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原位肝移植的替代治疗方法。小的肝细胞被称为肝细胞干细胞，将被认为是更适当的肝细胞移植。此外，如果通过基因转染小肝细胞来诱导一种特异性功能，将为肝功能衰竭患者提供一些益处。[目的]首先确定小肝细胞作为肝细胞移植的目标细胞来源。构建的肝细胞是否会发生从受体肝细胞到小肝细胞的交替变化，已经被研究过。因此，我们用腺病毒感染的方法将胰岛素分泌基因PDX 1转染到小肝细胞中，并对这些干细胞进行了体内外功能维持的分析。并初步探讨了利用这些细胞进行肝移植的可能性。[ ...更多信息 实验分类]本研究由以下三个实验组成。(1)胰岛素分泌基因转染方法的建立。利用腺病毒载体将胰岛素分泌基因PDX-1转染成年肝细胞和小肝细胞。免疫病理学观察转染细胞的转基因表达、胰腺内分泌激素的产生及肝细胞的特性。(2)转染细胞中胰岛素的表达和产生。对转染细胞的免疫组织学测定、蛋白质特异性mRNA的分析、转染细胞功能的维持进行了研究。(3)体内肝细胞移植。将转染或未转染该基因的小肝细胞或成年肝细胞通过脾内注射移植到肝脏中。[结果] 2001 ~ 2004年，上述研究计划均已完成。(1)在PDX-1转染后，在感染的细胞中测定几种转录因子的表达。培养液中胰岛素、胰高血糖素、生长抑素和胰多肽的定量测定成功。但要获得稳定的转染并成功重复该实验是非常困难的。(2)转染率极低，但在成年肝细胞中观察到的转染率高于小肝细胞。通过转染获得的功能的维持的局限性在本研究中留下了深刻的印象。(3)在非基因转染的小肝细胞实验中，观察到了在受体肝中高度成功的再增殖，并且注射的小肝细胞的增殖也是可信的。[结论]小肝细胞移植有望进入临床应用。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