARE
    Struttura:Dipartimento > Medicina clinica e sperimentale
    Soggetti:Area 05 - Scienze biologiche > BIO/13 Biologia applicata
    Parole chiave:HPV, CIN, colture cellulari, microarray
    Depositato il:26 Feb 2013 14:50

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