不同SOD1基因突变的肌萎缩侧索硬化患者皮肤成纤维细胞中突变蛋白表达及线粒体功能

Detection the mutated protein aggregation and mitochondriai function in fibroblasts from amyotrophic lateral sclerosis patients with SOD1 gene mutations

目的 针对携带不同SOD1基因突变的肌萎缩侧索硬化（ALS）患者皮肤成纤维细胞进行SOD1突变蛋白表达和线粒体功能的探索.方法 活检获取2008-2012年北京大学第三医院神经内科门诊确诊的携带SOD1-V14M、SOD1-G16A、SOD1-C 111Y基因突变的家族性ALS患者及年龄性别相匹配的健康对照皮肤组织,培养并建立成纤维细胞系;采用PCR扩增和直接测序的方法测定基因突变位点;共聚焦免疫荧光检测细胞内SOD1蛋白聚集量及线粒体膜电位变化;流式细胞仪检测活性氧（ROS）水平.结果 携带SOD1-V14M、SOD1-G16A、SOD1-C111Y基因突变的ALS病人成纤维细胞出现胞质SOD1蛋白异常聚集,三组突变的SOD1蛋白的浆核比与对照组相比分别升高2.54、2.80及3.25倍;成纤维细胞线粒体膜电位△Ψm明显降低,红/绿荧光强度的比值相比对照组分别减少了36％、124％、142％;且细胞内ROS水平均较对照组增高了3.33、3.65、6.87倍.结论 在携带SOD1基因突变的ALS病人成纤维细胞内出现胞质SOD1蛋白异常聚集、线粒体功能异常及ROS水平改变,患者的外周组织成纤维细胞可作为ALS发病机制研究的有力工具,更益于将来可能的临床应用.

Objectives To explore mutant superoxide dismutase （SOD）1 protein expression and mitochondrial function in amyotrophic lateral sclerosis （ALS） patients＇ fibroblasts carrying different SOD1 mutations.Methods SOD1 gene mutation was detected using PCR and direct sequencing.Skin fibroblasts of three familial ALS patients with mutations and age/gender matched controls obtained by a punch skin biopsy were cultured.We performed immunofluorescence staining and quantitative detection of SOD1 proteins and mitochondrial membrane potential.Also,we detected the intracellular ROS by flow cytometry.Results We found that fibroblasts from familial ALS patients carried SOD1-V14M,SOD1-G16A,SOD1-C111Y mutation,respectively.The cytoplasm abnormal SOD1 protein aggregates appeared in ALS patients carrying SOD1 mutations.And the cytoplasmic/nuclear ratio of SOD1 aggregates increased 2.54,2.80,3.25-fold for each mutations,respectively,compared to the control group.Three SOD1 mutant groups showed loss of mitochondrial membrane potential and the ratio of red / green fluorescence intensity decreased by 36%,124%,142%,respectively,compared to the control group.The intracellular ROS levels also increased 3.33,3.65,and 6.87-fold respectively.Conclusions This work highlights that ALS alters SOD1 protein expression,mitochondrial function,and increases the ROS level even in peripheral tissues outside the central nervous system.Fibroblasts might therefore represent a powerful and minimally invasive tool to investigate ALS pathogenic mechanisms,which might translate into considerable advances in clinical management of the disease.