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

患有或不患有先天性疾病的肝衰竭末期的细胞疗法的发展

• 批准号: 09671270
• 负责人: ENOSAWA Shin
• 金额: $ 2.3万
• 依托单位: NATIONAL CHILDREN'S MEDICAL RESEARCH CENTER
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09671270/
• 关键词: Hepatic failure; Cell therapy; Gene transduction; Hepatocyte growth; Hepatocyte differentiation; Hepatic stem cell; Amniotic epithelial cell; Immunosuppression

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

为了开发针对患有或不患有先天性疾病的肝衰竭末期的细胞疗法，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，以及4）移植细胞存活的免疫抑制。使用GUNN高胆红素血症大鼠（Crigler-Najjar综合征模型动物）和猪早熟肝细胞，异种猪细胞的肝内移植在FK506的免疫抑制下降低了胆红素。作为肝干样细胞的新来源，我们使用了人羊膜干细胞，该干细胞是从剖腹产获得的人胎盘中分离出来的（人体材料实验是在国家一级儿童医院伦理委员会的许可下进行的）。羊膜上皮的白蛋白呈阴性，而培养的羊膜上皮细胞的白蛋白、甲胎蛋白呈阳性。通过 ELISA 用培养物上清液显示白蛋白分泌。当羊膜上皮细胞被移植到SCID小鼠肝脏中时，细胞整合到肝结构中并存活至少2周，白蛋白和甲胎蛋白染色呈阳性。目前，我们正在研究使用转染*DP-葡萄糖醛酸基转移酶基因的羊膜上皮细胞对GUNN高胆红素血症大鼠进行细胞治疗。来自大鼠的肝实质细胞在细胞培养条件后很快就失去了分化功能。我们尝试用大鼠胎儿的匀浆或提取物（第16±1天）维持功能。提取物匀浆与大鼠血浆混合，形成凝胶，在凝胶内培养肝细胞，具有良好的形态特征。至于分化功能，从培养开始到第20天检测氨去除活性。 105,000 x g胎儿匀浆上清液仍具有保持肝细胞良好形态特征的活性。通过Fas-L基因转导研究移植细胞的免疫抑制作用。表达Fas-L的肝脏的存活时间比未处理的对照肝脏长约3倍。较少的

项目成果

Suzuki S et al.: "The induction of lymphocyte apoptosis in MRL lpr/lpr mice treated with FTY720." Clin Exp Immunol. 107. 103-111 (1997)

Suzuki S 等人：“用 FTY720 治疗的 MRL lpr/lpr 小鼠中淋巴细胞凋亡的诱导。”

Enosawa S et al.: "An attempt to add biological functions by genetic engineering in order to produce high-performance bioreactor cells for hybid artificial liver : Transfection of glutamine synthetase into chinese hamster ovary (CHO) cell." Cell Transplan

Enosawa S 等人：“尝试通过基因工程添加生物功能，以生产用于混合人工肝的高性能生物反应器细胞：将谷氨酰胺合成酶转染到中国仓鼠卵巢 (CHO) 细胞中。”

Li XL. Okuyama T, Tamura A, Enosawa S, Kaneda Y Takahara S, Funeshima N, Yamada M. Amemiya H, Suzuki S.: "Prolonged survival of rat liver allografts transfected with Fas-ligand expressing plasmid"Transplantation. 66(11). 1416-1423 (1998)

李雪莲.

Enosawa S et al.: "Higher efficiency of retrovirus transduction in the late stage of primary culture of hepatocytes from non-treated than from partially hepatectomized rat." Cell Transplantation. 7(4). 413-416 (1998)

Enosawa S 等人：“与部分肝切除大鼠相比，未处理的大鼠肝细胞原代培养后期的逆转录病毒转导效率更高。”

Yata Y, Enosawa S, Suzuki S, Li XL, Tamura A, Kimura H, Takahara T, Watanabe A: "In vivo Administration of Dichloromethylene Diphosphate (CL2MDP) to Obtain Highly Purified Hepatic Stellate Cells in Rats"Methods in Cell Science. (in press).

Yata Y、Enosawa S、Suzuki S、Li XL、Tamura A、Kimura H、Takahara T、Watanabe A：“体内施用二氯亚甲基二磷酸 (CL2MDP) 以在大鼠体内获得高度纯化的肝星状细胞”细胞科学方法。