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

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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