| Co-Investigator(Kenkyū-buntansha) |
OZONO Keiichi Osaka Medical Center and Research Institute for Maternal and Child Health, Department of Environmental Medicine, Professor, 部長 (20270770)
MUSHIAKE Sotaro Osaka University Graduate School of Medicine, Assistant Professor, 医学系研究科, 助手 (90291947)
大園 恵一 大阪府立母子保健総合医療センター研究所, 部長
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| Budget Amount *help |
¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1999: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1998: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
In this year (1999), we determined the conditioned medium in which rat hepatocytes can be cultured for more than 8 weeks. The medium was prepared by adding Fe, Zn, and Cu to that made for long term culture of rat small hepatocytes. Using this medium, we confirmed that the cells keep the exspression of CK8, AFP, and albumin constantly, and a part of them become to express CK 19 which is a marker of bile duct epithelium. The percentage of CK 19-positive cells increased as the cultivation term : 20% at 1 week, 25% at 2weeks, and 33% at 4 weeks.
We examined the expression of Jagged 1 mRNA in these cultured hepatocytes. Although the expression was detected by RT-PCR, no signal was observed on northern blot analysis. For further discussion, RNase protection assay or in situ hybridization should be necessary, but they are not performed yet.
On the other hand, we showed that thrombospondin-1 (TSP) is highly expressed in fibrotic lesion of the liver of biliary atresia. Studying the localization of TSP, TGF-β, and its receptor, we demonstrated that TSP plays a precipitative role for pseudo-bile ductule proliferation and fibrogenesis in the liver (Am J Pathol, in revise). From this viewpoint, we examined the effect of TGF-β on the phenotypic alteration of hepatocytes by adding it to the culture medium. In result, significant increment of the ratio of CK 19-positive cells and augmentation of signal intensity of CK8 and albumin were observed. Considering that TSP is a major activating factor of latent TGF-β, we are now studying the effect of TSP on the phenotypic change of the cultured hepatocytes, using inhibitory peptide against the activationg site of latent TGF-β.
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