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

本研究从肝干细胞的生物学特性、诱导肝细胞分化的因素、通过基因转导增加分化功能、免疫抑制对移植细胞存活的影响等方面进行了研究，以期为终末期肝衰竭的治疗提供新的思路。使用古恩高胆红素血症大鼠（Crigler-Najjar综合征模型动物）和猪早熟肝细胞，在FK 506免疫抑制下肝内移植异种猪细胞降低胆红素。作为肝干细胞样细胞的新来源，我们使用人羊膜干细胞，其通过剖腹产获得的人胎盘中分离（使用人材料的实验在NationeL 1儿童医院伦理委员会的许可下进行）。) .羊膜上皮细胞白蛋白阴性，培养的羊膜上皮细胞白蛋白、甲胎蛋白阳性。白蛋白分泌 关于我们 用培养上清液通过ELISA显示n。羊膜上皮细胞移植于SCID小鼠肝脏后，细胞与肝脏结构整合，存活至少2周，白蛋白和甲胎蛋白染色阳性。目前，我们正在研究用转 * DP-葡萄糖醛酸转移酶基因的羊膜上皮细胞对古恩高胆红素血症大鼠进行细胞治疗。用胎鼠（16 ± 1天）的匀浆或提取液维持其功能。将提取物匀浆与大鼠血浆混合后，形成凝胶，并在凝胶内培养出形态学良好的肝细胞。至于分化功能，从培养开始到第20天检测氨去除活性。105，000 × g胎肝匀浆上清液仍具有维持肝细胞形态的活性，用Fas-L基因转染法观察移植细胞的锡永抑制作用。表达Fas-L的肝脏存活时间比未处理的对照肝脏长约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