Development of hepatocyte bank and hepatocyte transplantation with Decoy receptor 3 gene transfer

我们在临床中证实了从切除的人肝组织中分离肝细胞。此外，根据Mitaka等人的描述，也从非实质肝细胞中分离出小肝细胞。我们对这些细胞进行了表征，并研究了基因治疗的可能性。1)分离成熟肝细胞和小肝细胞经胶原酶消化的肝组织，过滤后50xg离心。结果，成熟的肝细胞被沉淀，然而，小的肝细胞位于上清部分。结果;获得可生长3个月以上的小肝细胞，并能连续产生α 1抗胰蛋白酶。2)人离体肝细胞低温保存液的最佳成分：各种类型的低温保存液包括；a)细胞库，b)10%DMSO+UW, c)10%DMSO+10%FBS+UW, d)10%DMSO+FBS, e)UW比较。结果;10%DMSO+UW冷冻和解冻组肝细胞存活率最高（60%）。然而，这些冷冻解冻的肝细胞往往失去对细胞培养皿的粘附能力，表明这些细胞膜的完整性被破坏了。3) hvj包膜载体介导的基因转移到肝细胞我们初步研究了常规hvj脂质体在大鼠和猪肝细胞中的基因转移效果。在此基础上，我们利用LacZ和荧光素酶报告基因分析了hvj包膜载体转染肝细胞的效率。并对转染后的动物器官进行组织学和生化检查。结果;与门静脉注射相比，囊下注射报告基因的转染效率最高。根据组织学结果，其他器官未受此转染过程的损害。

We cinfirmed that the isolation of hepatocytes from resected human liver tissues in the clinics. In addition, small hepatocytes were also isolated from the fraction of non-parenchymal hepatocytes as described by Mitaka et al. We characterized these cells and investigated the possibility of gene therapy.1)Isolation of matured hepatocytes and small hepatocytesLiver tissues digested by collagenase were filtrated and centrifuged at 50xg. As a result, matured hepatocytes were precipitated, however, small hepatocytes located in the supernatant fraction.Result ; Small hepatocytes that could grow for more than 3 months were obtained which produced alpha1 anti-trypsin continuously.2)Optimal ingredients of cryopreservative solution for isolated human hepatocytesVarious type of cryopreservatice solution including ; a)Cell banker, b)10%DMSO+UW, c)10%DMSO+10%FBS+UW, d)10%DMSO+FBS, e)UW were compared.Results ; The highest viability (60%) of freezing and thawing hepatocytes were observed in 10%DMSO+UW group. However these freeze-thawed hepatocytes tended to lose adhesive capacity to the cell culture dishes, suggesting that the integrities of these cellular membranes were deteriorated.3)HVJ-envelope vector mediated gene transfer into hepatocytesWe initially investigated the efficacy of the conventional HVJ-liposome for gene transfer into hepatocytes in both rats and pigs. Based on these results, we analyzed the transfection efficiencies of HVJ-envelope vector into the hepatocytes by using LacZ and luciferase reporter genes. Also histological and biochemical examination of these transfected organs in the animals were performed.Results ; Subcapsular injection of the reporter genes shoed the highest transfection efficiency compared with portal vein injection. Other organs were not damaged by this transfection process according to the findings of histology.