诱导H-2半相合小鼠骨髓受体获得免疫赦免的体内研究

Induction of bone marrow receptor's immune privilege against graft-versus-host disease in vivo in an H-2 haplotype disparate mouse combination

目的 通过Fas配体 (FasL) Fas途径清除异体骨髓移植 (allo BMT)物中针对受体主要组织相容复合物 (MHC)的淋巴细胞 ,从而抑制受体发生移植物抗宿主病 (GVHD)。方法 实验组 (5组 )用磁性细胞分离系统分离BALB/C小鼠 (H 2d ,雌性 )早期造血细胞 (HC) ,经逆转录病毒法转移外源mFasLcDNA基因并扩增 1w后与BAC小鼠 (H 2d×b ,雄性 )骨髓单个核细胞 (BMMC)按 0 .6 2 5∶1混合培养 1w ,经尾静脉将 5× 10 6个混合细胞 (0 .5ml)注入经全身60 Coγ照射的BALB/C小鼠。同时设立 :1组 (空白对照组 ,未移植细胞 ) ;2组 (同基因BMT组 ) ;3组 (转染外源FasL的同基因BALB/CHC +正常BALB/C小鼠BMMC混合培养后移植组 ) ;4组 (H 2单倍型不同小鼠的allo BMT组 )。观察移植鼠的造血重建及细胞来源、GVHD、生存率和造血重建后的免疫学特征。结果 BMT后 +10d、+2 0d ,实验组、3和 4组的外周血白细胞、血小板计数均低于 2组 (P <0 .0 1) ,但 +30d后以上各组则均恢复正常。实验组中存活 2个月的 8只BALB/C小鼠 ,其BMMC中供体来源Y染色体出现率为 (81.14± 5 .3) % ,其中 1例脾细胞基因组DNA经PCR检出Neor 和mFasLcDNA整合 ;各组移植 2个月后的生存率为 :实验组 80 %、1组 0 %、2、3组均为 10 0 %、4组 2 0 % 。

ObjectiveTo induce bone marrow receptor's immune priviledge against graft versus host disease (GVHD) by specific elimination of mouse aloreactive T cells through Fas ligand (FasL) Fas way. Methods irst, the stem cell antigen 1 (sca 1) + early hematopoietic cells (EHC) of BALB/C mouse (H 2d, female) were isolated by using a magnetic cell sorting syste.The exogenous mFasL cDNA gene was transferred to these cells by retrovirus gene transfer system and expanded in liquid culture system for one week. Then, in the experimental group these expanded hematopoietic cells (HC) were co cultured with bone marrow mononuclear cells (BMMC) form BAC mouse (H 2?d×b, male) at the ratio of 0.625 to l for another one week. Last, 5×10 6 ( 0.5?ml) mixed viable cells were injected into whole bodily irradiated ( 60Co γ) mice (6 to 8 week old female BALB/C) via the tail vein. The following four groups of grafted BALB/C mice were simultaneously used: (1) the mice transplanted with 0.5?ml culture medium; (2) the mice transplanted with BMMC from normal ALB/C mice; (3) the ice transplanted with mixed cells, in which exogenous mFasL gene transferred BALB/C HC were cocultured with BALB/C BMMC for one week; (4) the mice transplanted with BMMC from H 2 haplotype disparate BAC mice. The hematopoietic reconstitution, the origion of bone marrow cells responsible for the reconstitution, GVHD, the survival rate and immunological character after the reconstitution for the recipient mice were observed after bone marrow transptantation (BMT). ResultsThe counts of leukocytes and platelets in recipient mice's blood of group two on+10 day and +20 day after BMT were higher than in the experimental group, group three andgroup four(P< 0.01), but on +30?d after BMT the counts for all groups were at their normal levels. The Y chromsome from donor mice was discovered in 81.14%± 5.3% BMMC of recipient mice having survived over two months after BMT in the experimental group. The genomic integration of exogenous mFasL and Neo r gene were found in spleen cells from one recipient mouse having survived over two months in the experimental group. The survival rate of the recipient mice two months after BMT in all groups wereseparately 80% for experimental group, 0% for group one, 100% for group two and three, and 20% for group four. The total survival rate of recepient mice in the experimental group were obviously longer than that of group four(P< 0.01)by the test of kaplan Meier survival curve with long rank test. Grade Ⅱ to Ⅲ GVHD signs were discovered on histology of liver, the skin of claw and small intestine from death mice after BMT in the experimental group and group four, and the mice having survived over two months in the groups four. Grade I or no GVHD signs were found on the histology from the mice having survived over two months after BMT in the experimental group. After hematopoietic reconstitution of recipient mice (BALB/C) the proliferation reaction of their spleen cells against allogeneic donor and syngenic mice spleen cells were not observed, but the proliferation reaction against xenogeneic spleen cells from Wistar rat was obviously discovered. ConclusionsAfter the recipient mice HC transferred exogenous mFasL gene and higher expressing the membrane protein of the gene were cocultured with the donor mice BMMC, the transplantation of the mixed cells into recipient mice made recipient mice to get hematopoietic reconstitution from donor BMMC, and immume privilege of donor lymphocyte against recipient mice with no obvious injure of Fas + organ, so asto prolong the survival rate of recipient mice after BMT.