【作者】 王华；

【导师】 韩明哲；

【作者基本信息】 中国协和医科大学 ， 内科学血液， 2008， 博士

【摘要】 【目的】1.研究供者KIR和供受者HLA配体对单倍体造血干细胞移植（HSCT）预后的影响;2.研究供者KIR和受者HLA配体对同胞HLA相合HSCT预后的影响;3.探讨异基因造血干细胞移植（allo-HSCT）中移植物和移植后一个月患者外周血NK细胞和T细胞上四种抑制型受体（CD158a、CD158b、NKB1及CD94/NKG2A）的表达与造血重建及急性移植物抗宿主病（aGVHD）的关系;4.研究同种反应性NK细胞用于小鼠单倍体骨髓移植（BMT）中的抗白血病（GVL）作用;5.探讨同种反应性NK细胞对小鼠单倍体BMT免疫重建的可能有益作用。【方法】1.采用特异性序列引物聚合酶链反应（PCR-SSP）方法进行供者KIR及供受者HLA-Cw基因分型;2.流式细胞仪检测NK及T细胞上四种抑制型受体的表达;3.免疫磁珠分选法制备供鼠NK细胞及同种反应性NK细胞;4.建立C57BL/6×BALB/c（BCF₁）→BALB/c小鼠（荷EL9611红白血病）单倍体BMT模型,观察同种反应性NK细胞的GVL作用;5.应用C57BL/6×BALB/c（BCF₁）→BALB/c小鼠单倍体BMT模型,观察同种反应性NK细胞对免疫重建的可能影响:胸腺病理组织学观查;流式细胞仪检测外周血CD3e的表达;脾细胞增殖实验;酶联免疫吸附试验（ELISA）测定外周血血清及脾细胞培养上清IL-4、IFN-γ浓度。【结果】1.在单倍体HSCT中,供者KIR2DS3、KIR3DS1显著延迟中性粒细胞重建,同时KIR3DS1显著延迟血小板重建;KIR2DS1、KIR2DS3、KIR3DS1显著降低慢性GVHD（cGVHD）;受者HLA-Bw4阳性显著增加aGVHD发生率（以上各组与相应组比较,P均＜0.05）;GVH方向HLA配体不合组与相合组、KIR受体—HLA配体不合组与相合组比较,造血重建、DFS、复发、TRM没有显著性差别（P均＞0.05）。2.在同胞HLA相合HSCT中,供者KIR2DS5显著延迟中性粒细胞重建;KIR2DS2显著增加aGVHD和CMV病毒血症的发生率（P均＜0.05）;C1纯合组与C1/C2杂合组相比,cGVHD发生率显著降低,而且4例广泛型cGVHD均发生在C1/C2杂合组,同时C1纯合组败血症/肺感染的发生率显著降低（P均＜0.05）;KIR受体—HLA配体不合组DFS显著高于相合组[移植后600天DFS分别为（88.89±7.41）%、（53.85±13.83）%,P＜0.05],同时不合组复发率有低于相合组的趋势（P=0.08）。3.移植物NK细胞上CD158a、NKB1、CD94/NKG2A的表达在Ⅱ～Ⅳ度aGVHD组明显高于0～Ⅰ度aGVHD组;而CD8⁺T细胞上CD94/NKG2A的表达在0～Ⅰ度aGVHD组明显高于Ⅱ～Ⅳ度aGVHD组（P均＜0.05）。4.同种反应性NK细胞显著延长接受单倍体BMT的荷白血病BALB/c小鼠的生存期,降低复发率（与非同种反应性NK细胞组和单纯BMT组比较,P均＜0.05）。5.同种反应性NK细胞促进单倍体BMT小鼠的外周T细胞免疫重建,体内有效诱导Th1型免疫反应的偏移。【结论】1.在单倍体HSCT中,供者激活型KIR2DS3、KIR3DS1延迟造血重建,KIR2DS1、KIR2DS3、KIR3DS1降低cGVHD;受者Bw4增加aGVHD;GVH方向HLA配体是否相合、KIR受体—配体是否相合对造血重建、DFS均无显著影响。2.在同胞HLA相合HSCT中,供者激活型KIR2DS5延迟造血重建,KIR2DS2增加aGVHD和CMV病毒血症的发生;C1纯合状态降低cGVHD发生率和严重程度,降低感染;KIR受体—HLA配体不合可能降低复发,改善DFS。3.Allo-HSCT中移植物中NK细胞高表达CD158a、NKB1、CD94/NKG2A时增加Ⅱ-Ⅳ度aGVHD,而CD8+T细胞上高表达CD94/NKG2A降低Ⅱ-Ⅳ度aGVHD。4.同种反应性NK细胞用于单倍体BMT的荷白血病BALB/c小鼠,延长生存期,降低复发率,具有GVL的作用。5.同种反应性NK细胞促进单倍体BMT小鼠的外周T细胞免疫重建,体内有效诱导Th1型免疫反应的偏移。更多还原

【Abstract】 Objective1. To explore the impact of donor killer immunoglobulin-like receptors（KIR） and donor/recipient HLA ligands on outcome in haploidentical hematopoietic stem cell transplantation（HSCT）.2. To explore the impact of donor KIR and recipient HLA ligands on outcome in HLA-matched, sibling donor HSCT.3. To investigate the correlation of four types of inhibitory receptors （CD158a, CD158b, NKB1 and CD94/NKG2A） on NK cells and T lymphocytes from grafts and recipients peripheral blood cells 1m after allogeneic HSCT （allo-HSCT） with hematopoietic reconstitution and acute graft-versus-host disease（aGVHD）.4. To study antileukemia effect by alloreactive NK cells mediated in murine undergoing haploidentical bone marrow transplantation（BMT）.5. To investigate the promotion of immune reconstitution of alloreactive NK cells in murine undergoing haploidentical BMT.Methods1. Donor KIR and donor/recipient HLA-Cw genotype was determined by PCR -sequence-specific primer analysis（PCR-SSP）.2. Four types of inhibitory receptors on NK cells and T lymphocytes were analyzed the expression by flow cytometry.3. Murine NK cells and alloreactive NK cells were prepared by immunomagnetic separation.4. The murine haploidentical BMT model was established by using C57BL/6×BALB/c （BCF₁） mice as donor, and BALB/c mice bearing EL9611 leukemia as recipient, and GVL effect was observed by infusion of grafts with or without alloreactive or non-alloreactive NK cells.5. The effect of alloreactive NK cells on immune reconstitution was observed in C57BL/6×BALB/c （BCF₁）→BALB/c murine haploidentical BMT model.Results1. In haploidentical HSCT, the presence of donor KIR2DS3, KIR3DS1 delayed neutrophil reconstitution, and KIR3DS1 delayed platelet reconstitution. KIR2DS1, KIR2DS3 and KIR3DS1 reduced cGVHD. The presence of recipient HLA-Bw4 increased aGVHD （P<0.05, respectively）. Hematopoietic reconstitution, disease-free survival（DFS） and relapse were not significantly different between those with and without HLA ligands incompatibility, and those with and without KIR receptor-HLA ligand incompatibility（P>0.05, respectively）.2. In HLA-matched sibling HSCT, the presence of donor KIR2DS5 delayed neutrophil reconstitution. KIR2DS2 significantly increased aGVHD and CMV（P<0.05）. The incidences of cGVHD and septicemia /pneumonia were significantly lower in patients homozygous for group C1 allele than in those heterozygous for group C1/C2 allele（P<0.05）. The DFS was significantly higher in patients with KIR receptor-HLA ligand incompatibility than in patients without（P<0.05）, and the former group had a lower relapse trend （P=0.08）.3. The percentage of CD158a, NKB1 and CD94/NKG2A expressing on NK cells in grafts was significantly higher in grade II～IV aGVHD group compared with in grade 0^I aGVHD group. The percentage of CD94/NKG2A expressing on CD8-positive T cells in grafts was significantly higher in grade 0～I aGVHD group compared with in grade II～IV aGVHD group（ P<0.05, respectively）.4. Alloreactive NK cells significantly prolonged survival of BALB/c mice bearing leukemia that accepted haploidentical BMT and reduced relapse （compared with nonalloreactive NK cells and no NK cells, P<0.05, respectively）.5. Alloreactive NK cells significantly promoted peripheral T cell reconstitution and induced Th1 immune response in vivo in BALB/c mice received haploidentical BMT. Conclusion1. In haploidentical HSCT, the presence of donor activating KIR2DS3, KIR3DS1 delays hematopoietic reconstitution. KIR2DS1, KIR2DS3 and KIR3DS1 reduces cGVHD. The presence of recipient Bw4 increases aGVHD.2. In HLA-matched sibling HSCT, the presence of donor activating KIR2DS5 delays hematopoietic reconstitution. KIR2DS2 increases incidence of aGVHD and CMV viremia. The incidence and severity of cGVHD are significantly reduced in patients homozygous for group C1 allele. KIR receptor-HLA ligand incompatibility may reduce relapse and improve DFS.3. The higher percentage of CD158a, NKB1 and CD94/NKG2A expressing on NK cells in grafts increases grade II～IV aGVHD. The higher percentage of CD94/NKG2A expressing on CD8-positive T cells reduces grade II～IV aGVHD.4. Alloreactive NK cells significantly prolong survival of BALB/c mice bearing leukemia that accepted haploidentical BMT , and reduces relapse and has a graft versus leukemia（GVL） role.5. Alloreactive NK cells significantly promote peripheral T cell reconstitution and induce Th1 immune response in vivo in BALB/c mice received haploidentical BMT.更多还原