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Bertalot, Thomas (2014) Microenviroment modulation on plasticity of Enteric Nervous System derived cells. [Tesi di dottorato]

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Abstract (inglese)

Growth factors such as EGF, bFGF and GDNF play an essential role in the ENS development and homeostasis. In vivo conditions which provide a reduction or absence of these factors promote the development of diseases such as intestinal agangliosis. Thus two models of in vitro culture that simulate the physiological condition (SM) and that of agangliosis (BM) was evaluated. ENS-derived cells (ENSc) were isolated from the myenteric plexus of the Sprague Dawley rats [Schaefer et al., 1997]. Particular attention was given to the regulation mechanisms mediated by TLR4 and Wnt signalling. At time of isolation, immunophenotypical characterization by flow cytometry showed the expression of stem cell (SOX2, NANOG, and CD34), neuronal and glial (p75, Nestin, GFAP) markers. Culturing in SM and BM showed a specific modulation of neuronal and glial differentiation and a greater responsiveness mediated by Frizzled 9 (SM) and TLR4 (BM) was observed. Moreover, a neuronal subpopulation co-expressed the receptors TLR4 and Frizzled-9 suggesting that this cell population may be involved in the maintenance of homeostasis and in the regulation of inflammatory processes. Furthermore, only SM cultures formed neurosphere-like structures. Wnt3a stimulation activated the canonical Wnt pathway through Frizzled-9 and qRT-PCR analysis demonstrated anti-inflammatory activity. In addition, a cross-talk between LPS/TLR4 and Wnt pathway was demonstrated by western blotting. Differentiation processes are also influenced by the extracellular matrix (ECM). In this study, the modulatory effect induced by ECM was evaluated assessing an in vitro model: ENS-derived cells cultured on a decellularized ECM of adult rat jejunum. Acellular matrixes (AMs) were provided using a modified enzyme detergent decellularization protocol [Meezan et al., 1975]. Histological study, SEM and quantification of residual DNA verified the complete decellularization. Immunofluorescence and western blotting demonstrated that the structural proteins such as collagen I, III , IV and laminin were preserved. After culturing ENSc on AMs for 7 and 14 days, the ECM demonstrated to influence the ENSc spatial organization, exerting a synergic effect with the factors present in the culture medium. In fact, only the AM cultures with SM, showed ganglion-like structures partially interconnected and positive for βIII tubulin. ENSc cultured on acellular matrix may represent a useful in vitro model for toxicological and pharmacological studies as well as a possible tissue scaffold in regenerative medicine.

Abstract (italiano)

E’ noto che i fattori di crescita quali EGF, bFGF e GDNF giocano un ruolo essenziale nello sviluppo e nell’omeostasi del sistema nervoso enterico (SNE). Condizioni in vivo che prevedono un calo o un’assenza del loro apporto, favoriscono lo sviluppo di patologie quali agangliosi intestinale. In questo lavoro di tesi, allestendo due modelli di coltura in vitro che simulano la condizione fisiologica (SM) e quella di agangliosi (BM) mediante coltura in presenza (SM) o meno di fattori di crescita (BM) è stata oggetto di studio la risposta differenziativa di cellule isolate da plesso mienterico di ratto Sprague Dawley (ENSc) [Schaefer et al., 1997]. In particolare, veniva prestata attenzione ai meccanismi di regolazione della risposta cellulare mediata dal segnale TLR4 e Wnt. Lo studio di caratterizzazione dell’immunofenotipo mediante citofluorimetria evidenziava nelle popolazioni estratte l’espressione di marcatori di staminalità (SOX2, Nanog e CD34) e di linea neuronale e gliale (p75, Nestina, GFAP). Inoltre, si evidenziava la presenza di una sottopolazione con caratteristiche neuronali che co-esprimeva i recettori TLR4 e Frizzled-9, suggerendo un ruolo nella regolazione del processo infiammatorio. La coltura in terreno SM e BM dimostrava di modulare in maniera specifica il differenziamento neuronale e gliale delle ENSc e di conferire una maggiore reattività mediata dal Frizzled 9 (coltura SM) e dal TLR4 (coltura BM). Inoltre, l’analisi di microscopia ottica evidenziava la formazione di strutture del tipo neurosfere solo nelle colture trattate con terreno standard. Lo stimolo indotto dal Wnt3a risultava efficace nell’attivare la via di segnale canonica di Wnt attraverso il recettore Frizzled 9 e, all’analisi di espressione genica mediante qRT-PCR, dimostrava un’attività di tipo anti-infiammatorio. Inoltre, mediante uno studio di western blotting, si dimostrava che la via pro-infiammatoria del TLR4 cross-reagiva con il segnale Wnt attivandolo. E’ noto che il processo differenziativo è fortemente condizionato dalla matrice extracellulare. In questo studio l’effetto modulatorio indotto dalla matrice sulla risposta differenziativa delle cellule ENSc è stato valutato utilizzando matrice acellularizzata (AM) di tessuto intestinale di ratto. La preparazione dello scaffold ha previsto 5 cicli ripetuti di decellularizzazione del trattamento modificato detergente enzimatico di Meezan [1975]. La completa decellularizzazione del tessuto veniva verificata mediante studio istologico, analisi di microscopia elettronica a scansione (SEM) e quantificazione del contenuto di DNA residuo. All’analisi di immunofluorescenza e western blotting, le proteine strutturali quali collagene I, III, IV e laminina risultavano preservate al termine della decellularizzazione. Dopo coltura per 7, 14 giorni delle cellule ENSc sulla matrice, AM dimostrava di condizionare l’organizzazione spaziale delle cellule ENSc esercitando un effetto specifico differenziativo in sinergia con i fattori di crescita. Infatti, solo le matrici mantenute in terreno SM mostravano una caratteristica organizzazione delle cellule ENSc in strutture interconnesse di tipo simil-gangliare esprimenti il marcatore neuronale βIII tubulina. Le colture di ENSc su matrice acellulare possono rappresentare un valido modello in vitro per studi tossicologici ed un possibile sostituto tessutale nella medicina rigenerativa.

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Tipo di EPrint:Tesi di dottorato
Relatore:Conconi, Maria Teresa
Correlatore:Di Liddo, Rosa
Dottorato (corsi e scuole):Ciclo 26 > Scuole 26 > BIOLOGIA E MEDICINA DELLA RIGENERAZIONE
Data di deposito della tesi:30 Gennaio 2014
Anno di Pubblicazione:30 Gennaio 2014
Parole chiave (italiano / inglese):LPS, Frizzled, Wnt, ENS, NF-kb
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/16 Anatomia umana
Struttura di riferimento:Dipartimenti > Dipartimento di Scienze del Farmaco
Codice ID:6762
Depositato il:14 Nov 2014 14:08
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