初代培養肝細胞の形態学的研究特に細胞接着と毛細胆管様構造の出現について
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We studied the mechanisms involved in reconstruction of cell surface polarity in cultured hepatocytes using rhodamine-phalloidin staining and 2D2 : a monoclonal antibody against an antigen located on the bile canalicular (BC) surface. Rat hepatocytes were isolated by a collagenase-perfusion technique and cultured on type I collagen-coated cover glasses under serum-free conditions. First, the effects of Ca concentration of culture medium on cellular polarity were examined using the incidence of BC-like structures as a parameter. Under low Ca concentrations of 0 and 0.2 mM, very few BC-like structures were detected even after 24 hrs in culture, whereas under high concentrations of 0.8 and 1.8 mM Ca, such structures were often detected at sites of cell-cell contact. Second, distribution of the antigen was examined in hepatocytes cultured under low (0.2 mM) and high (1.8 mM) Ca conditions by using a monoclonal antibody (2D2) which recognizes the bile canalicular domain of rat hepatocytes. Under low Ca conditio, the antigen detected by 2D2 was distributed over the entire surface of the hepatocytes. On the other hand, under high Ca concentration the antigen was shown to localize at the sites of cell-cell contact between adjacent cultured hepatocytes. Phase contrast microscopy and scanning electron microscopy revealed that under low Ca concentration, hepatocytes scarcely make contact with each other even at 24 hrs after plating, whereas under high Ca concentration, most hepatocytes connected with adjacent hepatocytes. These observations showed that low Ca concentration in the medium inhibits cell-cell contact between cultured rat hepatocytes, indicating the importance of cell-cell contact in reconstruction of cell surface polarity and BC reorganization. Scanning electron microscopy also showed that the BC-like structures found between adjacent hepatocytes are intercytoplasmic cysts and the structures have no opening to the cell surface.
- 札幌医科大学の論文
- 1989-10-01
札幌医科大学 | 論文
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