Estabishment Of Non-myeloablative Bone Marrow Cell Transplantation Model In Mice And Its Effects On Immune Tolerance And Anti-liver Cancer

Background:Liver transplantation has substantial therapeutic effects for liver cancer, but the paramount complications after long term use of immunosuppressants have been being the greatest headache. Some previous studies show that solid organ and donor non-myeloablative bone marrow cells combined transplantation can induce reciporal tolerance. The living donor liver transplantation makes the donor bone marrow cells available, so it is possible to perform the living donor liver transplantation and donor non-myeloablative bone marrow cell combined transplantation which is a promising therapeutic model to induce immune tolerance. The roles of Treg and Th17 cells in this process are elusive. The non-myeloablative allogeneic bone marrow cell transplantion has substantial therapeutic effects for renal cancers and breast cancers, but the evidences for liver cancer are lack.Objective:To establish reciprocal immune tolerance via non-myeloablative allogeneic bone marrow cell transplantation (N-Allo-BMT), and to explore the possible roles of Treg and Th17 cell in this process. To test the possible anti murine live cancer effects of N-Allo-BMT, and to clarify the possible mechanisms.Methods:Section-1:The non-myeloablative regiment consists of low dose of Co radiation (3Gy) and high dosage of cyclophosphamide (CTX,200mg/Kg, i.p).The cells were injected by retro-orbital injection (ROI). Wright staining and FCM were used to indicate the survival of the allograft cells. The GVHD was recorded in detail. After acquisition of the immune tolerance recipients, allogeneic skin transplantation was adopted to testify the reciprocal tolerance state. The expression of IL-17, IL-10, foxp3 and TGF-beta in spleen were also tested by RT-PCR. Section-2:After establishment of the N-allo-BMT, murine H-22 liver cancer cells were inoculated subcutaneously, and then the time of palpable tumor, the tumor growth curve, final tumor volume and Kaplan-merrier survival estimation were recorded. As well, the intratumoral lymphocytes, vascular density and VEGF-A expression were examined.Results:The success rate of retro orbital injection (ROI) for 50 mice is 100% without any complication. There is no mortality in sham group (8 mice) which only accepted non-myeloablative regimen without cell transplantation. The N-Allo-BMT group reconstructs hematopoietic system within 10 days.15 days later, the FCM (flow cytometery) indicates full donor chimerism in all recipients.18.9%(4/22) of the recipients survive more than 120 days, and 9.1%(2/22) of the recipients,with mixed chimerism state, achieve induced reciprocal immune tolerance verified by allogeneic skin transplantation. RT-PCR indicates the IL-17 expression is significantly high in severe GVHD recipients (p<0.01), to the contrary, FOXP3 and TGF-beta expressions are significantly high in long survivors (P<0.01). Section-2:The time of palpable tumor of each group has no statistical differences. The tumor of N-Allo-BMT group grows much more slowly than the other groups and the final tumor volume is significantly small. But the overall survival time showes no difference. In N-Allo-BMT group, more CD4 and CD8 lymphocytes infiltrated into tumor tissues and the vascular density and VEGF-A expression of tumor tissues significantly decline (P<0.01)Conclusions:Section-1:Low dose of radiation plus single dosage of CTX i.p injection can be an economic and reliable non-myeloablative regimen for mice. ROI is much easier to handle than tail vein injection and it can be a simple, safe and reliable approach to deliver cells in vivo. Through this N-Allo-BMT model,9.1% of the recipients successfully achieve reciprocal immune tolerance, and for the first time we discover the important roles of Treg and T17 cells play important roles in this process. Section-2:N-Allo-BMT has substantial anti murine liver cancer effects, but it cannot improve the overall survival time because of severe GVHD. We propose that the anti-liver cancer effects may results from the inhibition of neo-vessels, rather than direct killing of the cancer cells themselves. We hypothesize that living donor liver transplantation-donor non-myeloablative bone marrow cell transplantation can be a promising "one stone kills two birds" alteration for the unresectable liver cancer patients.