输注供者自然杀伤细胞对小鼠单倍型相合造血干细胞移植的影响

目的 探讨输注供者自然杀伤(NK)细胞对小鼠单倍型相合造血干细胞移植的影响.方法 选取C57BL/6(H-2b)雄性小鼠为供者、CB6F1(H-2d/b)雌性小鼠为受者.移植前制备供者的骨髓细胞(BMC)、脾细胞(SC)及脾NK细胞,NK细胞经体外培养扩增和激活;所有受者均接受直线加速器X线全身照射(TBI)预处理.TBI后将受者分为4组(每组10只),分别进行单倍型相合造血干细胞移植.单纯TBI组:TBI后不输注细胞,仅作为对照;单纯BMC输注组:输注5×106个BMC;诱发GVHD组:输注5×106个BMC+1.5×107个SC;NK细胞输注组:输注5 x 106个BMC+1.5×107个SC+1×107个NK细胞,并腹腔注射100 ng重组人白细胞介素2(rhIL-2)和1μg rhIL-15,持续7 d.移植后观察各组受者GVHD的发生情况,并对各组受者进行组织病理学、供者细胞嵌合度和免疫功能重建等检测.另取TBI后受者20只,设白血病复发组和白血病治疗组,每组10只.白血病复发组:输注5×106个BMC+1×107个SC+2×106个白血病细胞株EL9611;白血病治疗组:在白血病复发组的基础上再输注1 x 107个NK细胞,并腹腔注射100 ng rhIL-2和1μg rhIL-15,持续7 d.观察两组受者白血病复发情况和移植后100 d的存活率.结果 单纯BMC输注组受者无GVHD发生,NK细胞输注组受者GVHD的评分和组织病理学改变均较诱发GVHD组轻(P<0.05)f诱发GVHD组的免疫功能重建较NK细胞输注组延迟.白血病复发组和白血病治疗组移植后100 d的存活率分别为20%和90%,两组比较,差异有统计学意义(P<0.01).结论 输注激活的供者NK细胞可以减轻小鼠单倍型相合造血干细胞移植后的GVHD,减少白血病复发,促进免疫功能重建.

Effectiveness of donor natural killer cell infusion in haplo-identical hematopoietic stem cell transplantation

Objective To explore the effectiveness of natural killer (NK) cell infusion on engraftment, immune reconstitution, graft-versus-host disease (GVHD) and graft-versus-leukemia Bone marrow cells (BMC), splenocytes (SC) and NK cells were prepared from C57BL/6 mice before transplantation. NK cells were cultured and activated for 10 days in vitro. CB6F1 mice were lethally irradiated with 11.5 Gy using an X-ray source, then, assigned to TBI control, BMC control, NK cell treatment and GVHD control groups randomly and reconstituted with PBS control, 5 × 106 BMC, or 5 × 106 BMC and 1.5 × 107 SC with or without activated 1 × 107 NK cells via caudal vein, respectively. Graft-versus-host disease, lineage-specific chimerism, TRBV families were studied after transplantation. In the leukemia model, 20 CB6F1 mice were randomly assigned to leukemia control group and NK cell infusion group. CB6F1 mice were reconstituted with 5 × 106 BMC, 1 × 107 SC and 2 ～ 106 EL96! 1 cells with or without 1 × 107 NK ceils via lateral vein. Additionally, The mice in NK cell infusion group received 100 ng IL-2 and 1 μg IL-15 mixture treatment via intraperitoneal injection post transplantation for 7 days. Survival and leukemia relapse were analyzed during the 100-day post transplantation. Results CB6F1 mice that reconstituted with BMC did not suffer any GVHD. The mice in NK cell infusion group had lower clinical GVHD scores than GVHD control group (P<0.05). The mice in NK cell infusion group suffered less severe GVHD - associated weight loss than GVHD control group (P<0.05). Lymphoid immune reconstitution was delayed in GVHD control group as compared with NK cell infusion group. In the leukemia model, most of mice in control group died of leukemia relapse. The mice in NK cell infusion group survived longer than control group with 100-day survival rate being 90% and 20%, respectively (P<0.01). Conclusion Donor activated NK cell infusion and IL-2, IL-15 treatment post haplo-identical hcmatopoietic stem cell transplantation could alleviate GVHD, reduce leukemia relapse and promote lymphoid immune reconstitution.