瘢痕疙瘩成纤维细胞线粒体功能障碍及其对细胞代谢功能的影响

摘要

目的 比较瘢痕疙瘩成纤维细胞(KFb)与正常皮肤成纤维细胞(NFb)在常氧和低氧培养状态下的线粒体功能和能量代谢状态,以探讨瘢痕疙瘩成纤维细胞线粒体功能在瘢痕疙瘩发病中的作用.方法 采用组织块贴壁法原代培养KFb和NFb.常氧和低氧(2％O2)条件下培养KFb与NFb,CCK-8检测细胞增殖情况,分光光度法测定细胞氧消耗率和乳酸生成,化学发光仪检测细胞ATP生成情况,流式细胞仪检测细胞线粒体数量和活性氧含量,透射电镜观察细胞线粒体超微结构,RT-PCR检测线粒体融合/分裂相关基因MFN1、MFN2、FIS1的表达.结果 常氧培养1～5d,KFb与NFb的细胞数之比分别为0.98、1.04、1.18、1.08和1.31,2％O2条件下培养1～5d,KFb与NFb细胞数之比分别为1.15、1.13、1.13、1.30和1.31.常氧下KFb的细胞耗氧率和ATP含量分别比NFb低12.95％和45.70％ (P ＜0.05),低氧培养48 h后KFb耗氧率和ATP含量分别增加25.92％和56.24％ (P ＜0.05),而NFb耗氧率和ATP变化不明显.常氧条件下KFb乳酸生成较NFb少,2％低氧条件下培养1～5d的KFb乳酸生成量较常氧下的KFb分别增高19.87％、34.51％、92.64％、75.31％、125.38％,低氧培养1～5d的NFb乳酸生成与常氧下相比并无明显变化.常氧条件下KFb与NFb中活性氧含量相差不大;2％低氧条件下KFb和NFb中活性氧均随时间逐渐增加,NFb在低氧6h后活性氧升高到稳定水平(6h、12h、24 h分别增加86.30％、79.04、94.83％),KFb在低氧6h后活性氧仍持续升高(6h、12h、24 h分别增加42.68％ 、115.25％、209.11％).KFb中线粒体数量比NFb多15.33％(P＜0.05),电镜下观察发现KFb线粒体明显增大,嵴融合消失,RT-PCR检测发现KFb中MFN1、MFN2、FIS1表达比NFb分别升高33.27％、113.39％和20.34％(P＜0.05).结论 KFb可能存在线粒体功能障碍,引起细胞代谢模式改变,使得KFb能适应低氧下细胞的持续增殖.%Objective To compare the differences of mitochondrial functions between keloid fibroblasts and normal skin fibroblasts and explore its relationship with cell proliferation.Methods Keloid fibroblasts (KFb) and normal skin fibroblasts (NFb) were isolated by explants culture method.KFb and NFb were cultured under normoxia or hypoxia (2％ O2).Differences of cell proliferation were detected by CCK-8.Flow cytometer was used to detect the content of mitochondria and reactive oxygen species (ROS) in KFb and NFb.Ultra-structures of mitochondria in KFb and NFb were observed by transmission electron microscope (TEM).Mitochondria fusion/fission related genes MFN1,MFN2 and FIS1 were detected by RT-PCR.Oxygen consumption rate,lactate production and ATP contents were determined by spectrophotometry.Results KFb showed a higher proliferation rate compared with NFb,especially under hypoxia.The oxygen consumption rate,ATP content,lactate production and ROS of KFb were lower than NFb under normoxia.After incubated under hypoxia,there was a significant increase in oxygen consumption,ATP content,lactate production and ROS in KFb,while NFb showed less increase compared with KFb.KFb had 15.33％ more mitochondrion than NFb,and expressions of MFN1,MFN2,FIS1 in KFb were 33.27％,113.39％ and 20.34％ higher compared with NFb.Under TEM,KFb showed an increase of enlarged mitochondrion,with disrupted inner membrane and loss of cristae.Conclusions KFb may have dysfunctions of mitochondrion which lead to changes of cell metabolism and continuous proliferation of KFb.