IMMUNOPHARMACOLOGICAL STUDIES ON FULMINANT HEPATITIS MODEL USING THE SPHEROID CULTURE OF RAT LIVER CELLS

将细菌脂多糖(LPS)注入痤疮丙酸杆菌(P.acnes，P.acnes)预处理的大鼠体内，建立实验性急性肝损伤模型。通过血清转氨酶活性的升高来判断肝细胞的大量坏死。在这项研究中，我们通过使用原代共培养的大鼠肝细胞在体外模型中产生了这种坏死。利用旋转式培养系统成功地在24 h内成功地制备了一种新的方法，该方法由经痤疮杆菌预处理的厚壁和非厚壁肝细胞组成，虽然使用胶原结合的热反应聚合物(如细胞基质)需要7天才能形成球体，但在24 h内就成功地形成了球体。10µg/mlLPS作用48h后，转氨酶活性、肿瘤坏死因子-α和CINC-1/GRO/KC(IL-8)从这些球体渗入培养上清液后明显升高。这些结果表明，这种大鼠肝细胞旋转共培养体系是一种替代重型肝炎动物实验的有用模型。因此，我们筛选出了硫普罗宁、布比拉明、儿茶素、戈米菌素A和锌卟啉等几种肝脏保护剂。这些药物在该肝炎模型中以及在活体中都显示出明显的保护肝脏的活性。另一方面，利用LEC大鼠肝细胞的多细胞球体来检测铜在球体中的分布。当LEC大鼠球体暴露于浓度大于50微米的CuC12 24小时后，停用CuC12后LEC大鼠球体中铜含量仍维持在24小时内，且高于SD大鼠球体中铜含量。

An experimental acute liver injury model can be produced by the injection of bacterial lipopolysaccharide (LPS) into Propionibacterium acnes (P.acnes) pretreated rats. The massive liver cell necrosis is estimated by elevation of serum transaminase activities. In this study, we produced this necrosis in an in vitro model by using primary co-cultured rat liver cells. A novel method for the preparation of spheroids consisting of P.acnes pretreated parechymal and nonparechymal liver cells has been successfully developed quickly by the rotary culture system within 24 h although it takes 7 days to form the spheroid using a collagen-conjugated thermo-responsive polymer such as a cell substratum. Clear elevations of transaminase activities, TNF-a and CINC-1/gro/KC (IL-8) leaked from these spheroids into the medium caused by the exposure of 10 mug/ml LPS for 48 h were observed. These results suggest that this rotary co-culture system of rat liver cells is useful model as an alternative to animal tests for fulminant hepatitis. Therefore, several hepato-protective agents, tiopronin, bucillamine, catechin, gomisin A,and zinc porphyrins, were screened. These agents showed obvious hepato-protective activities in this hepatitis model as well as in vivo.On the other hand, the distribution of copper in spheroids was examined using multicellular spheroids of LEC rat liver cells. When LEC rat spheroids were exposed to CuC12 at concentrations of more than 50 muM for 24h, copper contents in LEC rat spheroids were maintained for 24h after the withdrawal of CuC12 and higher than those of the SD rat spheroid.