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

肝细胞移植有望成为治疗先天代谢障碍患者的新一代疗法。器官移植现在是对那些通过饮食或药物治疗难以控制的患者进行的。然而，器官移植对患者的侵袭性很强，供体器官短缺或终身免疫抑制治疗等问题严重。目前对18例先天性代谢异常患者进行了肝细胞移植，但临床改善不够理想，部分患者后来又进行了器官移植。在动物模型中研究了几种方法以获得较高的受体肝脏复发率。然而，已报道的方法或使用的药物太具侵入性或毒性，不适用于临床。本研究的目的是建立一种新的诱导肝细胞移植后再增殖的方法。众所周知，鹅去氧胆酸可诱导…更多的ES通过Fas信号通路诱导肝细胞的凋亡。我们计划用CDCA诱导受体肝细胞的凋亡，用CDCA预防供体肝细胞的凋亡，用慢病毒载体转导抑制caspase1和8的CrmA基因，将CrmA基因插入肠胆汁酸结合蛋白(I-BABP)启动子下游，供体肝细胞在肝细胞移植后只有在大剂量CDCA的作用下才能抵抗凋亡，其中供体肝细胞用慢病毒载体转导I-BABP-CrmA基因片段，CDCA计划用于受体。另一方面，供体细胞由于CrmA蛋白的表达而对凋亡产生抵抗力，并在受体肝脏中重新繁殖。1)从C57BL/6小鼠基因组中克隆了I-BABP启动子。2)CDCA激活I-BABP启动子。3)构建了含I-BABP启动子、EGFP和CrmA基因的慢病毒载体。4)将该基因片段导入人肝癌细胞系HepG2中。5)荧光显微镜下观察EGFP-CrmA蛋白的表达，Western印迹法检测其表达情况。6)转导该基因片段的HepG2细胞在没有CDCA的情况下对凋亡敏感，但在CDCA的存在下细胞对凋亡产生抵抗。较少

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