In vivo Hybridisierung einer Mausleber - Vergleich der Transplantation humaner Hepatozyten und Progenitorzellen aus explantierten Lebern versus Leberteilresektaten im Mausmodell
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Beschreibung
"In vivo hybridisation of a mouse liver - Comparison transplantation of human hepatocytes and progenitor cells from explanted livers versus partial liver resectates in a mouse model"
So far orthotopic liver transplantation is the standard treatment for terminal liver failure. Due to shortage of donors, the risk of rejection and the side effects of immunosuppression, there is still a need for optimizing the transplant procedure and for alternative treatments like liver cell transplantation. This work is part of a project that aims to repopulate allogen transplants with autologous hepatocytes and hepatic progenitor cells to create a so-called neohybrid liver graft. The main focus of the present dissertation is to analyse whether cell engraftment and neohybridization are dependent on the underlying disease of the donor. Additionally, it will be evaluated whether the extension of the animal experiment up to 30 days is feasible. Cells were isolated from three „healthy“ livers from patients who had undergone hepatic resections for liver metastasis and three diseased livers of patients after liver explantation. Hepatocytes and hepatic progenitor cells were isolated, purified via Percoll density gradient centrifugation and stored at 4°C for 12 to 24 hours until transplantation. Viability, total cell yield and cell culture were compared. After successful isolation, animals (12 FOX CHASE SCID/beige® per sample tissue) where pretreated with monocrotaline (200 mg/kg) intraperitoneally to initiate liver damage. 12 to 24 hours later, a 50 % partial hepatectomy was performed and 1 million hepatocytes and progenitor cells were transplanted by intrasplenic injection. Three mice of each group were euthanized on day 7, 15 and 30 after surgery and the liver was removed for further diagnostics. To test repopulation, the liver samples were evaluated histopathogically by immunhistochemical staining (anti-human-albumin) and by detection of human hepatocytes in the liver tissue via real time PCR (human albumin, B2M and ACTN4). The isolation of hepatocytes and progenitor cells was successful from both healty and diseased liver tissue, but total cell viability was significantly higher in healthy liver tissue. Cold storage for 12 to 24 hours was feasible with a small loss of viability only. The cells from diseased liver tissue seemed to tolerate cold storage better and total cell yield and viability of both groups where almost the same after cold storage. In a cell culture, both cell lines grew well. The established mouse model was successful with a survival rate of 86% of animals. The explant group that received cells from damaged liver tissue suffered from moderately higher animal losses. In 92 % of the mice immunhistochemistry showed engraftment of human hepatocytes in equal amounts in animals of the explant and resection group for the first 15 days, on day 30 in a significantly higher rate in the explant group. Confirmation of the immunohistological findings via PCR was only partly successful. The results of this study show that successful isolation, cold storage and transplantation of hepatocytes and progenitor cells from healthy liver tissue as well as from diseased, cirrhotic livers is possible. Also there are hints that cells from diseased livers are more stable in cold storage and in Long term repopulation. To compare both cell lines, the modified mouse model is suitable, however, the duration time of further studies needs to be longer than 30 days in order to properly evaluate the capacity of repopulation.
Autor*in
Katharina Venz
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Leberzellen Labortiere Leber Mäuse Transplantation Tiermodelle