Basic research of the treatment for hepatic failure by hepatocyte transplantation

肝细胞移植治疗肝衰竭的基础研究

• 批准号: 13557107
• 负责人: HIRATA Koichi
• 金额: $ 8.83万
• 依托单位: Sapporo Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13557107/
• 关键词: hepatic failure; hepatosyte; stem cell; small hepatocyte; transplantation; cholestasis; bile canalicutus; sepsis; 重症感染症; 非閉塞性胆汁うっ滞; Cooplet細胞; トランスポーター; ヒト肝細胞; 遺伝子導入; 細胞移植; IPF-1; アデノウイルスベクター; インスリン; 放射線照射; 肝再生置換; ミニ肝組織; hepatic failure; hepatosyte; stem cell; small hepatocyte; transplantation; cholestasis; bile canalicutus; sepsis; 重症感染症; 非閉塞性胆汁うっ滞; Cooplet細胞; トランスポーター; ヒト肝細胞; 遺伝子導入; 細胞移植; IPF-1; アデノウイルスベクター; インスリン; 放射線照射; 肝再生置換; ミニ肝組織

[Background] It is envisaged that hepatocyte transplantation and extracorporeal liver support systems may be a future alternative therapy to orthotopic liver transplantation. Small hepatocytes known as liver hepatocyte stem cells would be considered to be more adequate in hepatoyte transplantation. In addition, if a kind of specific function by a gene transfection to small hepatocytes was inducted, some benefits would provide for the patients with hepatic failure.[Object] At first, small hepatocytes were determined as the objective cell source in hepatocyte transplantation. Whether alternative changes of contructed hepatocytes from recipient hepatocytes to small hepatocytes might occur or not has been studied. Therefore, PDX 1 gene, an insulin-producing gene, was tranfected to small hepatocytes by adenovial infection and those stem cells were analysed about the functional maintenance in vitro and in vivo. And the possibility of hepatosyte transplantation by those cells has been tried.[ … More Experimental Classification] This research is composed of three kinds of experiments as shown as followings.(1) Establishment of the transfection method of insulin-production gene. The transfection of PDX-1 gene, as an insulin-producing gene to adult hepatocytes and to small hepatocytes had been studied by the infection of adenoviral vectors. The expression of transfere genes, the production of pancreatic endocrine hormones and the characteristics of the hepatocytes were immurohis topathologically made sure in the transfected cells.(2) Expression and production of insulin in the transfected cells. Immunohistologic determination of the transfected cells, analysis of m-RNA specific to the proteins, maintenance of function in the transfected cells were studied.(3) Hepatocyte transplantation in vivo. Small hepatocyte or adult hepatocytes with or without transfection of the gene were transplanted into liver via the injection into spleen.[Results] All of above research plans have been performed during 2001 to 2004, as shown in followings.(1) Expression of several transfere factors were determined in the infected cells after the transfection of PDX-1. The quantitations of insulin, glucagon, somatostatin and pancreatic polypeptide in the cultured solution were succeeded. But it was very difficult to get the stable transfection and to repeat this experiment successfully.(2) The transfection rates were extremely low but was observed higher in the adult hepatocytes than in small hepatocytes. The limitation of the maintenance of the function gained by transfection was strongly impressed on this research.(3) Highly succeeded repopulation in the recipient liver was observed in the experiment with non gene transfected small hepatocytes and the proliferation of small hepatocyte in injected was also confident.[Conclusion] This pilot research suggest that small hepatocytes transplantations would invite an entrance of the actuarial utilization of clinics. Less

[背景]可以预见，肝细胞移植和体外肝支撑系统可能是原位肝移植的未来替代疗法。被称为肝细胞干细胞的小肝细胞被认为在肝移植中更为足够。此外，如果通过基因转染向小肝细胞的一种特异性功能，则可以为肝衰竭的患者提供一些好处。[对象]首先，小肝细胞被确定为肝细胞移植中的客观细胞源。是否可能发生从受体肝细胞到小肝细胞的肝细胞的替代变化。因此，PDX 1基因是产生胰岛素的基因，通过腺丝感染转染至小肝细胞，并分析了这些干细胞的体外和体内功能维持。并且已经尝试了这些细胞肝移植的可能性。[…更多的实验分类]这项研究由三种实验组成，如下所示。（1）建立了胰岛素产生基因的转化方法。 PDX-1基因的转化，作为成年肝细胞和小肝细胞的产生胰岛素基因的转化已被腺病毒载体的感染进行了研究。转移基因的表达，胰腺内分泌恐怖的产生以及肝细胞的特征是免疫霍尼在翻译的细胞中确保的。（2）翻译细胞中胰岛素的表达和产生。对翻译细胞的免疫组织学测定，对蛋白质特异的M-RNA分析，在翻译细胞中的功能维持。（3）体内肝细胞移植。小型肝细胞或成年肝细胞有或没有基因翻译的情况下，通过注射将基因转移到肝脏中。[结果] [结果]在2001年至2004年期间，所有上述研究计划均在下面的表达中进行。培养溶液中胰岛素，胰高血糖素，生长抑素和胰多肽的定量成功。但是，很难获得稳定的转染并成功地重复该实验。（2）转化率极低，但在成年肝细胞中观察到的转化速率比小肝细胞高。这项研究对通过转化获得的功能维持的局限性印象深刻。（3）在非基因翻译的实验中观察到了受体肝脏中的高度成功再播种，而小型肝细胞的增殖也很有信心。较少的