Abstract:

Objective To construct a pig-mouse liver cell chimeric model，and to discuss the relevant conditions for the transplantation and regeneration of the porcine hepatocytes without T lymphocyte， B lymphocyte， and NK lymphocyte mediated rejection， and to evaluate the settlement， proliferation， and secretion of functional factors of the porcine hepatocytes in xenograft receptors， and to provide the basis for improving the reconstruction efficiency of the porcine hepatocytes in pig derived mouse models. Methods The porcine hepatocytes were transplanted into the Fah^－/－Rag2^－/－ IL2Rg ^－/－ （FRG） mice by using intrasplenic injection method to construct the transplantation model. Sixteen FRG mice were randomly divided into control group， blank control group， experimental group， and green fluorescence protein（GFP） gene experimental group， and there were four mice in each group.The administration of nitisinone 2-2-nitro-4-（trifluoromethyl）cyclohexane-1，3-dione（NTBC） of the mice in blank control group was stopped，and the body weight was monitored； the mice in control group were injected with the porcine hepatocytes， the administration of NTBC was stopped one week before transplantation， and the body weight was monitored after transplantation. When the body weight was decreased by 20%， the NTBC was administered for 3 d. The mice in experimental group were injected with the porcine hepatocytes， and the administration of NTBC was stopped one week before transplantation； the body weight was monitored after transplantation， and when the body weight was decreased by 20%， the NTBC was administered for 3 d. The mice in GFP gene experimental group were injected with the porcine hepatocytes carrying the GFP gene， the administration of NTBC was stopped one week before transplantation， and the body weight was monitored after transplantation； when the body weight was decreased by 20%， the NTBC was administered for 3 d. The body weights and survival status of the mice in various groups were observed. The levels of alanine aminotransferase （ALT） in serum of the mice in various groups were detected；the expressions of CD19+， CD3+， and NK1.1+cells in peripheral blood of the mice in various groups were detected by flow cytometry；the expression levels of porcine albumin in serum of the mice in various groups were detected by ELISA assay； the regeneration efficiencies of the porcine hepatocytes of the mice in various groups were detected by immunohistochemistry. Results Compared with method of collagenase digestion after shearing， the digestion process of the two-step perfusion method was milder and had less damage to cells， and the cell activity achieved 92%. After stopping the administration of NTBC， the body weight of the mice in control group was gradually decreased and the vitality was gradually decreased. Death began to occur in the mice on the 28th day after continuous stopping the administration.The irreversible cellular damage in liver tissue of the mice could be seen such as liver cell enlargement， cytoplasmic looseness and transparency， and nuclear lysis. The ALT levels in serum of the mice in blank control group was gradually increased after stopping the administration. After re-administration of NTBC， the body weight of the mice in control group was gradually returned to the normal levels. The flow cytometry results showed that the CD19+， CD3+， and NK1.1+cells in peripheral blood of the mice could not be seen after stopping the administration of NTBC. The immunohistochemistry staining results showed that no dark Fah+positive cells were found in liver tissue of the mice in experimental group， while the dark Fah+positive cells were observed in liver tissue of the transplanted mice； the regeneration efficiency of porcine hepatocytes was （11 ± 4）%. The regeneration efficiency of porcine hepatocytes of the mice in GFP gene experimental group was （10 ± 2）% when stimulated by three color lasers in the same field of view. Conclusion The pig derived FRG mouse liver chimeric models are successfully constructed， and the mouse models for long-term stable expansion of the porcine hepatocytes are established.

Key words: Hepatocyte xenotransplantation, Fah, Hepatocyte, Pig-mouse liver cell chimeric model, Immunodeficient mice