Cellular Immune Rejection Mechanism In The Orthotopic Transplantation Of Human Hepatocytes In Neonatal Mice

Background and PurposeXenogeneic transplantation has been a hot research topic world widely for it may solve the shortage of homogenetic cell.It has already been proved efficiently that xenohepatocyte transplantation is useful for the treatment of liver congenital azymia, hematological system disease and liver senescence in animal experiment.To improve the survival and functional conditions of xenograft,the main problem encountered is elucidating the rejection mechanism.The main problems exist in immunological rejection of xenogenic transplantation research are as follows:First of all,allograft and xenotransplantation have different mechanisms of immunological rejection.The xenograft will face a fiercer and more rapid immunological rejection,and may lose their functions completely or disappear in a couple of hours.Therefore,the mechanism of immunological rejection in xenotransplantation can not be studied step by step.Second,most studies on xenotransplangtation have dealt with the humoral immunity rejection.Little is known about the mechanism of cellular rejection in the xenotransplantation.At last,the rejection types are always different,depending on the character of xenograft,amount and intension of the antigen implanted and the immunity situation of accepter. Especially to a certain organ like live,the immunological rejection has its own characteristic.So,it has become a key work to select appropriate xenograft as well as accepter,and establish a model of xenohepatocyte transplantation for the directly mechanism research of cellular immunological rejection. According to the existing problems,a feasibility analysis has been made.First, the liver is called 'Immunological Preferential Organ' for its special tolerance.During a long-term clinical research,it is found that the rate and level of acute rejection are much lower than other organs after the transplantation of liver.Embryonic liver is the main synthesis and secretion organ of AFP(α-fetoprotein),which is a kind of protein related to the embryonal carcinomas.It is usually expressed in a higher level after the birth of mouse.AFP can function as a kind of suppressive factor on the immune system and help hepatocarcinoma cells escape from immune surveillance.Besides, according to the phenomenon of "the immune tolerance induced by external antigens", some researches showed that the level of immune tolerance caused by external antigens was relevant to the immaturity of immune system.If the xenogenic antigen was transplanted into the acceptor during early development stage of immune system, it would slow down the acceptor's xenogenic immunological rejection,even to be tolerant directly.In conclusion,we selected the liver of newborn mouse as the transplant acceptor.Furthermore,Research has confirmed that xenogenic antigens that the acceptor did not have arose the immunological rejection after transplantation.The xenogenic antigen mentioned was mainly the MHC(Major Histocompatibility Complex) molecular.The hepatocellular carcinoma cells not only had xenogenic antigens as normal human cells,but also they have a more powerful vitality.For these reasons,three types of human hepatocellular carcinoma cells,one kind of normal human hepatocyte and lung cancer cells were used as the candidate for xenogenic transplantation.At last base on this model,we tried to investigate the cellular immune rejection mechanisms of xenogenic hepatocyte transplantation.Methods1.Establishment of human hepatocyte orthotopic transplantation model in neonatal mice(1) According to the normal establishment way for animal model,human cell was injected into the liver of mice directly.(2) Xenogenic cells were traced by fluorescent reactive dye Dil in vivo.(3) Transplanted cells in mice were observed by autopsy.(4) Survival conditions and histological characteristics of human cells in each group were analyzed by general pathologic methods.(5) The expression of AFP and Survivin were detected by immunohistochemistry.2.Particularity of neonatal mouse liver microenvironment in xenogenic hepatocyte transplantation(1) Three-day-old mice were used for subcutaneous injections.6-week-old KM mice were used for intrahepatic implantation.(2) Transplanted cells were traced and located by fluorescent dye Dil.(3) The different immune pathologic progress of transplanted cells in different location was analyses by general pathologic methods.(4) Serum AFP concentration of KM mouse before and after transplantation was detected by quantitative ELISA;3.Studies on the cellular immunological rejection mechanisms of human hepatocyte orthotopic transplantation in neonatal mice(1) Immuno-regulated medicaments(CsA,PolyI:C) were used to set two different immune institution as controls:immunological activation and immunological suppression.(2) Establishment of human hepatocyte orthotopic transplantation model in neonatal mice.(3) The immunological situation of mice was analyzed after transplantation by histopathology assessment. (4) Killing activity of NK cells in each experimental group was tested.(5) T lymphocyte activity induced by Con A was measured.(6) Distribution of T cell subsets in each experimental group were detected by FCM assay.(7) Serum concentration change of IL-2 and IL-10 after transplantation was detected by quantitative ELISAS.ResultsThe first partAutopsy analysis of tumor locationMost of the hepatoma cells were transplanted successfully,and several cases were accompanied with certain transfer inside of the liver.Human lung carcinoma cells had a lower transplantation rate,along with the tendency of fading away.Ectopic changes were found in only two cases of human normal liver cell transplantation group.Location analysis with fluorescent dye DiI in vivoThe injected hepatoma cells generated externally visible single orthotopic nodules in all mice,obviously located and stably marked.But human lung cancer cells had a smaller location.There was no cellular structure in the ectopic change of normal liver cells group.Analysis of immune pathological procedureWithin the first 5 days,human hepatocarcinoma cells kept an immature and malignant appearance.At about the 8th day,lymphocytes infiltration and necrosis appeared,but there was not any lymphocytes infiltration and transplanted cell necrosis in the dissemination cells to the end of experiment.There was evident lymphocytes infiltration and necrosis occurred at the 5th day in the human lung cancer cell group. Immunohistochemical detectionAll the cells kept the original protein expression after transplantation:Be17402 and HepG2:AFP⁺;SK-Hep-1 and A549:AFP^-,all the cell lines had a positive expression of Survivin.The second partSubcutaneous transplantation of human hepatocarcinoma HepG2 cells into neonatal miceOn the second day after transplantation,lymphocytes infiltration and xenograft necrosis took place,and xenograft cells faded away completely after 2 weeks.Intrahepatic transplantation of human hepatocarcinoma HepG2 cells into adult miceThe injected tumor cells generated externally visible nodules in only 10%cases. Obvious lymphocytes infiltration and innflammatory cell encapsulation were found 24hs after transplantation.The transplanted cells showed an abundant,foamy, eosinophilic cytoplasm form,and were necrotized on the subsequent days.Cell in large quantity did not relieve the intensity and the pace of immunological rejection.Serum concentration change of AFPThe serum concentration of APF kept at a high level within the first 3 days after birth,but returned to a normal trace level after 2 weeks in KM mouse.The special re-expression of AFP was initiated by the transplantation of human hepatocarcinoma cells,making its serum concentration kept at a high level for 5-9 days,which showed distinct differences with normal mice.The third partEffects of immunomodulator Group CsA:The xenograft occupied larger space,a low tendency lymphocytes infiltration appeared on the 14^th day after transplantation.No obviously necrosis appeared until the 35^th day after transplantation.Group PolyI:C:The xenograft was smaller compared with the normal group. Lymphocytes infiltration appeared obviously on the second day after transplantation, and serious necrosis was found at the fifth day.Killing activity of NK cellsTransplantation group:The killing activity of NK cells increased markedly on the 4^th day after transplantation.Group CsA:The killing activity of NK cells increase slowly after transplantation, but it was lower than the transplantation group.Group PolyI:C:The killing activity of NK cells kept a high level all through the experiment,with prominent differences with the other two groups.T cell Transformation ActivityTransplantation Group:T cell transformation activity increased slowly at first, but it markedly increased at day 7 after transplantation.CsA Group and Poly I:C group:T cell transformation activity increased gradually without any significant changes,Poly I:C Group had the highest value and the CsA Group had the lowest.Distribution of T cell SubsetsMouse T cells did not developed adequately after birth,the number of CD4⁺ and CD8⁺ T cells increased as time goes by.Compared with normal mice group,the level of CD4⁺ and CD8⁺ T cells of the transplantation group was higher at any time point,and the number of CD4⁺ T cells grew faster.The ratio of CD4⁺/CD8⁺ T cells reached the highest point at day 11 after transplantation,which was significantly higher than other groups.CsA Group showed the smallest percentage of CD4⁺ T cells among all the groups,and its ratio of CD4⁺/CD8⁺ T cells reversed.The percentage of CD4⁺ and CD8⁺ T cells in Poly I:C group were both increased,and the ratio of CD4⁺/CD8⁺ T cells was slightly higher than the normal mice group.Concentration of IL-2 and IL-10Transplantation Group:On the 3^rd day after transplantation,IL-2 concentration decreased to the bottom while IL-10 concentration reached to the peak,then the situation reversed,on the 7^th day,IL-2 concentration arrived at its summit and IL-10 concentration reached the lowest point.In CsA Group,IL-2 was observably inhibited,and IL-10 concentration was higher than other groups.In Poly I:C group,IL-10 concentration was lower than transplantation group,but IL-2 concentration was much higher than other groups.Conclusion1.This research analyzed quantitatively the expression characteristic of AFP after the birth of KM mouse for the first time.Our results showed it had a similar expression pattern as BALB/c/J,BALB/c/BOM and C3H/He mice.2.Orthotopic transplantation of human hepatoma cells caused a high level re-expression of AFP,which was relevant to the formation of immunological suppression microenvironment in neonatal mouse.3.The orthotopic transplantation of human HepG2 cells in neonatal mouse could be used as a useful model for the study of immunological rejection mechanism of xenohepatocyte transplantation.4.The first event in xenotransplantation immunological rejection is the activation of NK cells.Yet this process did not cause strong immunological rejection to the xengraft. 5.The increase in T cell activity is vital for immunological rejection to the xengraft.CD4⁺ T cells are the main subset in cellular rejection.Imbalance or even reverse in the ratio of CD4⁺/CD8⁺ caused immunological rejection.6.During the differentiation of activated CD4⁺ T cells,an immune deviation took place and caused the transform of T0 to T1 instead of T0 to Th2,which turned mouse liver microenvironment from immune-tolerance to immune-rejection.7.CsA alone could not completely inhibit immunological rejection in xenotransplantation,only delay the process.