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Gonzalez Campos, Abril (2014) Isolation of human parathyroid cell type as a tool for investigating the mechanisms of human primary aldosteronism. [Tesi di dottorato]

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Abstract (inglese)

Background. The detection of the mineralocorticoid receptor in parathyroid cells alongside recent evidences implicating the parathyroid hormone (PTH) in overproduction of aldosterone in primary aldosteronism in spite of suppression of the renin-angiotensin system indicate a relationship between parathyroids and the adrenocortical zona glomerulosa. The investigation of this relationship has been hampered due to lack of parathyroid cell line suitable for in vitro studies.
Objective. To develop a technique to obtain a primary culture of human parathyroid cells with high yield and purity and to obtain subpopulations of chief and oxyphil cells.
Results. After digestion of tissue from parathyroid adenomas, we isolated cells using an immunomagnetic bead method to remove fibroblasts and filtration devices to isolate cells by size. real-time qPCR evidenced expression of specific markers of parathyroid cells in cultured cells until day 10. At protein level, we found that some cells showed marked PTH immunostaining, whereas the remaining cells showed only a faint signal, suggesting the presence of two cell types. After loading cells with tetramethylrhodamine-methyl-ester (TMRM), we found two cell types, which differed for the fluorescent signal intensity.
Conclusions. We found an optimal protocol to obtain a culture of human parathyroid cells that includes immunobinding-based technique and cellular filtration. Cultured cells retain their characteristic expression profile until day 10. Analysis of the mitochondrial uptake of TMRM and cell markers allows identification of two cell types forming the parathyroid tissue, i.e. oxyphil and chief cells

Abstract (italiano)

Nelle cellule di paratiroide è espresso il recettore mineralcorticoide Inoltre, nell’iperaldosteronismo primario i livelli sierici di ormone paratiroideo (PTH) sono aumentati. Questi dati unitamente al fatto che il PTH stimola la secrezione di aldosterone suggeriscono una relazione tra paratiroidi e zona glomerulosa del surrene. L'analisi di tale relazione è stata ostacolata finora dalla mancata disponibilità di una linea cellulare di paratiroide idonea per gli studi in vitro.
Obiettivo. Investigare la posibilita di sviluppare una linea cellulare di paratiroide umana che permetta di investigare se, e attraverso quali meccanismi, la secrezione di PTH sia regolata dall’aldosterone.
Risultati. Dopo digestione di tessuto ottenuto da adenomi di paratiroide sono state isolate le cellule utilizzando due metodi: 1) immunoseparazione mediante biglie incubate con CD90, anticorpo di membrana specifico per i fibroblasti, 2) separazione mediante filtrazione. L’espressione di marcatori propri delle cellule di paratiroide è stata confermata tramite real time qRT-PCR, microscopia elettronica e immunocitochimica nelle cellule in coltura fino al decimo giorno.
Su la base del grado di immunostaining per il PTH e all'intensità di segnale dopo caricamento delle cellule con tetramethylrhodamine-metil-estere (TMRM), una sonda specifica per misurare il potenziale di membrana dei mitocondri.,due fenotipi cellulari sono stati identificati.
Conclusioni. Con il protocollo messo a punto nel nostro laboratorio per isolare e coltivare cellule di paratiroide umana si è potuto accertare che le cellule della coltura primaria mantengono il loro fenotipo, caratterizzato dall'espressione dei marcatori propri delle cellule di paratiroide, fino al decimo giorno. Un processo di filtrazione, seguito dall'espressione dei marcatori cellulari e dall’analisi della captazione di TMRM mitocondriale consentono la separazione e l'identificazione delle due sottopopolazioni di cellule che formano il tessuto paratiroideo, cellule ossifile e cellule principali

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Tipo di EPrint:Tesi di dottorato
Relatore:Seccia, Teresa Maria
Dottorato (corsi e scuole):Ciclo 26 > Corsi 26 > Ipertensione Arteriosa e Biologia Vascolare
Data di deposito della tesi:29 Gennaio 2014
Anno di Pubblicazione:29 Gennaio 2014
Parole chiave (italiano / inglese):paratiroide/parathyroid cell type
Settori scientifico-disciplinari MIUR:Area 06 - Scienze mediche > MED/50 Scienze tecniche mediche applicate
Struttura di riferimento:Dipartimenti > Dipartimento di Medicina
Codice ID:6598
Depositato il:14 Nov 2014 10:45
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