| Natural killer cell (NK cell) is an important part of human body’s natural immune. It can effectively remove the mutated cells, including malignant transformed cells and infected cells by pathogenic microorganisms. NK cells can identify normal cells and abnormal cells directly, without antigen-presenting cells’help compared with antigen-specific T cells, and then they would respond quickly to the body’s abnormal signal. Generally believed, NK cells distinguish normal and abnormal cells through the NK cell receptors on NK cells. NK cells mainly express the killer cell immunoglobulin-like receptor (KIR) on their surface, and KIR can recognize and bind the major histocompatibility complex I molecular (MHC I). KIRs are divided into two types, inhibitory and activating types transmitting inhibitory and activating signals respectively, and regulate the cytotoxic activity of NK cells together. At present the widely accepted theory is "Missing Self" mechanism:the normal cells in our body express a large number of MHC I molecular, binding to the inhibitory KIR on the NK cells’surface and inhibit the NK cell activity. The MHC I molecules expression reduce when the cells malignant change or infected by virus, these cells will be eliminated by activated NK cells because the cytotoxicity of NK cells cannot be inhibited effectively. This mechanism is considered to be the effective means in the treatment of benign and malignant blood system diseases in bone marrow transplantation (BMT). According to the report, the allo-reactive natural killer cells (allo-NK cell) from allogeneic hematopoietic stem cell transplantation donors can remove the leukemic cells in recipients. Our previous study showed that NK cells can reduce the incidence of graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation, and the effect was related with the matching degree of NK cell KIRs and recipients’MHC I, because NK cells’ biological effect depends on the degree of matching and recognition between KIR and receptor.This study focuses on the influence of matching degree between KIRs & MHC I on the hematopoietic reconstitution after bone marrow transplantation. As a lymphocyte subsets, the NK cells in peripheral blood and spleen are very few. We need to isolate and culture in vitro purification NK cells, in order to guarantee adequate treatment dosage. We get a lot of high purity of NK cells through Density gradient centrifugation and immunomagnetic beads and vitro amplification culture using platelet lysate(PL). We intend to use the animal model to obtain the findings of the matching degree of NK cell receptor and host MHC I’s influence and mechanism on graft implantation after bone marrow transplantation, for the further study of allo-NK cells in bone marrow transplantation. We establish the bone marrow transplantation model first. Pretreatment before transplantation is a necessary condition for donor implantation, and pretreatment with different strength has significant effect on the prognosis of bone marrow transplantation. In this study, we used total body irradiation (TBI) as the main means of pretreatment. We used TBI of 3Gy and 6Gy 60Coy ray on the BALB/c and CB6F1 female mice to prepare the myeloablative and nonmyeloablative models. Then we input the bone marrow cells of C57BL/6J male mice through their tail vein, and observed the survival and chimerism rate of recipients to determine the optimal pre strength of bone marrow transplantation animal model. Finally, the control group of mice pretreated with 6Gy all died during the observation period, and more than 90% experimental group mice which were treated with bone marrow transplantation still survived. The peripheral white blood cells and platelet levels of the mice pretreated with 6Gy and treated with Bone marrow transplantation were suit for observation for hematopoietic reconstitution in mice.In order to observe the effects of the matching degree of NK cells from the donor mice and MHC I molecules from receptor mice on hematopoietic reconstitution after bone marrow transplantation. We isolated bone marrow cells and NK cells from 30 C57BL/c mice, and infused them to the mice pretreated with BALB/c and CB6F1.Recipient mice were divided into simple irradiation group, bone marrow cells infused group, bone marrow cells and NK cells infused group,6 mice per group. We observed the changes of the blood routine index, survival time and pathological indexes histopathological indexes at different times, then compared the difference among groups. The results showed that:mice from bone marrow cells infused group and bone marrow cells and NK cells infused group survived for a long time, there was no GVHD pathology. On the fourteenth day after transplantation, the peripheral white blood cell counts in BALB/c mice from bone marrow cells infused group and bone marrow cells and NK cells infused group in which the MHC Ⅰ molecules were completely mismatched were (1.35±0.33)×109/L and (1.80±0.18) × 109/L, and there was significant difference between them, and the platelet counts were (79±19) ×109/L and (117 ± 13)×109/L with significant difference between them. The peripheral white blood cell counts in CB6F1 mice from bone marrow cells infused group and bone marrow cells and NK cells infused group in which the MHC Ⅰ molecules were half-matched were (1.52±0.26)×109/L and (1.85±0.34)×109/L, with no significant difference between them, and the platelet counts were (90±12) ×109/L and (113±15)×109/L with significant difference between them. The improve of BALB/c mice’s white blood cells were better than the CB6F1 mice. We conclused that:the allo-NK cells can promote the recovery of hematopoietic function after transplantation, and the degree of recovery of hematopoietic function was influenced by the matching degree of surface receptors and MHC Ⅰ molecules. And the effects on hematopoietic function only in the early hematopoietic reconstitution after transplantation, this may be related to the half-life of NK cells. |
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