Development of Cell theraypy for the end Stage of Hepatic Failure with or without Congenital Disease

为了探索终末期肝衰竭合并或不合并先天性疾病的细胞治疗方法，我们研究了1)肝干细胞的生物学，2)诱导肝细胞分化的因素，3)通过基因转导增加分化功能，4)免疫抑制对移植细胞存活的影响。利用Crigler-Najjar综合征模型动物Gunn高胆红素血症大鼠和猪未成熟肝细胞，异种猪细胞肝内移植在FK506免疫抑制下降低胆红素。作为一种新的肝干细胞来源，我们使用了从剖宫产获得的人胎盘中分离的人羊膜干细胞(人材料实验是在国家一级儿童医院伦理委员会许可下进行的)。)。羊膜上皮细胞白蛋白阴性，培养的羊膜上皮细胞白蛋白、α-胎儿蛋白阳性。白蛋白分泌…用培养上清液进行酶联免疫吸附试验可显示更多的n。将羊膜上皮细胞移植到SCID小鼠肝脏后，细胞已整合到肝脏结构中，存活至少2周，白蛋白和α-胎儿蛋白染色呈阳性。目前，我们正在研究用转*DP-葡萄糖醛酸基转移酶基因的羊膜上皮细胞治疗Gunn高胆红素血症大鼠。我们尝试用大鼠胎鼠的匀浆或提取物(16±1天)维持其功能。提取物匀浆与大鼠血浆混合后形成凝胶，凝胶内有肝细胞培养，具有良好的形态特征。在分化功能方面，从培养开始到第20天，一直检测到脱氨活性。105,000×g胎儿匀浆上清液仍具有使肝细胞保持良好形态的活性。免疫抑制：Fas-L基因转导对移植细胞的免疫抑制作用。表达Fas-L的肝脏存活时间约为未经处理的对照肝脏的3倍。较少

With an aim of the development of cell therapy for end stage of hepatic failure with or without congenital disease, we studied on 1) biology of hepatic stem cells, 2) factors that may induce hepatocyte differentiation, 3) addition of differentiated function by gene transduction, and 4) immunosuppression for the survival of transplanted cells. Using GUNN hyperbilirubinemia rats, a model animal of Crigler-Najjar syndrome, and porcine premature hepatocytes, intrahepatic transplantation of the xenogeneic porcine cells lowered the bilirubin under immunosuppression of FK506. As a new source of hepatic stem like cells, we used human amnniotic stem cells, which are isolated from human placenta obtained by Caesarean section ( Experiments with human material was performed under the permission of Ethics Committee of NationeL1 Children's Hospital. ) . While amniotic epithelialium was negative for albumin, cultured amniotic epithelial cells were positive for albumin, α-fetoprotein. Albumin secretio … More n was shown by ELISA with culture supernatant. When the amniotic epithlial cells were transplanted into SCID mouse liver, the cells were integrated in the hepatic structure and survived at least for 2 weeks, positively stained with albumin and α-fetoprotein. Now, we are investigating on the cell therapy for GUNN hyper bilirubinemia rat using amniotic epithelial cells which was transduced with *DP-glucuronosyltransferase gene .Hepatic parenchymal cells from rat lost their differentiated functions soon after the cell culture condition. We tried to maintain the function with the homogenate or extract of rat fetus (day 16 ± 1). When the homogenate of extract was mixed with rat plasma, gel formed and hepatocytes were cultured inside the gel with good morlphorogic feature. As for the differentiated function, ammonia removal activity was detected until day 20 from the beginning of culture. The 105,000 x g supernatant of the fetus homogenate still hadthe activity that kept hepatocytes in good morphorogic feature.Immunosuppres:sion for the transplanted cells were investigated with transduction of Fas-L gene. The liver expressing Fas-L survived for approximately 3 times longer period than non-treated control liver. Less