| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2001: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2000: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1999: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Research Abstract |
To investigate the regulation of tissue formation by extracellular matrix (ECM), we co-cultured isolated, rat hepatic pareiichymal and stellate cells on or in the different kinds of extracellular matrix, i.e. in non-coated polystyrene culture dishes, on/in type I collagen gel, or on Matrigel, in the presence of a long-acting vitamin C derivative (L-ascorbic acid 2-phosphate) and examined tliree-dimensional morphology of the hepatic tissue by scanning and transmission electron microscopy (SEM and TEM). The tissue of co-cultured cells in type I collagen gel formed striking similar three-dimensional hepatic cord-like structures, as seen in vivo, and bile canaliculus-like structures with complete set ofjunctional complexes, including tight junctions, adherens junctions, and desrnosornes, in addition to gap junction. However, neither the three-dimensional hepatic cord-like structures nor the complete set of thejunctional complex were developed in the co-cultures on the other ECM. These resu
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lts indicate that the extracellular matrix components regulate the three-dimensional organization of the co-cultured cells. The co-cultures of parenchymal cells with stellate cells in/on type I collagen gel formed the junctional complexes between them. However, formation of intercellular junctions between, the co-cultured stellate cells had not yet been elucidated. We cultured the stellate cells alone on the different kinds of ECM and examined the cultured cells by TEM with special reference to their intercellular adhesive structures. A larger number of intercellular adhesive structures were formed between the cultured stellate cells on type I collagen gel or in polystyrene dishes, when compared to the stellate cells on Matrigel. The intercellular adhesive structures were identified as adherens junctions by their characteristic iiltrastructural appearances. To clarify molecular components of these intercellular junctions, we cultured stellate cells on type I collagen gel and examined the localization of cadherin and catenin by immunofluorescence microscopy using a con focal laser scanning microscope. Immunofluorescence for pan-cadherin antibody, and α- and β- catenin antibodies was localized at the sites of contact regions between the cultured stellate cells. Thus, these data indicate the possibility that ECM regulates the tissue formation of cells through the formation of intercellular junctions. This report is the first description of the presence of intercellular junctions between hepatic stellate cells in mammals at the fine structural and molecular level. Less
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