体外扩增造血细胞重建小鼠造血功能的实验研究

Engineering of hematopoietic cells and the hematopoietic reconstitution of expanded cells in SCID mice

目的探讨体外扩增造血祖细胞和定向诱导功能血细胞生成的条件及扩增细胞重建长期造血能力的维持。方法应用免疫磁珠法（ＭＡＣＳ）分离纯化脐带血ＣＤ３４＋造血干或祖细胞，在体外液体培养体系中经不同细胞因子的组合进行扩增和定向诱导，并将扩增细胞移植经亚致死剂量照射的ＳＣＩＤ小鼠。结果ＦＬ、干细胞因子、血小板生成素（ＴＰＯ）等细胞因子的不同组合可分别扩增细胞总数、粒系及巨核系造血祖细胞、ＣＤ３４＋ＣＤ３８－早期造血祖细胞达（２１３０±５７）、（７０±７）、（１１８±１１）和（４６±５）倍；树突状细胞的比例也由（０．４±０．３）％增至（２４．３±２．１）％；ＦＬ和ＴＰＯ具有明显的协同扩增早期祖细胞的作用；ＣＤ３４＋ＣＤ３８－细胞亚群的扩增也可明显减缓细胞分化的速率；扩增细胞可顺利重建亚致死剂量照射ＳＣＩＤ小鼠的造血，移植存活６周的ＳＣＩＤ小鼠骨髓中仍可检测到人造血细胞。结论合理的细胞因子组合在大量扩增造血细胞的同时可保留造血干／早期祖细胞的自我更新和造血重建能力。造血细胞工程这一新的细胞治疗策略将广泛应用于造血调控研究、造血干细胞移植、造血支持治疗、肿瘤免疫治疗、基因治疗等领域。

Objective To elucidate the roles of cytokines for ex vivo expansion and orderly differentiation of hematopoietic progenitor cells, and the capacity of hematopoietic reconstitution of the expanded cells. MethodsCD34+ cells were isolated from umbilical cord blood by using a highgradient magnetic cell sorting system(MACS),and expanded with the different combinations of cytokines in a liquid culture system. The expanded cells were then transplanted into sublethally irradiated SCID mice. Results The combination of cytokines including FL, SCF, TPO, etc. increased total cells, progenitor cells(CFUGM and CFUMK), and CD34+CD38 early progenitor cells by (2 13057), (707), (11811) and (465) folds, respectively. The percentage of dendritic cells (24.32.1)% was also much higher than the control(0.40.3)%. The CD34+ CD38- subsets and the combination of FL and TPO were identified as the most potential for expanding early progenitor cells. The expanded cells could smoothly engraft SCID recipients and reconstitute their hematopoiesis. Furthermore, human hematopoietic cells could be detected in marrow cells from SCID mice transplanted 6 weeks late. Conclusions It is possible to expand hematopoietic cells ex vivo efficiently and maintain the hematopoietic reconstitution capacities of hematopoietic stem/early progenitor cells by an appropriate combination of cytokines. The engineering of hematopoietic cellsthe new generation of cellular therapeutics are now underway in the applications of stem cell transplantation, immunotherapy of cancers, and gene therapy.