小鼠脐血联合骨髓c-kit(+)细胞移植加快造血重建的研究

Earlier Hematopoietic Reconstitution by Mouse Cord Blood Transplantation Combined with Bone Marrow c-kit(+) Hematopoietic Progenitor Cells

本研究探讨小鼠脐血与骨髓造血祖细胞联合移植以加快移植后早期造血重建作用,并达到脐血来源细胞长期嵌合的程度。采用MACS间接免疫分选法纯化BDF1小鼠骨髓中lin-sca-1-c-kit+(c-kit+)及lin-sca-1+(sca-1+)细胞群,用半固体集落培养结合细胞形态学方法比较二者的体外生物学特性。将60Coγ射线照射的BDF1小鼠(CD45.2)作为受体鼠;接受来自同基因小鼠(CD45.1)骨髓c-kit+细胞群与DBA/2新生小鼠外周血(NPB)的联合移植作为实验组,以NPB干细胞单独移植以及其与sca-1+细胞的联合移植作为对照组,比较移植后22天内的白细胞及血小板变化。用流式细胞术检测移植60周内受鼠外周血粒细胞、T细胞、B细胞中供、受鼠来源细胞比例的动态变化。结果显示:①与sca-1+细胞比较,c-kit+细胞胞体大,胞浆多,核仁明显,其体外集落体积明显小于sca-1+组,未见高增殖潜能集落(HPP)形成;②与NPB干细胞单独移植比较,当NPB干细胞与1×104、2.5×104或5×104细胞量的c-kit+或sca-1+细胞联合移植时,可使白细胞>1×109/L及血小板>100×109/L所需恢复时间从17天提前至12天;③联合c-kit+细胞的移植实验组至移植后60周时脐血来源粒细胞、B细胞和T细胞分别为(96.68±2.68)%、(92.55±3.04)%和(67.96±7.91)%,比第2周时有明显增加(p<0.01),与此相反,联合sca-1+细胞的移植对照组来自同基因供髓和残髓的细胞逐渐增多,而脐血来源细胞逐渐减少,至移植后60周时,脐血来源的粒细胞,B细胞和T细胞仅占(28.46±18.98)%、(17.75±13.17)%、(6.19±7.62)%,比第2周时明显减少(p<0.01)。结论:脐血与受鼠同基因骨髓c-kit+细胞联合移植可加快移植后早期造血恢复,并达到粒细胞、B细胞、T细胞中脐血来源细胞的长期完全或主要嵌合。

This study was purposed to investigate the accelerating effect of neoborn mouse cord blood transplantation combined with hematopoietic progenitor cells of bone marrow（BM） on the early hematopoietic reconstitution after transplantation,and the long-term chimerism of cord blood-derived cells,so as to develop a combined transplantation method for accelerating the early hematopoietic reconstitution.The lin-sca-1-c-kit＋（c-kit＋）cells and lin-sca-1＋（sca-1＋） cells in the bone marrow of BDF1 mice were isolated by MACS method.Biological characteristics in vitro of isolated fractions were observed and compared by semisolid colony culture combined with Giemsa staining.After transplantation of cord blood（CB） alone,or together with graded numbers of either c-kit＋ or sca-1＋ cells isolated from BDF1 mice（CD45.1） bone marrow into lethally irradiated CD45.2 congenic BDF1 mice,numbers of WBC and platelet were measured within 22 days of post-transplantation.The proportion of chimerism on granulocyte,T and B cell was dynamically measured by flow cytometry within 60 weeks of post-transplantation.The results showed that the number of colony from BM c-kit＋ cells cultured in semi-solid agar medium was significantly smaller than that from BM sca-1＋ population,which showed low proliferative potential in vitro and morphological characteristics of medium-or large-sized blast-like cells.The co transplantation of CB and BM c-kit＋ cells or sca-1＋ cells at the dosages of 1×104 or 2.5×104 or 5×104 to recipient mice leads to the quantity of WBC and platelets increased to 1×109/L and 1×1012/L at day 12,whereas the injection of CB alone resulted at day17.When mice were transplanted with CB together with BM c-kit＋cells,and the CB-donor type cells in the peripheral blood increased progressively,while congenic donor BM-derived stem cells decreased gradually.After cotransplantation with CB and BM c-kit＋ cells for 60 weeks,a frequency of complete chimerism in CB-derived cells was continually maintained in granulates（96.68±2.68）% and B lymphocytes（92.55±3.04）%,while T lymphocytes（67.96±7.91）% were dominantly derived from CB.On the other hand,congenic bone marrow or residul-derived cells were the dominant population,and the ratio of CB-derived cells in the peripheral blood was less than 10%（6.19±7.62）% after cotransplantation with CB and sca-1＋cells for 60 weeks.It is concluded that the cotransplantation of CB and BM congenic c-kit＋ cells is able to accelerate early hematopoietic reconstitution of recepient mice due to congenic marrow cells.Complete or main chimerism of cord blood is formed in long-term multilineage reconstitution of granulocytes,B cells and T lymphocytes.