To study the stimulating effects of retinoic acid on the primary culture of immature rat hepatocyte, we examined the changes in the bile canaliculi formation by observing the cytokeratins 55 kDa and 49 kDa. The formation of the bile canaliculi sheaths was our end point of differentiation. We obtained monolayer hepatocyte culture from 14-day old male Wistar Charles River rats, using the two-step collagenase perfusion method of Seglen (1976), as adapted to preweanling rats by Deschenes et al (1980). All-trans retinoic acid 10$\sp{-5}$M dissolved in 0.1% dimethyl sulfoxide was added to the treated cultures. All cultures were maintained for 48 hours. The organization of cytokeratin was visualized by immunofluorescence using monoclonal antibodies to cytokeratins 55 kDa and 49 kDa. Under the influence of retinoic acid, the number and size of bile canaliculi formed were significantly increased. The bile canaliculi formed and a more complex architecture, such as ramification. Theses observations were confirmed by un-embedded whole mount electron microscopic studies on detergent extracted preparations. In retinoic acid treated colonies, the bile canaliculi appear to be functional as demonstrated by the cellular uptake followed by polarization and secretion of the fluorescein diacetate dye. Gel electrophoresis showed an apparent disparity in the relative quantity of cytokeratin 55 kDa and 49 kDa, especially in the control group. This inequality may be attributed to a selective increase in proteolysis during preparation. It is concluded that the addition of retinoic acid to the media stimulates the differentiation in hepatocyte monolayer cultures. It is possible that retinoic acid induces modification in the hepatocyte gene expression and the formation of bile canaliculi could be associated with changes in the expression of the cytokeratin genes. The two cytokeratins would be coexpressed in response to the treatment with retinoic acid.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/6953 |
| Date | January 1993 |
| Creators | Claude, Annie. |
| Contributors | French, S., |
| Publisher | University of Ottawa (Canada) |
| Source Sets | Université d’Ottawa |
| Detected Language | English |
| Type | Thesis |
| Format | 204 p. |
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