胰岛β细胞铁过载模型的建立及铁过载对胰岛β细胞损伤的研究  

Establishment of pancreatic β cell iron overload model and β cell injury induced by iron overload

作　　者：张丽娜[1] 侯乐乐[1] 刘祖霖[1] 李平甘[1] 黄思琪 孟哲[1] 欧辉[1] 江转南 梁立阳[1] ZHANG Li-na;HOU Le-le;LIU Zu-lin;LI Ping-gan;HUANG Si-qi;MENG Zhe;OU Hui;JIANG Zhuan-nan;LIANG Li-yang(Department of Pediatrics,Sun Yat-sen Memorial Hospital,Sun Yat-sen University,Guangzhou 510260,China)

机构地区：[1]中山大学孙逸仙纪念医院儿科

出　　处：《中国病理生理杂志》2019年第12期2150-2155,共6页Chinese Journal of Pathophysiology

基　　金：中华医学会金磊儿科内分泌中青年医师成长科研基金资助项目(No.PEGRF201607012);中山大学逸仙纪念医院逸仙临床研究培育项目(No.SYS-C-201703)

摘　　要：目的:通过枸橼酸铁铵(FAC)与大鼠胰岛素瘤细胞株INS-1共培养,构建铁过载INS-1细胞模型,并观察铁过载对胰岛细胞铁沉积、存活、胰岛素分泌、氧化应激以及线粒体损伤的影响。方法:体外培养INS-1细胞,不添加FAC作为对照组,加入不同浓度(5、10、20、40、80、160和320μmol/L)的FAC干预INS-1细胞,分别培养24 h、48 h和72 h,建立铁过载INS-1细胞模型,测定细胞内的不稳定铁池(LIP),CCK8法分析细胞活力的改变,筛选出后续实验组所用FAC浓度,ELISA法检测胰岛素分泌功能,活性氧簇(ROS)探针染色流式细胞术检测ROS的生成,JC-1试剂盒检测线粒体膜电位,透射电子显微观察线粒体的变化。结果:加入不同浓度FAC处理后,铁过载组INS-1细胞内的LIP水平明显高于对照组(P<0.05)。随着FAC浓度的升高及干预时间的延长,INS-1细胞的活力逐渐降低(P<0.05)。选取80、160和320μmol/L FAC作用48 h作为后续实验的铁过载组。胰岛素分泌随FAC浓度的增加出现先升高后降低,160和320μmol/L组与对照组相比差异具有统计学意义(P<0.05)。INS-1细胞内ROS的水平较对照组均显著增加(P<0.05)。线粒体膜电位随着铁浓度的增加而降低(P<0.05)。铁过载后INS-1细胞的线粒体肿胀,内嵴扩张,失去正常结构,随着FAC浓度增加,线粒体结构破坏更加明显。结论:与FAC共培养48 h可成功建立INS-1细胞铁过载模型。铁过载可显著破坏线粒体结构和功能,增加细胞内ROS的水平。胰岛β细胞的存活对铁敏感,即使低剂量的铁也损伤胰岛β细胞,但只有细胞数量减少到一定程度,才会减少胰岛素的分泌。AIM:To establish rat insulinoma INS-1 cell iron overload model,and to study the effect of iron overload on the viability,insulin secretion,mitochondrial defect and oxidative stress change in the INS-1 cells.ME-THODS:INS-1 cells were cultured with ferric ammonium citrate(FAC)at different concentrations(0,5,10,20,40,80,160 and 320μmol/L).Labile iron pool(LIP)were calculated by detecting calcein-AM fluorescence in 24 h,48 h and 72 h.The cell viability was measured by CCK8 assay.Iron overload model was established by screening for the best combination to ensure both high LIP level and cell viability.Reactive oxygen species(ROS)level was further analyzed by flow cytometry after fluorescent probe staining.The function of insulin secretion was measured by ELISA.The mitochondrial membrane potential was detected by JC-1 kit,and the mitochondrial changes were observed by transmission electron microscopy.RESULTS:Intracellular LIP levels were significantly increased in FAC groups in a concentration-dependent manner(P<0.05).The viability of INS-1 cells was suppressed with increasing FAC concentration or culture time(P<0.05).The highest LIP level and cell viability over 50%were observed with the condition of exposure to FAC for 48 h,indicating that INS-1 cell iron overload model was established.With the increase in the FAC concentrations,the insulin secretion was also increased and then decreased,and that in 160 and 320 mol/L groups showed statistical difference compared with control group(P<0.05).The ROS level was significantly increased by FAC exposure as compared with control group(P<0.05).Mitochondrial membrane potential was decreased with the increase in the iron concentration(P<0.05).After iron overload,the mitochondria of INS-1 cells were swollen,the internal cristae were expanded,and the normal structure was lost.With the increase in the FAC concentration,the mitochondrial structure was destroyed more obviously.CONCLUSION:Co-culture of INS-1 cells with FAC for 48 h successfully establish the iron overload model.Iron o

关 键 词：铁过载 胰岛Β细胞 氧化应激 

分 类 号：R363.2[医药卫生—病理学] R329.2[医药卫生—基础医学]