氧化应激对铁过载造血干祖细胞造血功能的影响

Effects of oxidative stress on hematopoiesis of hematopoietic stem and progenitor cells with iron overload

目的 体外诱导脐血来源的造血干祖细胞铁过载,检测参与氧化应激的活性氧物质（ROS）的水平以及对造血干祖细胞造血功能的影响.方法 通过体外培养脐血单个核细胞（MNC）的过程中添加枸橼酸铁铵（FAC）,建立铁过载造血干祖细胞模型.实验分组：对照组、FAC组、FAC＋N-乙酰半胱氨酸（NAC）组、FAC＋谷胱甘肽（GSH）组.检测各组细胞内ROS水平,并用抗氧化剂处理,检验这一过程中ROS、细胞内可变铁池（LIP）、凋亡水平、造血集落形成和脐血各系造血细胞数目的变化.结果（1）在培养液中加入不同浓度FAC（50、100、200、400μmol/L）培养不同时间（6、12、24 h）,发现ROS水平随着FAC的浓度和培养时间变化,且在200μmol/L FAC的培养液中培养24h时ROS水平达到最高.（2）用抗氧化剂（NAC、GSH）联合FAC处理细胞发现,抗氧化剂处理组细胞内总的ROS以及髓系细胞和红系细胞内的ROS明显低于FAC组（均P＜0.05）.（3）在200 μmol/LFAC的浓度下,培养脐血MNC 24 h后细胞内总的LIP、髓系细胞和红系细胞内LIP水平均显著高于对照组（均P＜0.05）,但抗氧化剂NAC和GSH对铁过载细胞内LIP没有明显影响.（4）对脐血造血干祖细胞造血功能的检测发现：FAC组细胞凋亡比例[（20.90±3.45）％]明显高于对照组[（9.20±1.29）％]（P＜0.05）；造血集落形成单位（CFU-E、BFU-E、CFU-GM、CFU-mix）计数明显低于对照组（前3项两组间差异具有统计学意义,P ＜0.05）；CD34＋、CD33＋、GlyA＋细胞比例及细胞计数均显著低于对照组（均P＜0.05）；这些损伤都可以通过NAC或GSH处理部分恢复.结论 氧化应激在铁过载造血干祖细胞的损伤中扮演着重要角色,可以通过升高细胞内ROS水平抑制其造血功能,清除细胞内多余的ROS能减轻这些损伤.研究结果可为治疗铁过载患者因氧化应激诱导的造血功能低下提供新的靶点和研究思路.

Objective To establish a model of hematopoietic stem and progenitor cells with iron overload derived from umbilical cord blood（UCB）cells and explore the effects of reactive oxygen species （ROS）on the hematopoiesis of hematopoietic stem and progenitor cells with iron overload.Methods The model was established by adding different concentrations（50,100,200,400 μmol/L）of ferric citrate（FAC）into mononuclear cells from UCB and culturing for different times（6,12,24 h）.The UCB cells were divided into 4 groups：control group,grup FAC,group FAC ＋ N-acetyl-L-cysteine（NAC）and group FAC ＋ L-Glutathione（GSH）.Then the changes of ROS,labile iron pool（LIP）,apeptosis,the capacity of hematopoietic clony forming（CFU-E,BFU-E,CFU-GM,CFU-mix）and the percentage and the numbers of CD34 ＋,CD33 ＋,GlyA＋ cells were detected.And the changes of these indices were tested after the treatment of iron overload UCB with antioxidants（NAC and GSH）.Results UCB cells were cultured with the addition of FAC at different concentrations for different times.The level of total ROS increased in time and concentration-dependent manners.The intracellular level of ROS peaked when cultured at 200 μ mol/L of FAC for 24 hours.Cells were treated with antioxidants NAC or GSH after cultured with 200 μmol/L FAC for 24 hours.Then the ROS levels of total cells,myeloid cells and erythroid cells decreased markedly versus normal controls.The LIP of total cells,myeloid cells and erythroid cells increased markedly when cells were cultured at 200 μmol/L of FAC for 24 hours versus normal controls（P 〈 0.05）.NAC and GSH had no effect on the level of LIP.The apoptotic rates of FAC-treated cells[（20.90 ± 3.45）%]increased significantly versus normal controls[（9.20 ± 1.29）%]（P 〈 0.05）.The capacity of hematopoietic colony forming in FAC treated cells decreased markedly versus normal controls.The percentage and numbers of CD34 ＋,CD33 ＋,GlyA ＋ cells of FAC-treated cells also decreased significantly versus normal controls（P 〈0.05）.And these changes could be recovered by the addition of NAC or GSH.Conclusion Oxidative stress plays an important role in the injuries of hematopoiesis of hematopoietic stem and progenitor cells with iron overload by inducing the generation of ROS.These findings may help us find a specific target and improve the therapeutic efficacy of ineffective hematopoiesis in patients with iron overload.