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

用痤疮丙酸杆菌（P.acnes）预处理大鼠，经腹腔注射细菌脂多糖（LPS），可造成实验性急性肝损伤模型。大量肝细胞坏死通过血清转氨酶活性升高来估计。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这种坏死。尽管使用胶原缀合的热响应聚合物如细胞基质需要7天来形成球状体，但是已经成功地通过旋转培养系统在24小时内快速开发了用于制备由痤疮丙酸杆菌预处理的实质和非实质肝细胞组成的球状体的新方法。观察到由10 μ g/ml LPS暴露48小时引起的转氨酶活性、TNF-α和CINC-1/gro/KC（IL-8）从这些球状体泄漏到培养基中的明显升高。这些结果表明，这种大鼠肝细胞的旋转共培养系统是一个有用的模型，作为一种替代动物实验的暴发性肝炎。因此，几个肝保护剂，硫普罗宁，布西拉明，儿茶素，戈米辛A，和锌卟啉，进行了筛选。这些药物在该肝炎模型中以及在体内显示出明显的保肝活性。另一方面，使用LEC大鼠肝细胞的多细胞球体检查铜在球体中的分布。当LEC大鼠球状体暴露于浓度大于50 μ M的CuC 12 24 h时，LEC大鼠球状体中的铜含量在CuC 12停药后24 h内仍保持高于SD大鼠球状体的铜含量。

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.