骨髓腔内输注脐带血及间充质干细胞对大鼠免疫重建影响

Effect of intra-bone marrow infusion of umbilical cord blood and mesenchymal stem cells on immune reconstitution in rat models

目的探讨骨髓腔内(IBM)输注脐带血及骨髓间充质干细胞(BMSC)对移植后大鼠免疫重建的影响。方法雌性F344胎鼠及新生鼠外周血(FNPB)及雄性F344大鼠BMSC共移植经致死量60Coγ射线辐照的雌性Wistar大鼠,其中FNPB均由IBM输注,BMSC则通过IBM或尾静脉(IV)注射。观察受鼠存活状况、供体造血干细胞(HSC)植入水平、免疫细胞及功能恢复、移植物抗宿主病(GVHD)表现,并用PCR检测受鼠供体BMSC来源Y染色体。结果两共移植组60d均100%存活,生存率和供体HSC植入水平差异无统计学意义,但明显优于单纯FNPB移植组;骨髓腔共移植组外周血白细胞及粒单系细胞集落形成单位(CFU-GM)、混合集落形成单位(CFU-GEMM)集落产率恢复较快,14d显著高于其他组(P<0.05)。而单纯FNPB移植组各检测时点集落计数均明显低于共移植组(P<0.05),30dCD19+细胞比例亦明显低于正常;30d各移植组PHA、LPS诱导淋巴细胞增殖实验及单向混合淋巴细胞培养无明显差异;60d两共移植组存活受鼠均检测到供体BMSC来源Y染色体。结论 HSC与BMSC联合输注可促进受者免疫重建,尤以IBM输注为佳。

Objective To investigate the effect of intra-bone marrow（IBM） infusion of umbilical cord blood and mesenchymal stem cells（BMSC） on immune reconstitution in rats.Methods Female Wistar rats conditioned with lethal dose 60Co γ-ray irradiation were co-transplanted with F344 female fetal and neonatal peripheral blood（FNPB） and BMSC separated from bone marrow mononuclear cells of F344 male rats.The donor BMSC were infused by IBM injection in bilateral tibia or intravenous（IV） injection,while the FNPB were all infused via IBM route.The survival rate,engraftment level of hematopoietic stem cells（HSC）,GVHD,recovery of immune cells and their functions of recipients were monitored.The Y chromosomes were examined by polymerase chain reaction（PCR）.Results The recipient rats of the 2 co-transplantation groups were all survival at day 60.There was no significantly different between 2 groups on the survival rates or the engraftment levels of HSC,but each of them was much better than that of the FNPB group.The recovery of peripheral white blood cells and bone marrow hematopoietic stem/progenitor cell colonies of the FNPB（IBM） ＋ BMSC（IBM） group was excellent in experiment groups,especially at day 14（P 0.05）.The counts of colony-forming unit-granulocyte/macrophage and colony-forming unit mix in FNPB（IBM） group were lower than that of co-transplantation group at each time point（P 0.05）,and the CD19＋ cells were still below the normal level at day 30.At the same time,the experimental groups had similar immune response on lymphocyte proliferation induced by PHA and LPS,and showed normal responsiveness in one-way mixed lymphocyte culture.At the day 60,the Y chromosomes could be detected in all of the recipient rats in 2 co-transplantation groups.Conclusion It is demonstrated that the co-transplantation of HSC and BMSC can accelerate immune reconstitution in recipient.The effect of IBM route infusion is superior to IV route.