Establishment of the novel method for induction of repopulation after hepatocyte transplantation

肝细胞移植后诱导增殖新方法的建立

• 批准号: 18591181
• 负责人: OGAWA Atsushi
• 金额: $ 2.53万
• 依托单位: Chiba University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18591181/
• 关键词: Hepatocyte transplantation; Apoptosis; CrmA

Hepatocyte transplantation is expected to be a next generation of treatment for the patient with inborn error of metabolism. Organ transplantation is now performed to the patient who is difficult to be controlled with diet or drug therapy. However organ transplantation is very invasive to the patients and other problems such as a shortage of donor organ or life long immunosuppresive therapy are serious. Hepatocyte transplantation was performed to 18 patients with inborn error of metabolism so far now, but the clinical improvement was not enough, some of the patients were received organ transplantation later. Several methods were investigated in animal model to get a higher rate of repopulation in recipient liver. However the reported methods or drugs used were too invasive or toxic to apply to clinical settings.The purpose of this study was to establishment of a novel method for induction of repopulation after hepatocyte transplantation It is well known that chenodeoxycholic acid induc … More es apoptosis to hepatocytes via Fas signal pathway. We plan to use CDCA to induce apoptosis to hepatocytes of recipient, To prevent the donor hepatocytes from apoptosis with CDCA, CrmAgene, that inhibits caspase 1 and 8, was planed to be transduced with Lentivirus vector CrmA gene was inserted downstream of intestinal bile acid binding protein (I-BABP) promoter donor hepatocytes were expected to be resistant for apoptosis only under the high dose administration of CDCA After hepatocyte transplantation, in which donor hepatocytes were transduced with I-BABP-CrmA gene fragment with Lentivirus vector, CDCA is planed to be administered to the recipient. It is expected that apoptosis is induced to hepatocytes of recipient, on the other hand, donor cell become resistant to apoptosis because of expression of CrmA protein and get repopulated in the recipient liver.The results are as follows. 1) I-BABP promoter was cloned from the genome of C57bl/6 mouse. 2) I-BABP promoter was activated with CDCA. 3) Lentivirus vector was produced containing of gene fragment, I-BABP promoter, EGFP and CrmA gene 4) this gene fragment was transduced to HepG2 cells 5) Expression of EGFP-CrmA protein was observed under the fluorecent microscopy and checked by western blot as well. 6) HepG2 cells transduced the gene fragment were susceptible for apoptosis without CDCA, however, the cells become resistant to apoptosis under the presence of CDCA. Less

肝细胞移植有望成为治疗先天性代谢缺陷患者的新一代方法。器官移植现在是对难以通过饮食或药物治疗控制的病人进行的。然而，器官移植对患者具有很大的侵入性，而且供体器官短缺或终身免疫抑制治疗等问题也很严重。目前对18例先天性代谢错误患者行肝细胞移植，但临床改善不够，部分患者后续接受器官移植。在动物模型上研究了几种方法，以获得较高的受体肝脏再生率。然而，所报道的方法或使用的药物过于侵入性或毒性，无法应用于临床环境。本研究旨在建立一种诱导肝细胞移植后再生的新方法。众所周知，鹅去氧胆酸可通过Fas信号通路诱导肝细胞凋亡。我们计划用CDCA诱导受体肝细胞凋亡，为了防止供体肝细胞凋亡，我们计划用慢病毒载体转导抑制caspase 1和caspase 8的CrmAgene，将CrmA基因插入肠胆汁酸结合蛋白（I-BABP）启动子下游，在肝细胞移植后，仅在高剂量CDCA的作用下，供体肝细胞才有凋亡抗性。其中，用慢病毒载体将I-BABP-CrmA基因片段转导给供体肝细胞，计划将CDCA施用于受体。预计受体肝细胞会发生凋亡，另一方面，供体细胞因表达CrmA蛋白而对凋亡产生抗性，并在受体肝脏中重新填充。结果如下：1)从C57bl/6小鼠基因组中克隆I-BABP启动子。2) I-BABP启动子被CDCA激活。3)制备含有基因片段、I-BABP启动子、EGFP和CrmA基因的慢病毒载体，4)将该基因片段转导至HepG2细胞，5)荧光显微镜下观察EGFP-CrmA蛋白的表达，并进行western blot检测。6)转染该基因片段的HepG2细胞在没有CDCA的情况下易发生凋亡，而在CDCA存在的情况下，细胞对凋亡产生抗性。少

项目成果

先天代謝異常症に対する肝細胞移植治療

肝细胞移植治疗先天性代谢缺陷

• 发表时间: 2007
• 作者: Yoneshige;A.;Kubo;N.;Suzuki;K.;Matsuda;J;小川 真司
• 通讯作者: 小川 真司