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PLoS ONE 2013

Hypoxia pathways and cellular stress activate pancreatic stellate cells: development of an organotypic culture model of thick slices of normal human pancreas.

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Вход / Регистрация
Линкът е запазен в клипборда
Vinciane Rebours
Miguel Albuquerque
Alain Sauvanet
Philippe Ruszniewski
Philippe Lévy
Valérie Paradis
Pierre Bedossa
Anne Couvelard

Ключови думи

Резюме

Pancreatic stellate cells (PSC) are involved in fibrogenesis and oncogenesis by modulating the extracellular matrix.

OBJECTIVE

To evaluate the effect of cellular stress on PSC activation using a model of normal human pancreatic tissue slices culture preserving the microenvironment.

METHODS

Thin sections (300 μm) of normal human pancreas were cultured under hyperoxia (90% O2) during 72 hours. Viability and morphological analysis were performed at baseline, H24, H48 and H72. Cell differentiation (insulin, trypsin, CA9 and CK7), hypoxia (HIF1-α), apoptosis (caspase-3), proliferation (Ki67), TGF-β expression and PSC activation (smooth muscle actin (SMA), nestin) were assessed using immunostaining, longitudinally. Control experiments were performed under normoxic conditions (21% O2).

RESULTS

Thirty sections per specimen (n=10) were cultured. Hypoxia pathways were activated by the higher expression of HIF1-α at H48 and H72. Apoptosis was limited with only rare acinar cells expressing of the caspase-3 at 48 and H72 (NS). Morphological analysis showed gradual appearance of acinoductal metaplasia, proven by CK7 expression and ductal phenotype of dedifferentiated acini. Transdifferentiation of PSC was shown by de novo SMA immunochemistry at H24 and H48. Expression of Ki67 index identified significant proliferation of activated PSC (double immunostaining Ki67-SMA) at H48 and H72 (p=0.02). In vitro culture of normal human pancreas thin sections is feasible with optimized cell viability at 72 hours. This model of culture in hyperoxic conditions provides evidences that cellular stress may rapidly induce transactivation of PSC with ducto-acinar metaplasia.

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