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

为了发展有或无先天性疾病的终末期肝功能衰竭的细胞治疗，我们研究了1)肝干细胞的生物学，2)可能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。以高胆红素血症大鼠（Crigler-Najjar综合征模型动物）和猪早产肝细胞为实验对象，在免疫抑制FK506的情况下，异种猪细胞肝内移植可降低胆红素水平。作为肝干细胞样细胞的新来源，我们使用的人羊膜干细胞是从剖宫产获得的人胎盘中分离出来的（人体材料实验经国家第一儿童医院伦理委员会许可）。． 羊膜上皮细胞白蛋白阴性，培养的羊膜上皮细胞白蛋白、α-胎蛋白阳性。培养上清酶联免疫吸附法测定白蛋白分泌量。将羊膜上皮细胞移植到SCID小鼠肝脏后，细胞与肝脏结构融合，存活至少2周，白蛋白和α-胎蛋白染色阳性。目前，我们正在研究利用* dp -葡萄糖醛基转移酶基因转导的羊膜上皮细胞对GUNN型高胆红素血症大鼠的细胞治疗。大鼠肝实质细胞在细胞培养后不久就丧失了分化功能。我们尝试用大鼠胎儿匀浆或提取液（第16±1天）维持其功能。将提取液匀浆与大鼠血浆混合，形成凝胶，并在凝胶内培养肝细胞，具有良好的形态特征。分化功能方面，从培养开始到第20天检测氨去除活性。105,000 x g的胎儿匀浆上清液仍有活性，使肝细胞保持良好的形态特征。通过Fas-L基因的转导研究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处理对照肝脏的3倍。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