| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1988: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1987: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
Retinoids have long been recognized as an essential nutrient, but the molecular mechanism of their functions has not been fully understood. Retinoids are potent agents controlling cellular differentiation. The local application of retinoic acid (RA) to chick limb bud, for example, mimics the function of polarizing region. Lack of appropriate experimental system, however, had hindered further analysis at molecular level. Of many embryonal carcinoma cell lines, F9 is special since its differentiation is triggered only after treating with RA. RA-primed F9 cells differentiate into parietal endoderm-like cells when exposed to dibutyryl cAMP, and into visceral endoderm-like cells when allowed to aggregate. In this study, functions of retioids in cell differentiation were analyzed with F9 cells as a model system.
We previously showed that the differentiation of RA-primed cells into parietal endoderm-like cells is reversible dependind on the presence of dibutyryl cAMP. The reversibility was fur
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ther confimed by northern hybridizarion of RNA exracted from variously treated F9 cells with 6 cDNA clones screened from libraly of parietal endoderm-like F9 cells. Among these clones, a clone coding laminin b mRNA was included. The differntiation of F9 cells, as monitored by the secretion of basement membrane proteins, was biphasic depending on the dose of RA used. Secretion of laminin a reached a plateau at 10 nM and increased again at more than 100 nM RA. This biphasic pattern of differentiation was stable for a long period of time but the addition of 1 mM dibutyryl cAMP changed it to monophasic. Fractionation of F9 aggregates, grown by culturing clumps of 1-50 RA-primed F9 cells in a sppiner bottle , on parcoll density gradients revealed that only aggregates with diameter larger than 0.18 mm developed embryoid dodies and secreted a-fetoprotein(AFP). This size is comparable to that of egg cylinders when they first develop AFP-producing vieceral endoderm. This suggests that visceral endoderm maturation is triggered when the size of core cell mass exceeds a critical threshold. Less
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