阵发性睡眠性血红蛋白尿症患儿骨髓CD34+CD59+细胞的体外扩增及克隆变化趋势的研究

Ex vivo expansion and clonal variation of CD34+CD59+ cells from bone marrow in children with paroxysmal nocturnal hemoglobinuria

摘要:

目的：研究阵发性睡眠性血红蛋白尿症（PNH）患儿骨髓CD34⁺CD59⁺细胞的分离、纯化及体外扩增的条件，并对扩增后细胞的长期造血能力进行评估，为探索PNH患儿新的治疗途径提供实验依据。方法：采用磁珠-流式细胞仪二步分选法，从PNH患儿骨髓中分选出CD34⁺CD59⁺细胞，在不同细胞因子组合条件下进行体外扩增，并培养集落形成细胞（CFCs）及长期培养始动细胞（LTC-IC）。结果：体外扩增最适宜的细胞因子组合为干细胞因子+白细胞介素（IL）-3 +IL-6+FLT3配基+巨核细胞生成素+红细胞生成素，最适宜的扩增时机为第7天，此时CD34⁺CD59⁺细胞的扩增倍数为30.4±6.7倍。CD34⁺CD59⁺细胞在扩增以后，仍为CD59⁺细胞，能保持较好的形成CFCs的能力，仍能形成 LTC-IC，与扩增前相比差异无统计学意义；但PNH患儿CD34⁺CD59⁺细胞的扩增能力低于正常对照的CD34+细胞（P<0.01）。结论：PNH患儿的CD34+CD59+细胞能够进行体外扩增，扩增后的细胞仍然具备长期造血重建的能力，并且无向PNH克隆转化的趋势，说明对PNH患儿进行自体移植在临床上有可行性。

Abstract:

OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.