| Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been shown to be the most efficient method for leukemia treatment. However, there still remained a lot of challenges, including graft-versus-host disease (GVHD), graft rejection and immune recovery, especially the dilemma to establish qualified strong graft versus leukemia (GVL) effect while under no GVHD condition.To focus on these allo-HSCT associated clinical problems, in this study, we established a method to proliferate and purify nature killer (NK)-cells in vitro, set up mouse bone marrow transplantation model, and carried out the research on the role and mechanism of NK cells in mouse allogeneic bone marrow transplantation.According to our experimental results, we presumed alloreactive NK cells could reduce the incidence of GVHD and graft rejection, augment hematopoietic stem cells engraftment level, promote hematopoiesis and immune reconstruction and increase recipient survival rate. We also found alloreactive NK cells graft-versus-tumor (GVT) inhibitory effects on murine B16-F1 cells. This study closely related to allo-HSCT clinical application, had theoretical significance on the study of NK cells effector mechanism, also presented instructive information on the NK cells application to allo-HSCT and allogeneic tumor treatment.Chapter 1 Preliminary study on proliferation and purification ofmurine nature killer cells in vitro Objective: To establish a method of nature killer(NK)-cells proliferation andpurification in vitro. Methods: NK-cells were isolated from splenic mononuclear cells(MNC) by a two step adherence system and magnetic microbeads actived cell sorting(MACS), follow-up these cells were cultivated with feeder cells and IL-2 in RPMI 1640 medium 5days, lOdays, 15days, and 20days. Enriched cells were counted and stained with FITC-anti-mouse CD3 and PE-anti-mouse NK1.1, then FACS measured the purity of NK-cells, the cytotoxicity to YAC-1 targets was detected by MTT. Results: In the two step adherence system, enriched cells cultivated 5days, 10days, 15days, and 20days was 0.5×107,1.4×107,2.6×107, and 3.0×107, respectively. The percent of CD3-NK1.1+ cells was 18.36%, 43.44%, 55.68%, and 60.03%, respectively. The cytotoxicity to YAC-1 targets was significantly higher than splenic MNC, and increased from 41.01 % up to 54.38%, 66.54%, 79.38%, 83.86%, respectively. When NK-cells were purified by MACS, although the purity reached 93.60%, enriched cells was only 1.5×106 from 1.0×108 total cells, and enriched cells was not prolific. Conclusion: The optimal time for NK-cells proliferation and purification in vitro was 2~3 weeks by using the two step adherence system, the obtainable cells was abundant, the purity of NK-cells was 55%~60%, and the cytotoxicity to YAC-1 targets was about 80%(Effector cell and targets ratio was 25:1).Chapter 2 Establishment of acute graft-versus-host disease model inmouse allogeneic bone marrow transplantation Objective: To establish murine acute graft-versus-host disease(aGVHD) model for the study of GVHD in allogeneic bone marrow transplantation. Methods: Lethally irradiated BALB/c (H-2d) mice were transplanted with C57BL/6 (H-2b) bone marrow containing variational quantity of donor peripheral lymphocytes. Evaluated the degree of GVHD according to clinical and pathological data. Results: 20% mice in the group of transplanted without peripheral lymphocytes survived long (>120 days), and aGVHD could not be induced in them. The degree of GVHD was different in that group of transplanted with variational quantity of donor peripheral lymphocytes. In the group of bone marrow cells (4×106) and peripheral lymphocytes ratio was 1:1, aGVHD could be induced in all recipient, and the degree of GVHD was nearly consistent, all mice survived 6 to 24 days after transplantation, and clinical signs of aGVHD such as hunched back were seen in 5 to 14 days. Conclusion: Lethally irradiated BALB/c mice were transplanted with 4×106 C57BL/6 mouse bonemarrow cells and peripheral lymphocytes, successfully establish lethal aGVHD mo...
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