铁过载巨噬细胞体外模型的建立及氧化应激对铁过载巨噬细胞的损伤作用  

Establishment of macrophage model of iron overload in vitro and the injury induced by oxidative stress on macrophage with iron overload

作　　者：曹小立[1] 赵明峰[1] 李德冠[2] 邢艺[1] 张宇辰[1] 陈洁[1] 贺小圆 崔蕊[1] 孟娟霞[1] 肖霞[1] 穆娟[1] 江嫣雨 武日茂 

机构地区：[1]天津医科大学一中心临床学院 天津市第一中心医院血液科,300192 [2]中国医学科学院放射医学研究所

出　　处：《中华医学杂志》2016年第2期129-133,共5页National Medical Journal of China

基　　金：国家自然科学基金（81400092）;天津市自然科学基金（13JCYBJC23400）;天津市卫生局科技基金攻关项目（13KG106）

摘　　要：目的通过铁与巨噬细胞共培养，建立体外铁过载巨噬细胞模型，检验铁过载对细胞氧化应激水平的影响以及氧化应激升高对巨噬细胞的影响。方法在巨噬细胞培养的过程中分别添加不同浓度（5、10、20、40、80μmol／L）枸橼酸铁铵（FAC）建立铁过载巨噬细胞模型（铁过载组），以不添加FAC作为对照组，检验这一过程中细胞数量及状态、细胞活性、细胞吞噬功能的变化，细胞生成活性氧、活性氮水平以及与之相关的氧化应激信号通路的变化。再用地拉罗司（DFX）去铁及抗氧化剂N-乙酰半胱氨酸（NAC）清除过多的氧化应激后，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，发现巨噬细胞内可变铁池（LIP）水平升高，且具有浓度依赖性，在含80μmol／LFAC的培养液中LIP水平达到最高。（2）随着FAC浓度的提高，巨噬细胞的活性逐渐降低，依次为对照组的51．58％、40．98％、16．23％、3．46％、0．05％（均P〈0．05）。选取活性降低至16．23％的FAC浓度（20μmol／L）作为后续实验铁过载组。（3）和对照组相比，铁过载组巨噬细胞的数量减少至对照组的32．80％（P〈0．05），细胞状态由贴壁变为部分悬浮；铁过载组巨噬细胞的吞噬功能降低至对照组的20．40％（P〈0．05）。（4）进一步的机制研究发现铁过载组的活性氧水平及活性氮水平分别是对照组的7．71及1．45倍（均P〈0．05）；经过荧光定量PCR检测发现，与对照组相比，参与活性氧生成的还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶4（NOX4）基因、参与活性氮生成的诱导型一氧化氮合酶（iNOS）基因的mRNA表达水平升高，参与活性氧清除的谷胱甘肽过氧化物酶1（GPX1）基因mRNA表达水平降低；氧化应激信号通路相关的磷脂酰肌醇-3-激酶（P13K）基因mRNA表达水平升高；负反馈调节氧化应激的Objective To establish macrophage iron overload model in vitro by co-culture macrophages with iron, and to explore the effect of iron overload on cell reactive oxygen species （ROS） and the impact of ROS on macrophages. Method Iron overload group were treated with different concentrations （0, 5, 10, 20, 40, 80 μmol/L respectively） of ferric ammonium citrate （FAC）. The control group was the group of macrophages without FAC treatment. We detected the number and state of cells, metabolic activity,the change of phagocytosis, the levels of ROS and reactive nitrogen, and changes of related oxidative stress signaling pathways in different groups. Changes in the above indexes were detected after application of deferasirox （DFX） to remove iron and the antioxidant N -acetylcysteine （NAC） to clear excess oxidative stress. Results （1）The levels of labile iron pool （LIP） in macrophages co-cultivated with iron was increased with the increase of iron concentration in a dose-dependent manner. The LIP levels was the highest in the macrophages treated with 80 μmol/L. （2） The increase of FAC concentration, the metabolic activity of macrophages in the 5 FAC-treated groups decreased to 51.58%, 40.98% , 16. 23%, 3.46% , and 0. 05% of the activity level of the control group （ all P 〈 0. 05 ）. The group with the metabolic activity decreased to 16. 23% （20μmol/L） was selected as the iron overload group for the following experiments. （3） Compared with the control group, the number of macrophages in the iron overload group reduced to 32. 80% （ P 〈 0.05 ） , and the state of cells changed from adherence to partial suspension. The phagocytosis of macrophages in the iron overload group reduced to 20.40% of the control group （ P 〈 0. 05 ）. （ 4 ） Our further experiment showed that the levels of ROS and the activity nitrogen in the iron overload group increased by 7.71-and 1.45-fold compared with the control group （ both P 〈 0. 05 ）. The RT-PCR showed up-regulated mRNA expressi

关 键 词：巨噬细胞 铁超负荷 细胞存活 氧化性应激 信号传导 

分 类 号：R392[医药卫生—免疫学]