Expression of desmin, GFAP and ?-SMA in human and RAT pancreatic stellate cells / 解放军医学杂志

ABSTRACT

Objective To investigate the dynamic changes in expression of cell markers desmin, glial fibrillary acidic protein (GFAP), and (-smooth muscle actin (?-SMA) in primary cultures of human and rat pancreatic stellate cells (PSCs). Methods PSCs were isolated from human as well as rat pancreas using Nycodenz discontinuous density gradient centrifugation following digestion with combination of collagenase IV, Pronase E and DNase I, and purified by centrifugal elution techniques. Freshly isolated cells were examined by laser scanning confocal microscopy for vitamin A autofluorescence, by immunostaining for desmin, GFAP, and ?-SMA. Expression of ?-SMA was as well measured by Western analysis. Procollagen ?1(Ⅰ) mRNA expression was analyzed by Northern analysis. Results The purity of rat PSCs obtained by centrifugal elution were above 95%. More than 85% of either freshly-isolated human or rat PSCs displayed positive vitamin A autofluorescence. Rat PSCs stained positively for desmin and GFAP and negatively for ?-SMA, whereas human PSCs were negative for either desmin, GFAP or ?-SMA. During the process of primary culture, rat PSCs were positive for ?-SMA at 3d and completely transformed from quiescent state to myofibroblast-like phenotypes at 7d, which negatively or scarcely expressed desmin and GFAP, but fully expressed the ?-SMA protein and procollagen ?1(Ⅰ) mRNA, similarly to the settings of human PSCs. Conclusions Human and rat PSCs could be successively isolated in above 95% purity by combining gradient centrifugation with following centrifugal elution techniques. The results show some species differences in desmin and GFAP expression between freshly-isolated human and rat PSCs. Both of which, however, acquire a myofibroblast-like phenotype largely expressing ?-SMA protein and procollagen ?1(Ⅰ) gene in culture.