A Study of Expression and Function of Jagged 1 Gene Relating the Development of Intrahepatic Bile Ducts and Bile Ductule Proliferation in Hepatic Fibrosis.

在这一年（1999年），我们确定了培养大鼠肝细胞超过8周的条件培养基。在大鼠小肝细胞长期培养培养基中加入铁、锌、铜配制成培养基。使用该培养基，我们证实细胞持续表达CK8、AFP和白蛋白，部分细胞转为表达胆管上皮标志物ck19。随着培养时间的延长，CK 19阳性细胞的比例增加：1周时为20%，2周时为25%，4周时为33%。我们检测了这些培养的肝细胞中Jagged 1 mRNA的表达。虽然RT-PCR检测到表达，但northern blot分析未观察到信号。为了进一步讨论，RNase保护试验或原位杂交应该是必要的，但它们尚未进行。另一方面，我们发现血栓反应蛋白-1 （TSP）在胆道闭锁肝纤维化灶中高表达。通过对TSP、TGF-β及其受体的定位研究，我们发现TSP在肝脏伪胆管增殖和纤维化中起促进作用（Am J Pathol, in revise）。从这个角度出发，我们通过将TGF-β添加到培养基中来检测其对肝细胞表型改变的影响。结果显示，ck19阳性细胞比例显著增加，CK8和白蛋白信号强度增强。考虑到TSP是潜伏TGF-β的主要激活因子，我们目前正在研究TSP对培养肝细胞表型变化的影响，采用抑制肽对潜伏TGF-β激活位点的作用。

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.