| Allogenic hematopoietic stem cell transplantation (allo-HSCT) is the most efficient treatment for hematological malignancies. But the graft versus host disease (GVHD) induced by allogenic immune cells is one of the major causes of mortality in allo-HSCT. Although there is a variety of measures to prevent and treat GVHD, but there is still 30%~70% of patients after transplantation developed to GVHD.Currently consider the most effective prevention and treatment of GVHD after allogeneic bone marrow transplantation and improving the success rate for allo-HSCT lies in the set up immune tolerance between donor and receptor, so that donor and receptor cells can peaceful coexistence and maintain normal immune function. Set up a bone marrow transplant immune tolerance is the key for the immune cells, the donor immune cells can identified the receptor outside cells as the self, not to happen immune response. We select the embryonic animal as a donor, in immunological system forming period, induced its immune tolerance of the receptor by means of vaccination receptor MHC antigen in Yolk sac cavity. After the induction of tolerance to donor, hematopoietic stem cells was transplanted to the receptor provided the toleragen, thus preventing the occurrence of GVHD after transplantation, and ultimately improve the feasibility of hematopoietic stem cell transplantation. To overcome the restrictive theory that the hematopoietic stem cell transplantation must be HLA-matched laid the foundation, and for further study of adult stem cells and embryonic stem cells used in HSCT-induced immune tolerance study provides the experimental support. In this experiment normal female Wistar rats and SD fetal mice of pregnant 7.5-8.5d as experimental objects, then divided into four groups:①Simple exposure group: normal female Wistar rats only given lethal doses of irradiation.②Simple transplantation group: bone marrow cells from normal male SD rats (embryonic stage without any intervention)was transplanted into lethally irradiated Wistar rats.③Blank transplantation group: SD fetal mice of pregnant 7.5-8.5d yolk sac cavity only to give the 2.5ul PBS injected fluid, male SD fetal rat born to the adult rat bone marrow cells transplanted to the Wistar female rats after lethal dose irradiation.④Transplantation group after induced: peripheral blood mononuclear cells from Wistar rats (as the toleragen) about 1×105 was injected into the yolk sac cavity of SD fetal mice. When the male SD fetal rat born to the adult rat, the bone marrow cells about 5×107 harvested male SD rat was grafted to the female Wistar rat (the original provider of toleragen) via the tail vein, Wistar rats pre-transplant 4-6h accepted Co60 irradiation (γ-ray), with a total dose of 8.0Gy, dose rate 0.5Gy/min, removal of their own blood and immune system.Induced group was the experimental group, others were the control groups. After transplantation the general survival receptor in rats (including body weight changes and survival rates, etc.) were observed and recorded every day; each receptor rat was observed blood cell count changes in peripheral blood every 4d until post-transplant restore;the rats died post-transplant time and 30d, 105d observed bone marrow proliferation through the pathological bone marrow smears Detection; post-transplant 30d and 105d used PCR to assay receptor rat peripheral blood and bone marrow cells of donor origin Y gene expression; post-transplant 15d, 105d selected the liver, spleen, skin, intestinal GVHD Detect; the 30d receptor after transplantation of skin allografts in rats observed immune response after transplantation; post-transplant 30d, 105d, used one-way mixed lymphocyte reaction detection to observe the degree of the receptor immune tolerance.The results showed that: (1) rats after lethal dose 8.0Gy irradiation, the control group continued to drop weight until be death, no return, while the experimental rats about 10d after exposure weight has started to increase, about 20d after exposure the weight replied the former level before exposure.The experimental group and the control groups were significantly different, while between the control groups showed no differences; 12d after irradiation all exposure group rats died, and blank control group, transplantation control group, almost all the rats died within 16d, and the experimental group have 85% of the rats survived after post-transplant 105d. compared the survival rate of rats between the experimental group and the control groups were significantly different (P <0.05), while the transplant group and the blank group no difference (P> 0.05). (2) The peripheral blood white blood cell count of rats after irradiation continued to decline, the groups got a minimum at 8d after irradiation.When the exposure group at 12d after irradiation were deaths, all deaths white blood cell count <0.5×109/L. The experimental group and other control groups after irradiation 10-12d the reply has already begun, but the survival of rats in control groups died within 16d, when white blood cell count has not yet returned to normal levels.Tthe experimental rats at 30d after irradiation had been completely back to normal. (3) The irradiated group rats died of bone marrow hematopoietic failure.Their bone marrow smears and bone marrow biopsy showed hematopoietic failure,but after transplantation of 30d, 105d , the experimental group rats bone marrow smears and biopsy showed active bone marrow hyperplasia. Experimental rats at 30d, 100d after transplantation were detected sry-gene expression used PCR methods, and a survival rat of transplanted group was not detected sry-gene expression. (4) The①and②control groups rat liver, the spleen, the intestine and the skin pathology examines the GVHD phenomenon, but the 15d experimental group rat organization organ pathology inspection only sees mild GVHD or the non-GVHD phenomenon, the 105d organization organ pathology shows the organizational structure basically normal. (5) After the transplantation, the 30d acceptor rat grafting donor skin piece success ratio is obviously higher than third irrelevant supplies rat, the difference has statistics significance. After the transplant, 30d and 105d experimental group rats was one-way mixed lymphocyte reaction hyporeactivity, significantly lower than the control group (P<0.05). Conclusion: The donor embryo sac by means of vaccination after allogeneic mononuclear cells, normal birth, survival, the experimental method is safe and feasible; Bone marrow cells transplantation via tail vein is a convenient and feasible and effective method of hematopoietic stem cell transplantation; After Induced donor embryos immunotolerance, the donor marrow cells transplanted was able to rebuild the vivo hematopoiesis and immune system in the receptor; Induced embryos immune tolerance through the yolk sac approach applied to hematopoietic stem cell transplantation, to a certain extent, prevent GVHD after the hematopoietic stem cell transplantation.
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