阵发性睡眠性血红蛋白尿症患者骨髓CD34^+CD59^+细胞与正常人CD34^+细胞生存、增殖、扩增性能的比较

Comparison of Capabilities of Survival,Proliferation and Expansion between CD34^+CD59^+Cells from Patients with PNH and CD34 + Cells from Normal Control

目的探索阵发性睡眠性血红蛋白尿症(PNH)患者骨髓CD34CD59细胞体外扩增的方法,并对其与正常人CD34+细胞的生存、增殖及扩增性能进行比较,为PNH患者实现自体骨髓移植(ABMT)或自体外周血干细胞移植(APBSCT)提供重要的实验基础。方法应用免疫磁珠富集纯化CD34+细胞,然后用流式细胞仪分选出PNH患者的CD34+CD59+细胞及正常人CD34+细胞。对两种细胞在不同造血生长因子组合下,进行体外扩增液体培养2周。并对扩增前及扩增不同阶段的细胞进行体外半固体培养。结果(1)PNH患者CD34+CD59+细胞在体外能得到有效的扩增,在细胞因子合理组合作用下,第7天时CD34+CD59+细胞绝对数扩增约23.49倍。2PNH患者CD34+CD59+细胞与正常人CD34+细胞存在部分相同或相似的特性。在体外扩增过程中,对细胞因子的反应有相同的趋势,均为扩增第7天达到扩增高峰,均在SCF+IL-3+IL-6+FL+Tpo+Epo组合条件下达到最大扩增能力。并且扩增后的细胞仍有形成CFU的能力,均能很好地保持CD59抗原,无GPI锚连蛋白的丢失。(3)正常人CD34+细胞在生存、增殖、形成CFU的能力及扩增能力上均明显强于PNH患者的CD34+CD59+细胞。结论(1)PNH患者CD34+CD59+细胞在体外能得到有效的扩增,临床上应用具有一定的可行性,能满足大多数PNH患者ABMT或APBSCT的需要。(2)

Objective To explore in vitro expansion of CD34 + CD59 + cells from patients with PNH,and compare the capabilities of survival,proliferation and expansion between CD34 + CD59 + cells from patients with PNH and CD34 + cells from normal control.Methods CD34 + CD59 + cells from patients with PNH and CD34 + cells from normal control were selected from the bone marrow mononuclear cells by means of two -step sorting method with immunomagnetic microbead-flow cytometry,then underwent in vitro expansion for two weeks and semi -solid culture in vitro before and after expansion.Results(1)CD34 + CD59 + cells from patients with PNH can be expanded effectively in vitro,and the biggest expansion of CD34 + CD59 + cells was about 23.49fold on the7th day.(2)There were some similar characteristics between CD34 + CD59 + cells from patients with PNH and CD34 + cells from normal control,such as:the best combination of hematopoietic factors for in vitro expansion was SCF+IL-3+IL-6+FL+Tpo+Epo,and the seventh day was the most suitable in course of4-14days for in vitro expansion,and after in vitro expansion,the cells remained CD59positive and strong capability of performing colony-forming.(3)CD34 + cells from normal control had better proliferation,expansion and stronger potential to survive than CD34 + CD59 + cells from patients with PNH.Conclusions (1)In vitro expansion of CD34 + CD59 + cells from patients with PNH can be performed.The present study showed the possibility of performing ABMT or APBSCT clinincally for patients with PNH.(2)There were some similar characteristics between CD34 + CD59 + cells from patients with PNH and CD34 + cells from normal control,but the latter had better proliferation,expansion and stronger potential to surviv e than the former.CD34 + CD59 + cells from patients with PNH were not completely normal cells.