铁过载诱导活性氧物质生成对骨髓造血功能影响的体外实验研究

目的 建立体外铁过载骨髓造血细胞模型,检验铁过载对细胞活性氧物质(ROS)水平的影响以及ROS升高对骨髓造血功能的影响。方法 在骨髓单个核细胞培养的过程中添加枸橼酸铁铵(FAC),使细胞铁过载,检验这一过程中细胞ROS水平、细胞凋亡水平、造血细胞集落形成和CD 34+细胞计数的变化。再用去铁胺(DFO)祛铁或抗氧化剂N-乙酰半胱氨酸(NAC)清除过多的ROS后,检测上述指标的变化。结果 ①在培养液中加入不同浓度FAC培养不同时间,发现骨髓造血细胞内可变铁池(LIP)水平升高,且具有时间和浓度依赖性,在含400μmoL/L FAC的培养液中培养24h时LIP水平达到最高。②在400μmol/L FAC浓度下,培养骨髓造血细胞24h后骨髓造血细胞内总的ROS、粒细胞和红细胞内ROS显著升高,分别为对照组的1.77、1.75和2.12倍。与对照组比较,DFO和NAC处理后均能明显降低细胞内ROS水平(P＜0.05)。③对骨髓细胞造血功能的检测发现FAC组细胞凋亡比例(24.80±2.99)％]较对照组(8.90±0.96)％]显著升高;造血细胞集落形成单位(CFUE、CFU-GM、BFU-E和CFU-mix)计数明显低于对照组(P值均＜0.05);CD34+细胞比例(0.39±0.07)％]较对照组(0.91±0.12)％]也显著降低。且这些损伤都可以通过DFO和NAC处理而部分恢复。结论 铁过载通过诱导ROS生成影响骨髓造血功能,这种损伤可以通过祛铁和抗氧化处理减轻。可能为治疗铁过载患者骨髓造血功能低下寻找新的靶点。

In vitro effect of iron overload on bone marrow cell function by inducing the reactive oxygen species

Objective To investigate the in vitro effect of iron overload on the generation of reactive oxygen species (ROS) and of bone marrow (BM) cell function. Methods BM mononuclear cells (BMMNCs) were cultured with ferric citrate(FAC) at different concentrations and for different time to create iron overload and confirmed by the detection of cellular labile iron pool (LIP). The changes of ROS, apoptosis, hematopoietic colony formation ( CFU-E, BFU-E, CFU-GM and CFU-mix) and the percentage of the CD34 + cells percentage were analyzed. The differences of these index were tested after the iron overload treated with deferasirox (DFO) or antioxidants ( N-acetyl-L-cysteine, NAC). Results ①When BMMNCs were cultured with FAC, the LIP was found to increase in a time and concentration dependent manner. The intra cellular LIP reached maximum at 400 μmol/L of FAC for 24 hours. ② The ROS of total cells, leukocytes and erythrocytes increased to 1.77, 1.75 and 2.12 fold respectively compared with that of normal control when cells were cultured at 400 μmol/L of FAC for 24 hours . DFO and NAC could reduce the ROS efficiently (P ＜ 0.05 ). ③ The apoptotic rates of the FAC treated cells( 24.80 ± 2.99 ) ％]increased significantly compared with that of normal control(8.90 ± 0. 96) ％]. The capacity of hematopoietic colony formation in FAC treated cells decreased markedly compared with that of normal control ( P ＜ 0.05 ). The percentage of CD34 + cells of FAC treated cells(0.39 ± 0.07 )％]also decreased significantly compared with that of nor mal control(0.91 ±0. 12)％]. And these changes could be recovered by addition of NAC or DFO. Condusion Iron overload can affect the hematopoiesis by inducing the generation of ROS and this damage could be corrected by removing the excess iron and ROS of the BM cells. These findings might improve the treatment of dyshematopoiesis in patients with iron overload.