脐带血间充质干细胞的分离培养及诱导表达IDO对免疫介导再生障碍性贫血小鼠的治疗研究
Research on isolation and culture of umbilical cord blood mesenchymal stem cells and the effect of induced IDO expression on murine with immune mediated aplastic anemia
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摘要: 目的:从人脐带血中分离和培养脐带血间充质干细胞(umbilical cord blood mesenchymal stem cells,UCB MSCs),探讨其吲哚胺2,3-过氧化酶(indoleamine2,3-dioxygenase,IDO)活性对MSCs治疗再生障碍性贫血(aPlastic anemia,AA)的影响。方法:采集足月分娩儿脐带血,淋巴细胞分离法分离培养UCB MSCs。倒置显微镜观察细胞形态,流式细胞术检测细胞表面标志物表达,成骨与成脂诱导鉴定UCB MSCs分化潜能。建立免疫介导AA小鼠模型,尾静脉输注经-干扰素(IFN-)诱导的UCBMSCs,观察对AA小鼠的治疗效果。结果:UCB MSCs培养1周可见少量贴壁梭形细胞,于3周左右细胞生长形态较均匀,成纤维样、平行排列或漩涡状;传代细胞1周即可见80%左右融合。流式检测细胞表面标志物,不表达造血标志物CD34,CD44、CD73阳性表达率分别为94.36%和92.48%。UCB MSCs经IFN-诱导表达IDO活性,对AA小鼠造血恢复具有治疗作用。结论:脐血分离培养的MSCs在IFN-诱导作用下,其IDO基因表达对于免疫介导AA小鼠模型具有治疗作用。
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关键词:
- 再生障碍性贫血 /
- 间充质干细胞 /
- 脐带血 /
- 免疫介导 /
- 吲哚胺2,3-过氧化酶
Abstract: Objective: To isolate and culture of umbilical cord blood mesenchymal stem cells(UCB MSCs) from human umbilical cord blood and explore the effect of induction indoleamine2,3-dioxygenase(IDO) expresson on the treatment of immune mediated aplastic anemia(AA) in mice. Methods: UCB was collected from full-term deliveries. UCB MSCs were isolated and cultured using the lymphocyte isolation method. The cell morphologhy was observed under inverted microscope,and the surface markers of MSCs were analyzed by flow cytometry. Osteogenic and adipogenic induction was used to verify the differentiation potential of UCB MSCs. The murine model with immune mediated aplastic anemia were established. The caudal vein of mice was injected with UCB MSCs induced by IFN-, and the treatment effect was observed. Results: A little of adherent spindle cells presented at 1 week culture of UCB MSCs. About 3 weeks of culture, these uniformity fibroblast-like cells were found, and showed parallelly or whirlpool-shape arrangement. After passage,MSCs reached 80% confluence at 1 week. Immunophenotype analysis showed that MSCs expressed CD44(94. 36%) and CD73 (92. 48%) ,but no CD34 of hematopoietic marker. UCB MSCs that induced by IFN- promoted IDO activity, and had therapeutic effect on haematogenesis function recovery of mice with aplastic anemia. Conclusions: Under IFN- induction, the isolated and cultured UCB MSCs from umbilical cord blood can improve the IDO activity,which has therapeutic effect on murine immune mediated aplastic anemia. -
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