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Establishment of an hepatocyte cell line derived from human amnio-blast cells and the evaluation of its functions when transplanted and orientation to artificial bio-liver apparatus.

人羊膜母细胞来源的肝细胞系的建立及其移植时的功能评估和人工生物肝装置的定位。

基本信息

批准号：
16591274
负责人：
TABEI ISAO
金额：
$ 2.05万
依托单位：
Jikei University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16591274/
关键词：
HEPATOCYTE TRANSPLANTATION AMNION CELL CELL CULTURE ARTIFICIAL ORGAN 神経原基

项目摘要

[Object] Although liver transplantation has become the ultimate therapy for hepatic failure patients, the lack of absolute donor organ has opened the path of development to adjuvant and/or alternative methods such as cell transplantation and artificial liver apparatus. The research to develop the material for these methods has involved fetal, embryonic stem cells and transfection, transductions of hepatocyte to become immortalized cells. But most trials are stalled due to unanswered problems involving function, tumorigensis, immunogenetic and ethical questions. The placenta is usually disposed after birth and amnion obtained from this waste expresses little MHC class I and no class II. We focused on this material that also produces other immuno-regulartory factors, hypothesizing to find immature cells that would mature into functional hepatocytes. [Methods] Human-early embryo amnion was dissected in 0.1%trypsin-0.02%EDTA/PBS (-) and cultured in growth medium (GM) containing DMEMF12+ … More 20%FBS+0.1%NEA. Small circular cells developed within the single-layered cells were cloned colonially. The cells were named HEAC cells and were used in the following experiments. HEAC cells were maintained in GM with addition of 1ng/ml of LIF. Liver failure rats were created chemically using carbon tetrachloride with the survival rate of 33%. Cells were transplanted into these rats to investigate on their protrombin time, serum albumin levels and also survival rate. Also they were cultured in collagen type 1 to characterize the differentiated cells in 3-dimension culture. [Results] Amnion cells are fairly small cells with immature cytoplasm and active differentiation potency. After several regulations the cytoplasm becomes rich with various organelle, evolving the cells to differentiate. Within the collagen sponge 3-dimention culture the cells produced Albumin, a typical characteristic of a hepatocyte. When transplanted into chemically induced liver failure rats, it improved protrombin time, serum albumin levels and also survival rate up to 90%. Less

[目的]虽然肝移植已成为肝功能衰竭患者的终极治疗方法，但由于缺乏绝对供体器官，为细胞移植和人工肝器等辅助和/或替代方法的发展开辟了道路。为这些方法开发材料的研究涉及胎儿、胚胎干细胞和肝细胞的转基因，转导成为永生化细胞。但大多数试验由于涉及功能、肿瘤发生、免疫遗传学和伦理问题的悬而未决的问题而停滞不前。胎盘通常在出生后被处理，从这些废物中获得的羊膜表达很少的MHC I类和没有II类。我们专注于这种物质，它还会产生其他免疫调节因子，假设寻找能够成熟为有功能的肝细胞的未成熟细胞。[方法]人早期胚胎羊膜在0.1%胰酶-0.02%EDTA/PBS(-)中分离，在含有DMEMF12+…的生长液中培养。20%以上的FBS+0.1%的NEA。对单层细胞内发育的小环状细胞进行克隆。这些细胞被命名为HEAC细胞，并用于以下实验。在GM中加入1 ng/mlLIF维持HEAC细胞。用四氯化碳化学法复制大鼠肝衰竭模型，存活率为33%。将细胞移植到这些大鼠体内，观察它们的原凝血酶时间、血清白蛋白水平以及存活率。并将其在1型胶原中培养，在三维培养中对分化细胞进行鉴定。[结果]羊膜细胞体积较小，胞质未成熟，分化能力较强。经过几次调节后，细胞质变得富含各种细胞器，使细胞进化分化。在胶原海绵三维培养中，细胞产生白蛋白，这是肝细胞的典型特征。将其移植到化学性肝衰竭大鼠体内，可改善原凝血酶时间、血清白蛋白水平，存活率高达90%。较少