| Research objective: Hematopoietic stem cell transplantation(HSCT)not only can cure some malignant diseases of the blood system,but also it is widely used in non-hematological diseases.However graft-versus-host disease(GVHD)remains a leading cause of death related to transplantation.As early as 1966,Billingham defined three essential prerequisites for the development of GVHD: the presence of immunologically competent cells in the donor graft,histocompatibility difference between the donor and the recipient,and theinability of the immunosuppressed recipient to mount an effective immune response to reject the donor cells.In recent years,the study found that the removal of T cells in the allograft or inhibition of its function can reduce the incidence of GVHD or its severity,but increase the opportunistic infection and disease recurrence rate.In recent years,the immunoregulation cell of GVHD has become a new hotspot,such as CD4+CD25+Tregs,mesenchymal stem cells,NK cells,NK T cells,which can inhibit the immune attack for T cells to allogeneic antigen,so as to reduce the occurrence of GVHD.However,the isolation,purification and amplification of these cells require special conditions and equipment,which limits their wide application in clinic.Further studies have shown that the recipient's own cells such as bone marrow cells,peripheral blood mononuclear cells,spleen cells can also effectively reduce the incidence of GVHD.The recipient lymphocyte has been paid more and more attention because of its advantages such as convenient,easy to obtain,safe and convenient to use.The aim of this study is to investigate the effects on the production and development of GVHD by infusion of different doses of recipient spleen cells at different time after MHC haploidentical HSCT in mice and further explore the possible mechanism.Contents research:1,The establishment of GVHD model of MHC haploidentical hematopoietic stem cell transplantation in mice2,The effects on donor chimera and GVHD by infusion of different doses of recipient spleen cells at different time after HSCT3,The mechanism of reducing GVHD by infusion of recipient spleen cells 3 ×107 at the fourth day after HSCTResearch methods:1,The establishment of GVHD model of MHC haploidentical hematopoietic stem cell transplantation in miceThe recipient mice were randomly divided into 4 groups according to the number of cells,each group with 8 mice.The experiment was divided into A group: 8Gy TBI pretreatment.B,C,Dgroups: the same pretreatment,respectively by infusion of spleen cells was 1×107,3×107 and 6×107.A group was infused saline.Blood respectively via tail vein respectively at +1d,+4d,+7d,+14d and +21d,monitoring hematopoietic recovery and donor chimera.The changes of physical signs and histopathological changes were monitored,and the GVHD score and the diagnosis were performed.2.The effects on donor chimera and GVHD by infusion of different doses of recipient spleen cells at different time after HSCT(1)Recipients were respectively infused by spleen number 1×107,3×107 and 6×107at day1 after HSCT.The control group received the same amount of saline.(2)Recipients were respectively infused by spleen number 1×107,3×107 and 6×107at day4 after HSCT.The control group received the same amount of saline.(3)Recipients were respectively infused by spleen number 1×107,3×107 and 6×107at day7 after HSCT.The control group received the same amount of saline.The hematopoietic recovery,donor chimerism,changes of physical signs and histopathological changes were observed after HSCT.3,The mechanism of reducing GVHD by infusion of recipient spleen cells 3×107 at the fourth day after HSCTRecipients were injected into the same recipient cells 3×107 at day1,day4 and day7 after HSCT.The changes of the proportion of peripheral blood lymphocyte subsets and the expression of T lymphocytes in the peripheral blood of FasL were monitored.Results of research:1.Recipients were pretreated with 8Gy TBI irradiation,and the incidence of GVHD was only 50% when the number of cells was about 1×107,although the mice were able to achieve complete donor chimerism.Recipients given the cell number by 3×107 and 6×107,respectively,were able to form a complete donor chimerism.The incidence rate of GVHD was 100%,and the pathological spleen,intestine,skin and liver showed pathologic changes of GVHD.2.At day1 after HSCT recipients were respectively infused by the number of 1 ×107 and 3×107.The donor obtained complete chimerism(CC).The incidence and mortality of GVHD was 100%.The median survival time was 18d~21d.However the infusion of 6×107 has led to the graft rejection and all recipients for hematopoietic failure and death.The median survival time was 14 d.At day7 after transplantation,the recipients were transplanted with 1×107,3×107,and 6×107,respectively.The donor chimera was CC,and the incidence of GVHD and mortality were both in the range of 100%,and the median survival time was 23d~28d.At day4 after HSCT the recipients were transplanted with 3×107 and 6×107,respectively.The donor CC was obtained and its incidence rate of GVHD in 20%~30%,significantly reduce the incidence of GVHD,The median survival time was more than60 d,and prolonged the survival time of recipients.However,the incidence of GVHD was 80% and the median survival time was 45 d when recipients wereinfused the number of 1 × 107 at day4 after HSCT.3.The expression rates of CD3+CD4+FasL+ and CD3+CD8+FasL+ in normal donor(C57)were(0.3±0.06)%,(0.4±0.11)%.After HSCT,the expression rate of CD3+CD4+FasL+ and CD3+CD8+FasL+ in the peripheral blood donors in the positive control group was(72.12±19.05)%,(36.23±15.73)%,which was significantly higher than that in the normal donor(P<0.05).The expression of +4d infusion group CD3+CD4+FasL+,CD3+CD8+FasL+ respectively(10.14±7.26)% and(6.12±5.75)%,were significantly lower than the positive control group(P<0.01),and had statistical difference(P<0.05)compared to +1d infusion group: CD3+CD4+FasL+:(51.52±11.12)% andCD3+CD8+FasL+;(13.13±3.35)% P<0.05,and also had statistical difference(P<0.01)compared to +7d infusion group: CD3+CD4+FasL+:(68.16±13.25)%,CD3+CD8+: FasL+(36.14±9.64)% compared to P<0.01.Research conclusions:1,In this study,we established a H-2 haploidentical mouse model of GVHD,which provides an ideal platform for further study on the mechanism of GVHD;2,Infusion of recipient cells at day4 after HSCT can significantly reduce the incidence of GVHD and prolong the survival time in mice,but the incidence of GVHD can not be reduced at day1 and day7 after HSCT.3,Infusion of recipient cells at different time after HSCT will lead to the expression of T lymphocytes surface FasL showed a different state.The FasL expression of T lymphocytes is significantly decreased at day4 infusion than at day1 and on day7 infusion. |
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