铁过载及铁死亡在瘢痕疙瘩成纤维细胞中的作用及机制  

The role and mechanism of iron overload and ferroptosis in keloid fibroblasts

作　　者：龚玲 李宇[1] 李明轩 马娟[1] 迟宏羽 董祥林[1] Gong Ling;Li Yu;Li Mingxuan;Ma Juan;Chi Hongyu;Dong Xianglin(Department of Plastic Surgery,the First Affiliated Hospital of Xinjiang Medical University,Urumqi 830011,China;Department of Thoracic Surgery,the First Affiliated Hospital of Xinjiang Medical University,Urumqi 830011,China)

机构地区：[1]新疆医科大学第一附属医院整形科,乌鲁木齐830011 [2]新疆医科大学第一附属医院胸外科,乌鲁木齐830011

出　　处：《中华整形外科杂志》2023年第12期1299-1310,共12页Chinese Journal of Plastic Surgery

基　　金：新疆维吾尔自治区青年基金项目(2019DOIC308);新疆维吾尔自治区自然科学基金项目(2023D01C101)。

摘　　要：目的了解瘢痕疙瘩与正常皮肤组织中铁含量及转铁蛋白受体1(TfR1)表达水平,在体外构建Erastin诱导的瘢痕疙瘩成纤维细胞(KFB)铁死亡模型,检测Erastin和铁抑制素-1(Fer-1)对细胞活力、亚铁离子(Fe^(2+))含量及脂质过氧化物、铁死亡和纤维化相关调节因子的影响。方法收集2022年3至6月新疆医科大学第一附属医院6例瘢痕疙瘩组织与6例包皮组织,采用组织铁含量试剂盒测定2种组织真皮层中铁含量,Western blotting法检测2种组织中TfR1蛋白表达情况。采用组织块培养法获取原代KFB和正常皮肤成纤维细胞(NFB),使用Erastin诱导KFB铁死亡模型并用CCK-8法检测不同浓度的Erastin和Fer-1对细胞活性的影响,筛选合适的药物浓度。后续实验分为5组:NFB组、control组、Erastin(0.6μmol/L)组、Fer-1(1μmol/L)组、Erastin(0.6μmol/L)+Fer-1(1μmol/L)组,其中后4组采用KFB作为实验对象;采用划痕实验检测细胞迁移能力,荧光探针法和试剂盒检测各组细胞中丙二醛(MDA)、活性氧(ROS)和Fe^(2+)含量;Western blotting法检测各组细胞中TfR1、谷胱甘肽过氧化物酶4(GPx4)、溶质载体家族7成员11(SLC7A11)、α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原蛋白(COL-1)表达水平,免疫荧光检测KFB中TfR1、Gpx4蛋白表达及定位情况。采用GraphPad Prism 9.0统计软件,计量资料以±s表示,2组间比较采用独立样本t检验,多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。P<0.05表示差异具有统计学意义。结果与正常皮肤组织比较,瘢痕疙瘩中的铁含量及TfR1蛋白表达均明显较高(P<0.01)。不同浓度Erastin处理的KFB增殖率逐渐下降,IC50为0.61μmol/L,Fer-1在0.1~20μmol/L对KFB无明显毒性。划痕实验显示,control组迁移率显著高于NFB组(P<0.01);与control组相比,Erastin干预后KFB迁移率明显下降(P<0.01);与Erastin组相比,Erastin+Fer-1组KFB迁移明显加快(P<0.01)。control组ROS、MDA水平显著高于NFB�Objective To investigate the iron content and transferrin receptor 1(TfR1)expression levels in keloid and normal skin tissues.Erastin induced ferroptosis model of keloid fibroblasts(KFB)is constructed in vitro,and the effects of Erastin and Ferrostatin-1(Fer-1)on cell viability,ferrous ion(Fe^(2+))content and lipid peroxidation,ferroptosis and fibrosis-related regulatory factors are examined.Methods Six keloid tissues and six prepuces were collected from the First Affiliated Hospital of Xinjiang Medical University from March to June 2022.The tissue iron content kit was used to determine the iron content in the dermis,and TfR1 protein expression level was detected by Western blotting.Primary KFB and normal skin fibroblasts(NFB)were obtained by tissue cultivation,Erastin-induced KFB ferroptosis model and CCK-8 assay were used to detect the effects of different concentrations of Erastin and Fer-1 on cell viability,and to screen the appropriate drug concentration.The subsequent experiments were divided into five groups:NFB group,control group,Erastin(0.6μmol/L)group,Fer-1(1μmol/L)group,and Erastin(0.6μmol/L)+Fer-1(1μmol/L)group.KFB was used in the last 4 groups.Cell migration ability was detected by scratch assay.The contents of malondialdehyde(MDA),reactive oxygen species(ROS)and Fe^(2+)were detected by fluorescence probe and kits;the protein expression levels of TfR1,glutathione peroxidase 4(GPx4),solute carrier family 7 member 11(SLC7A11),α-smooth muscle actin(α-SMA)and type I collagen(COL-1)in each group of cells were detected by Western blotting;the protein expression and localization of TfR1 and Gpx4 in KFB were detected by immunofluorescence staining.GraphPad Prism 9.0 statistical software was used in the statistical analyses,and the measurement data were expressed as Mean±SD.Independent samples t-test was used for comparison between 2 groups,and one-way ANOVA was used for comparison between multiple groups,LSD-t test was used for pairwise comparison between groups.P<0.05 indicated statistical significa

关 键 词：瘢痕疙瘩 成纤维细胞 铁死亡 铁过载 活性氧 脂质过氧化 

分 类 号：R622[医药卫生—整形外科]