体外分阶段共培养法扩增的脐血造血干/祖细胞移植于NOD/SCID小鼠的实验研究  

Experimental study of ex vivo expanded hematopoietic stem/progenitor in the two step culture from human umbilical cord blood transplanted into NOD/SCID mice

作　　者：王金福[1] 项盈[1] 解纯刚[1] 贾冰冰[1] 

机构地区：[1]浙江大学生命科学学院,浙江杭州310012

出　　处：《浙江大学学报（理学版）》2005年第4期448-451,共4页Journal of Zhejiang University（Science Edition）

基　　金：浙江省科技厅重点计划项目(2003C23015).

摘　　要：研究用分阶段共培养法进行体外扩增的脐血造血干/祖细胞对NOD/SCID小鼠重建造血功能的作用,探讨新扩增技术体系对将来临床移植的可行性.分离脐血单个核细胞,分别用rhSCF+rhG-CSF+rhMDGF组合(非共培养组合)和rhSCF+rhG-CSF+rhMDGF+人骨髓间充质干细胞组合(共培养组合)进行脐血造血干/祖细胞的体外扩增,并对扩增细胞进行流式细胞术和细胞集落形成能力分析.在移植实验中,将经致死剂量射线照射后的NOD/SCID小鼠随机分为3组,每组10只.A组:每只移植8.5×106个用rhSCF+rhG-CSF+rhMDGF+人骨髓间充质干细胞组合扩增的细胞;B组:每只移植8.5×106个用rhSCF+rhG-CSF+rhMDGF组合扩增的细胞;C组:每只仅输注0.5 mL生理盐水.移植后定期眼眶底部采血,分析重建造血.12周后取存活小鼠骨髓,检测人特异Alu基因的存在率.实验结果表明,rhSCF+rhG-CSF+rhMDGF+人骨髓间充质干细胞组合的共培养法显著提高了造血干/祖细胞的扩增倍数,并且有利于造血干/祖细胞的自我更新和提高细胞集落形成能力.两种组合扩增的细胞移植小鼠12 d后,小鼠的白细胞类群开始回升,25 d后基本恢复到了基数水平.在45～55 d后小鼠细胞有明显的第2个恢复期,并且共培养组合扩增细胞移植的小鼠中第2次恢复明显快于非共培养组合.12周后在两种组合移植的小鼠中检测到人特异Alu序列片段的概率分别为87.5%(7/8)和88.9%(8/9).由此可推论,分阶段共培养扩增体系有效地提高了脐血造血干/祖细胞的扩增能力和重建造血能力.Effects of ex vivo expanded hematopoietic stem/progenitor cells (HSPCs) in two-step culture on hematopoietic reconstruction of irradiated NOD/SCID mice were studied. Mononuclear cells (MNCs) were isolated from human umbilical cord blood (UCB) and cultured in the non-coexpansion scheme of rhSCF+rhG-CSF+rhMDGF combination and the coexpansion scheme of rhSCF+rhG-CSF+rhMDGF+human bone marrow-derived mesenchymal stem cells (MSCs). Sublethally irradated NOD/SCID mice were transplanted with ex vivo expanded HSPCs in the dose of 8.5×10~6 cells. After transplantation, the dynamics of WBC in the transplanted mice was measured periodically, and the Alu sequence fragment special for human in the transplanted mice was inspected by PCR. Results showed that the coexpansion scheme increased ex vivo expansion of UCB-derived HSPCs. After transplantation with expanded HSPCs, the population of WBC in the transplanted mice increased in 12 d and reached the first peak in 25 d. It was showed the second increase of WBC in 45-55 d. Expanded cells from the coexpansion scheme appeared to be favorable for the second increase of WBC in the transplanted mice. After 85 d, the Alu sequence fragment was detected in the probability of 87.5% (7/8) for the no-coexpansion scheme and (88.9% (8/9) for the coexpansion scheme.)

关 键 词：脐血 造血干/祖细胞 体外扩增 NOD/SCID小鼠 移植 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]