Xiao Juan, Wu Yong-Ji, Han Bing, Dong Hong-Yan, Chen Shi-Ping
Department of Pediatrics, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing 100730, China.
Zhongguo Dang Dai Er Ke Za Zhi. 2013 Aug;15(8):627-32.
To investigate the isolation, purification and ex vivo expansion of CD34(+)CD59(+) cells from the bone marrow of children with paroxysmal nocturnal hemoglobinuria (PNH), to evaluate the capability of long-term hematopoietic reconstruction of the expanded CD34(+)CD59(+) cells, and to provide a laboratory basis for novel treatment of PNH.
CD34(+)CD59(+) cells were isolated from the bone marrow mononuclear cells of children with PNH using immunomagnetic beads and flow cytometer in sequence. The isolated cells were subjected to ex vivo expansion in the presence of different combinations of hematopoietic growth factors for two weeks. The colony-forming cells and long-term culture-initiating cells (LTC-ICs) were cultured and counted.
The optimal combination of hematopoietic growth factors for ex vivo expansion was stem cell factor+interleukin (IL)-3+IL-6+FLT3 ligand+thrombopoietin+ery-thropoietin, and maximum expansion (30.4 ± 6.7 folds) was seen on day 7 of days 4 to 14 of ex vivo expansion. After ex vivo expansion, CD34(+)CD59(+) cells remained CD59-positive, retained strong capability of forming colony-forming units, and could still form LTC-ICs. There was no significant difference in capability of forming LTC-ICs between CD34(+)CD59(+) cells before and after expansion. The expansion capability of CD34(+)CD59(+) cells from children with PNH was significantly lower than that of CD34(+) cells from normal controls (P<0.01).
The CD34(+)CD59(+) cells from children with PNH can be expanded in vitro. Post-expansion CD34(+)CD59(+) cells retain capability of long-term hematopoietic reconstruction. CD34(+)CD59(+) cells showed no trend towards PNH clone during culture. Ex vivo expansion of CD34(+)CD59(+) cells from children with PNH might be practical in performing autologous transplantation clinically for these children.
探讨阵发性睡眠性血红蛋白尿症（PNH）患儿骨髓中CD34(+)CD59(+)细胞的分离、纯化及体外扩增方法，评估扩增后的CD34(+)CD59(+)细胞长期造血重建能力，为PNH的新型治疗提供实验室依据。
采用免疫磁珠法和流式细胞仪依次从PNH患儿骨髓单个核细胞中分离CD34(+)CD59(+)细胞。将分离的细胞在不同组合的造血生长因子存在下进行体外扩增2周。培养并计数集落形成细胞和长期培养起始细胞（LTC-ICs）。
体外扩增的最佳造血生长因子组合为干细胞因子+白细胞介素（IL）-3+IL-6+FLT3配体+血小板生成素+促红细胞生成素，体外扩增第4至14天的第7天可见最大扩增倍数（30.4±6.7倍）。体外扩增后，CD34(+)CD59(+)细胞仍为CD59阳性，保留了较强的集落形成单位形成能力，且仍能形成LTC-ICs。扩增前后CD34(+)CD59(+)细胞形成LTC-ICs的能力无显著差异。PNH患儿的CD34(+)CD59(+)细胞扩增能力明显低于正常对照的CD34(+)细胞（P<0.01）。
PNH患儿的CD34(+)CD59(+)细胞可在体外扩增。扩增后的CD34(+)CD59(+)细胞保留长期造血重建能力。CD34(+)CD59(+)细胞在培养过程中未显示出向PNH克隆发展的趋势。PNH患儿CD34(+)CD59(+)细胞的体外扩增可能在临床上对这些患儿进行自体移植中具有实用性。
