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

反义基因调控肝硬化疗法及增殖性肝细胞移植的研究进展

• 批准号: 09557100
• 负责人: OHKOHCHI Nobuhiro
• 金额: $ 6.02万
• 依托单位: TOHOKU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09557100/
• 关键词: liver cirrhosis; liver regeneration; gene therapy; antisense; cell fusion; 肝切除; 増殖性肝細胞; 移植; ハイブリッド肝細胞; アンチ・センス; コラゲナーゼインヒビター; 遺伝子導入

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.

实验一：用四氯化碳和二甲基亚硝胺(DMNA)分别对小鼠和大鼠建立肝硬变动物模型。经CCl_4染毒的大鼠肝脏有明显的肝硬变，但实质肝细胞损伤严重。DMNA组大鼠肝脏出现严重的肝硬变，肝细胞基本正常。因此，DMNA可作为大鼠实验性肝硬变的动物模型。实验2：观察肝切除术后肝细胞生长因子和肿瘤生长因子b拮抗剂对大鼠肝纤维化模型的影响。HGF对再生进程有一定影响，而TGFb拮抗剂对再生无明显影响，可抵消HGF的作用。实验三：为了制作具有肝细胞功能和增殖功能的杂交瘤，我们尝试用激光制作细胞融合装置。利用这个装置，我们可以用小鼠骨髓瘤细胞和淋巴细胞制作杂交瘤。我们从该杂交瘤细胞中克隆，并对两个原始细胞的特性进行了检测。此外，我们还用骨髓瘤细胞和肝细胞制作了杂交瘤，目前正在对杂交瘤的特性进行研究。实验四：为了通过基因转导治疗细胞功能，我们进行了反义肿瘤坏死因子基因的脂质体包裹并转导Kupffer细胞的初步研究。反义治疗后，肿瘤坏死因子的产量下降到40%。现在利用伊藤细胞，反义治疗的效果正在研究中。胶原酶抑制因子反义基因治疗仍未见报道。