Development of therapy for regulation of liver cirrhosis by antisense gene and transplantation of proliferative hepatocyte

实验一：采用四氯化碳（CCl_4）和二甲基亚硝胺（DMNA）对小鼠和大鼠进行肝硬化动物模型的建立。CCl_4染毒组动物肝组织坏死，但实质肝细胞损伤严重。另一方面，DMNA组大鼠肝脏严重坏死，肝细胞似乎基本正常。因此，我们认为DMNA大鼠适用于肝硬化实验。实验二：在肝硬化模型中，肝切除后观察HGF和TGF β拮抗剂的治疗效果。HGF对再生有促进作用，而TGF β拮抗剂对再生无明显影响，且可抵消HGF的作用。实验三：为了制备具有肝细胞功能并能增殖的杂交瘤，我们尝试制作了激光细胞融合装置。利用该装置，我们可以从小鼠骨髓瘤细胞和淋巴细胞中制备杂交瘤。我们从该杂交瘤中克隆了两株原始细胞，并对其性状进行了检测。另外，我们用骨髓瘤细胞和肝细胞制备了杂交瘤，目前正在研究这种杂交瘤的特性。实验四：为了通过基因转导治疗细胞功能，我们进行了用HVJ脂质体包裹TNF反义mRNA转染Kupffer细胞的初步研究。用反义治疗，TNF的产生减少到40%。目前正在利用Ito细胞研究反义治疗的效果。利用胶原酶抑制剂mRNA的反义进行基因治疗尚未见报道。

Experiment 1 : For establishment of an animal model with liver cirrhosis, CCl4 and dimethyl nitrosoamine (DMNA) were administered to mouse and rat. Liver of animals with administration of CCl4 were cirrhotic but parenchymal hepatocyte was injured so severely. On the other hand, liver of rat with administration of DMNA was sever cirrhotic and hepatocyte seemed to be almost normal. Therefore, we conclude that rat with administration of DMNA was suitable for experiment of cirrhosis. Experiment 2 : In model with liver cirrhosis efficucies of HGF and antagonist of TGFb administration were examined after liver resection. HGF had an effect on progress of regeneration but antagonist of TGFb had no remarkable effect on regeneration and it counteracted the effect of HGF. Experiment 3 : For making hybridoma which has function of hepatocyte and proliferates, we tried to make cell fusion equipment using laser. Using this device, we could make hybridoma from mouse myeloma cells and lymphocytes. We made clone from this hybridoma and examined characters of both original cells. In addition we made hybridoma with myeloma cells and hepatocytes and are investigating the character of this hybridoma now. Experiment 4 : For treatment of cell function by gene transduction, we carried out the preliminary study that antisense for mRNA of TNF was encapsuled with HVJ liposome and was transfected to Kupffer cells. With antisense therapy, production of TNF decreased to 40%. Now using Ito cells, effect of antisense therapy is investigating. Gene therapy using antisense of mRNA of collagenase inhibitor is still not done yet.