Lombari, Patrizia (2006) Modulazione dell'espressione della Serpina Proteasi Nexina 1 (PN1) in sistemi cellulari neurali e gliali. [Tesi di dottorato] (Unpublished)

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Item Type: Tesi di dottorato
Language: Italiano
Title: Modulazione dell'espressione della Serpina Proteasi Nexina 1 (PN1) in sistemi cellulari neurali e gliali
Creators:
CreatorsEmail
Lombari, PatriziaUNSPECIFIED
Date: 2006
Date Type: Publication
Number of Pages: 96
Institution: Università degli Studi di Napoli Federico II
Department: Biochimica e biotecnologie mediche
PHD name: Biochimica e biologia cellulare e molecolare
PHD cycle: 17
PHD Coordinator:
nameemail
D’Alessio, GiuseppeUNSPECIFIED
Tutor:
nameemail
Pietropaolo, ConcettaUNSPECIFIED
Date: 2006
Number of Pages: 96
Uncontrolled Keywords: Proteasi Nexina I, Sistema Tet-Off, RNA Interference
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MIUR S.S.D.: Area 05 - Scienze biologiche > BIO/10 - Biochimica
Date Deposited: 04 Aug 2008
Last Modified: 29 Oct 2014 12:11
URI: http://www.fedoa.unina.it/id/eprint/626

Abstract

Le serpine (serine proteinase inhibitors) sono una superfamiglia di proteine caratterizzate da un comune ripiegamento e dal meccanismo d'inibizione che è del tipo substrato suicida. Il meccanismo di inibizione consiste nella formazione di un complesso irreversibile tra serpina e proteasi che una volta internalizzato viene degradato. Tra le serpine la Proteasi Nexina-1 (PN1), uno tra i maggiori inibitori della trombina, è presente principalmente nella matrice del sistema nervoso centrale. La sua funzione in questo distretto si pensa sia legato all’inibizione della trombina, che su cellule nervose differenziate causa retrazione dei neuriti. Inoltre, la PN1 è espressa con un particolare pattern spaziale e temporale durante lo sviluppo e l’organogenesi del sistema nervoso centrale, che fa supporre un suo coinvolgimento in questi processi. Per chiarire le funzioni della PN1 sono stati messi a punto un sistema per l’over-espressione inducibile e un sistema per il silenziamento genico di questa proteina. Nel primo caso è stato utilizzato come modello sperimentale il sistema tet-off in cellule PC12, cellule di feocromocitoma di ratto. Le cellule PC12 sono in grado di differenziarsi in neuroni mediante aggiunta di Nerve Growth Factor (NGF) o mediante deprivazione di siero. Il sistema tet-off permette di indurre l’over-espressione della proteina di interesse e studiarne gli effetti sulle cellule. Il plasmide per l’over-espressione inducibile contenente il cDNA della PN1 è stato cotransfettato insieme al plasmide contenente il gene per la resistenza all’igromicina nelle cellule PC12 tet-off. I cloni stabili sono stati isolati ed amplificati. Dall’analisi delle proteine, da lisati totali mediante western blot, e dall’analisi dell’mRNA, mediante RT-PCR semiquantitativa, alcuni dei cloni analizzati sono risultati inducibili. I cloni che hanno mostrato una maggiore produzione di PN1, successivamente all’induzione, sono stati analizzati morfologicamente durante il differenziamento, indotto sia dall’aggiunta di NGF al mezzo, sia da deprivazione di siero. Gli esperimenti condotti in presenza di NGF hanno mostrato un ruolo protettivo, nei confronti delle cellule differenziate, dell’over-espressione di PN1 in caso di aggiunta di trombina al mezzo di coltura. Gli esperimenti condotti in assenza di siero mostrano una diminuzione del numero di cellule differenziate in caso di over-espressione di PN1. Dai campioni analizzati morfologicamente durante il differenziamento sono stati preparati i lisati totali per osservare la produzione di PN1 mediante western blot. Da tale analisi è risultato un aumento di espressione della serpina indipendente dall’induzione del sistema tet-off. Tale aumento, da successivi esperimenti su cellule PC12 wild-type, è risultato dipendente sia dal tempo, sia dalla concentrazione di trombina. Per ottenere il silenziamento genico della PN1, è stato utilizzato l’RNA interference in cellule C6. A questo scopo sono stati clonati nel plasmide pAVU6+27 vari tipi di oligonucleotidi, in grado di attivare il processo di RNA interference, a valle di un promotore per piccoli RNA. I plasmidi ottenuti sono stati transfettati nelle cellule C6. A 48 ore dalla transfezione le cellule sono state raccolte per estrarre RNA totale e lisati totali. Dall’analisi dell’RNA mediante northern blot è risultata una diminuzione di mRNA di PN1 variabile a seconda dei costrutti utilizzati, comunque mai superiore al 60%. Dall’analisi dei lisati, mediante western blot, è risultata una diminuzione della proteina di circa 80% in tre dei campioni analizzati. Sebbene il processo di RNA interference non provochi un’efficiente degradazione del mRNA di PN1, c’è un efficiente blocco della traduzione

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