The Preliminary Study Of The Effect Of Ghrelin On The Thymus Regeneration Of Mice After Allogeneic Bone Marrow Transplantaion

Background and Objective:Immune dysfunction after allogeneic hematopoietic stem cell transplantation,which is one of the most important reasons of infection and disease recurrence.Long-term defects in T cell function caused by thymic atrophy and thymic functional defects is one of the leading causes of immune reconstruction delay.How to promote the recovery of thymus function after transplantation is a hotspot in this field.In recent years,the immune regulation of ghrelin(GRL)has received more and more attention,there are studies reported that GRL can reverse the the thymus atrophy of aging mice.However,there is still no research about the effect of GRL on thymus after hematopoietic stem cell transplantation.The aim of this study was to investigate the effect of GRL on the thymus of mice after bone marrow hematopoietic stem cell transplantation by constructing the model of myeloablative total body radiation and allogeneic hematopoietic stem cell transplantation.Methods:1.Taking C57BL/6 mice(H-2Kb)as donor and BALB/c mice(H-2Kd)as recipient to establish a model of myeloablative allogeneic bone marrow transplantation.We then investigated the does of total body irradiation(TBI)(7.0Gy,7.5Gy,8.0Gy)and the does of bone marrow mononuclear cells(BMNCs)(0.5 x107,1.0x107,2.0x107).Then observe the general clinical manifestations,GVHD performance,chimerism rate.2.In order to observe the effect of GRL on thymus injury in mice after myeloablative TBI,120 mice were randomly divided into 4 groups:In fusion of GRL 7 days before TBI(group A1)or 1 day after TBI(group B1),infusion of PBS 7 days before TBI(group C1)until the end of the experiment.Group D1 as a normal control,without any treatment.The thymus cells were sacrificed at 1,7,and 14 days after TBI.Thymus cells were counted by thymus tissue,HE staining and immunofluorescence were used to analyze the changes of thymic structure.Flow cytometry was used to detect the subgroups of thymus subpopulations proportion.3.In order to observe the effect of GRL on thymus regeneration after bone marrow transplantation(BMT),120 mice were randomly divided into 4 groups:In fusion of GRL 7 days before BMT(group A2)or 1 day after BMT(group B2),infusion of PBS 7 days before BMT(group C2)until the end of the experiment.Group D2 as a normal control,without any treatment.Thymus cells were counted by thymus tissue,HE staining and immunofluorescence were used to analyze the changes of thymic structure.Flow cytometry was used to detect the subgroups of thymus subpopulations proportion.The number of sjTREC levels in peripheral blood was detected by SYBR Green I method.Result:1.The mice received 7.5Gy TBI were all dead within 30 days,and the number of white blood cells was less than 0.5 × 109/L,which was hematopoietic failure state.In this study,7.5Gy was selected as the myeloablative pretreatment program.Infusion of 0.5 x107,1.0x107,2.0x107 BMNCs mice were able to survive for a long time,but the chimeric analysis showed that 0.5 x107 and 1.0x107 BMNCs were mixed chimeric,2.0x107 BMNCs were complete chimeric.2.The effect of GRL on thymus after TBI:(1)thymus structure:The degeneration and necrosis of the thymus epithelial cell was significantly reduced in mice with GRL treatment.The results shown that group A1 which with GRL protection of A1 group still remains normal structure at 1 day after TBI,while mice in group Ci has been significant atrophy and apoptosis.(2)T cells in thymus:Compared with group C1,the imbalance of T lymphocyte subsets in mice with GRL infusion was significantly reduced,and the absolute number of each group T lymphocyte subsets was higher.This difference is particularly evident in group A1.RL treatment of thymus T lymphocyte subsets of imbalance was significantly reduced,and the absolute number of T lymphocyte subgroups higher.The absolute numbers of CD4-CD8-,CD4 + CD8 +,CD4+,CD8 + T cells in A1 group and C1 group were 5.08±0.48)x106vs(2.86±0.57)x106,(P<0.05)、(3.62±0.69)x106vs(1.38±0.89)x106,(P<0.05)、(0.65±0.08)x106vs(0.222±0.09)x106,(P<0.05)、(0.55±0.30)x106vs(0.24±0.09)x106,(P<0.05)。3.The effect of GRL on thymus after BMT:(1)thymus structure:Compared with group C2,GRL-treated mice had rapid recovery of thymus and less thymocyte apoptosis.At 28 days after transplantation,the structure of group A2 was recover to normal structure and K5 + K8 + cells were significantly increased when compared with group C2.(2)T cells in thymus:T lymphocyte subgroup imbalance in mice with GRL treatment was significantly lower than mice in group C2,the proportion of immature double negative cells were relatively reduced,the proportion of double positive cells were significantly increased,and significantly improve the development of thymus mature CD4 + cell ratio,but no significant effect on CD8 + cell ratio.The absolute numbers of CD4 + in A2 and B2 groups were(9.51 ± 1.59)x106vs(6.96 ± 1.26)x106,respectively,which were significantly higher than those in group C2(4.90 ± 1.80)x106(P<0.05).The levels of TREC in the peripheral blood of group A2 were significantly higher than those of group C2(P<0.05)at 21 and 28 days after transplantation.Conclusion:GRL can protect the thymus from pretreatment of transplant,enhance the thymus resistance against pretreatment,promote the impaired thymus reconstruction and thymocyte proliferation,and can promote immature t double negative to double positive thymocytes differentiation,promote the differentiation of thymocytes to CD4+ SP,enhance thymus output function and accelerate the central immune reconstruction.