Basic research of the treatment for hepatic failure by hepatocyte transplantation

[背景]肝细胞移植和体外肝支持系统有望成为未来肝移植的替代疗法。被称为肝细胞干细胞的小肝细胞将被认为更适合于肝细胞移植。此外，如果通过基因转导小肝细胞来诱导一种特定的功能，将为肝功能衰竭患者提供一些好处。[目的]首先确定小肝细胞作为肝细胞移植的目的细胞来源。研究了结构肝细胞是否可能发生由受体肝细胞到小肝细胞的交替变化。因此，通过腺病毒感染将胰岛素分泌基因PDX-1基因导入小鼠肝细胞，并对干细胞在体内外的功能维持进行了分析。用这些细胞进行肝细胞移植的可能性已经尝试过了。[…]更多实验分类]本研究由三种实验组成，具体如下：(1)胰岛素产生基因转染法的建立。通过腺病毒载体的感染，研究了作为胰岛素分泌基因的PDX-1基因对成人肝细胞和小鼠肝细胞的转染作用。从病理上证实了转染区基因的表达、胰腺内分泌激素的产生以及肝细胞的特性。(2)转染组细胞中胰岛素的表达和分泌。对转基因细胞的免疫组织学检测、m-RNA蛋白特异性分析、转基因细胞的功能维持等进行了研究。(3)体内肝细胞移植。[结果]上述研究计划均于2001~2004年完成，主要结果如下：(1)PDX-1基因导入感染细胞后，检测到多种转导因子在感染细胞中的表达。对培养液中的胰岛素、胰升糖素、生长抑素和胰多肽进行了定量。但要获得稳定的转染率并成功重复实验是非常困难的。(2)成体肝细胞转染率极低，但成体肝细胞转染率高于小肝细胞。(3)非转基因小肝细胞移植实验中，受体肝脏再生成功，注射小肝细胞的增殖能力也较好。[结论]小肝细胞移植为临床精算利用开辟了新的途径。较少

[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