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Danesi, Andrea (2013) Studio della partecipazione di cellule staminali mesenchimali umane (hu-MSCs) nei processi di riparazione conseguenti al danno miocardico acuto in topi Balb/c Athymic Nude-Fox1nu. [Tesi di dottorato]

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Abstract (inglese)

Cardio Vascular Diseases (CVD) are the most common causes of death worldwide and, among these, the acute myocardial infarction (AMI) represents the leading one. Studies comparing the regenerative capacity of distinct stem cells populations suggest that bone marrow mesenchymal stem cells (BM-MSCs) could be the most indicated cells for these purpose. They have a multi-lineage differentiation, an immunomodulatory and self-renewal capacity as well as a high proliferative ratio.
3x106 BM-MSCs in 45 μl of D-MEM have been transplanted in Balb/c Athymic Fox1nu mice with different anesthetic e surgical protocols. The BM-MSCs has been characterized for mesenchymal (CD29, CD44, CD73, CD90 and CD105) and hematopoietic/endotelial (CD14, CD34 e CD45) markers, as well as evaluated for multi-lineage differentiation (osteogenic, chondrogenic and adipogenic) and immunomodulatory property. Before in vivo use, the BM-MSCs have been transfected with trans-activator of transcription (TAT)-GFP and characterized again with CD73, CD105, HLA ABC, CD40, CD54, CD80, CD86, CD106 and HLA-DR.
Supine position without intubation has reached the highest surgical successful ratio. AMI has been diagnosticated in 100% of hearts and atypical cells clusters have been observed in 13.85% (9/65) of cases. These atypical cells were positive for von Willebrand factor and α-SMA and negative for c-kit/CD117. Positive cells for human mitochondrial antigen and α-SMA has been observed in 2 cases by immunofluorescence technique (IF), whereas no positive cells were detected for GFP.
In conclusion, these study has refined the anesthetic and surgical protocols and increased the successful surgical ratio reducing the use of animals. Moreover, it has ameliorated the isolation and expansion protocols for BM-MSCs as well as transfection TAT-GFP technique without phenotype changes. BM-MSCs involvement in repair processes post-AMI has been evaluated with immunohistochemistry (IHC) even though the same results have not been reached with IF technique. This different sensibility suggests more investigations between IHC and IF in order to find out the best revealing technical options for BM-MSCs. Lastly, this project seems to confirm the participation of BM-MSCs in the reparative processes after AMI although their number is not adequate

Abstract (italiano)

Le malattie cardiovascolari (CVD) sono la prima causa di morte nel mondo e, tra queste, l’infarto miocardico acuto (IMA) ne è la prima causa singola. La ricerca sulla terapia rigenerativa dell’IMA ha evidenziato come le cellule staminali mesenchimali umane da midollo osseo (BM-MSCs) sono le più indicate a tale scopo in quanto presentano una multi-linearità differenziativa, una capacità immunomodulante, un’alta capacità auto-rigenerativa e un alto potenziale proliferativo.
In topi Balb/c Athymic Nude Fox1nu sono state trapiantate, a seconda dei gruppi e con diverse tecniche anestesiologiche di intubazione e di posizionamento dei soggetti ai fini del successo chirurgico, 3x106 BM-MSCs sospese in 45 μl di D-MEM prelevate da pazienti sani e caratterizzate per l’espressione dei markers mesenchimali (CD29, CD44, CD73, CD90 e CD105) e per la negatività dei markers ematopoietici/endoteliali (CD14, CD34 e CD45). Inoltre, sono state effettuate, e confermate, anche prove di differenziazione verso le linee mesenchimali (osteocitaria, condrocitaria e adipocitaria) e di immunomodulazione. Al momento dell’uso, le aliquote sono state trasfettate con GFP tramite la proteina trans-attivatore di trascrizione (TAT) e nuovamente caratterizzate per i marcatori CD73, CD105, HLA ABC, CD40, CD54, CD80, CD86, CD106 e HLA-DR, confermandone la staminalità.
Il miglior successo chirurgico si è avuto con l’animale in posizione supina anche in assenza di intubazione ma con anestesia gassosa via maschera.
L’IMA è stato confermato nel 100% dei casi mentre, nel 13,85% dei campioni (9/65) si sono osservati cluster cellulari atipici con colocalizzazione espressiva di von Willebrand factor e α-SMA e negatività per c-kit/CD117. L’IF ha evidenziato la presenza di cellule di origine umana in 2 casi a 24h, con una positività all’antigene mitocondriale umano e all’α-SMA. Inoltre la valutazione dell’espressione di GFP veicolato da TAT nei cuori trapiantati a 24h, 48h e 72h non ha permesso di evidenziare alcuna positività mediante IF.
In conclusione, questo progetto ha permesso di finalizzare una tecnica chirurgica di induzione dell’IMA aumentando il successo post-operatorio permettendo, di conseguenza, una riduzione degli animali utilizzati in accordo con i principi delle 3R (Replacement, Reduction, Refinement). Inoltre ha definito un’ottimale tecnica di prelievo, coltura ed espansione di BM-MSCs umane e una tecnica di transfezione con la TAT-GFP, che ne ha mantenuto invariato il fenotipo. Gli studi in IHC hanno permesso di valutare il successo del trapianto e la compartecipazione delle BM-MSCs umane in corso di AMI anche se il risultato può essere solo parzialmente confermato in quanto l’IF non ha evidenziato tali cellule nella stessa percentuale. Tale differente sensibilità suggerisce la necessità di un confronto approfondito tra IHC e IF come tecniche da impiegare per la rilevazione delle BM-MSCs GFP+. In ultimo, questo progetto sembra indicare la partecipazione delle BM-MSCs nei processi riparativi conseguenti a danno ischemico anche se non in quantità significativa

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Tipo di EPrint:Tesi di dottorato
Relatore:Zappulli, Valentina
Dottorato (corsi e scuole):Ciclo 23 > Scuole per il 23simo ciclo > SCIENZE VETERINARIE > SANITA' PUBBLICA E PATOLOGIA COMPARATA
Data di deposito della tesi:28 Gennaio 2013
Anno di Pubblicazione:31 Gennaio 2013
Parole chiave (italiano / inglese):acute myocardial infarction; mesenchymal stem cell; TAT protein; regenerative medicine
Settori scientifico-disciplinari MIUR:Area 07 - Scienze agrarie e veterinarie > VET/03 Patologia generale e anatomia patologica veterinaria
Struttura di riferimento:Dipartimenti > Dipartimento di Biomedicina Comparata ed Alimentazione
Codice ID:5582
Depositato il:14 Ott 2013 09:27
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