mTOR调控自噬在Duchenne肌营养不良症肌纤维损害中的研究

mTOR regulates autophagy in muscle fiber damage in Duchenne muscular dystrophy

目的探索哺乳动物雷帕霉素靶点(mTOR)相关自噬调节通路在mdx小鼠骨骼肌纤维中有关线粒体形态学异常以及肌肉萎缩和坏死过程中所处的地位和作用.方法选取8 w龄雄性mdx小鼠为实验组,同龄C57小鼠为对照组,每组各3只,取股四头肌组织,通过病理染色观察肌肉组织病理学改变,应用电镜技术观察线粒体的形态变化,应用免疫荧光染色技术观察自噬泡标志物微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)和线粒体标志物电压依赖性阴离子通道(voltage-dependent anion channel,VDAC)的共定位情况,利用Western blot技术分析mTOR相关自噬调控通路蛋白的表达,应用Real-Time PCR技术分析自噬相关基因的表达水平.结果与对照组相比,8w龄mdx小鼠股四头肌组织萎缩,肌纤维内可见核内移、肌再生及炎性细胞浸润;电镜下可见线粒体体积变小,数量增多,并出现线粒体肿胀、嵴消失现象.免疫荧光可见线粒体标记物VDAC和自噬泡标记物LC3的团块状聚集,两者的共定位增加.自噬相关基因蛋白及基因表达上升、线粒体自噬相关基因明显上升,mTOR通路下游相关基因蛋白表达水平下降.结论8w龄mdx小鼠股四头肌组织内肌纤维存在变性和坏死,线粒体形态学异常.mTOR的活性降低,自噬相关蛋白表达水平升高,该阶段自噬通路处于激活状态.

Objective To explore the role of mTOR-related autophagy regulatory pathway in mitochondrial morphological abnormalities,muscle atrophy and necrosis in skeletal muscle fibers of mdx mice.Methods Eightweek-old male mdx mice were selected as the experimental group,and C57 mice of the same age were selected as the control group,with 3 mice in each group.The quadriceps femoris tissue was collected to observe the pathological changes of muscle tissue by pathological staining,and the morphological changes of mitochondria were observed by electron microscopy;immunofluorescence staining was used to observe the co-localization of autophagy vesicle marker microtubule-associated protein 1 light chain 3(LC3),and mitochondrial marker:voltage-dependent anion channel,(VDAC).Western blot was used to analyze the expression of mTOR-related autophagy regulatory pathway proteins,and real-time PCR was used to analyze the expression levels of autophagy related genes.Results Compared with the control group,the quadriceps femoris tissue of mdx mice showed nuclear migration,muscle regeneration and inflammatory cell infiltration.Under electron microscopy,the size of mitochondria became smaller,the number increased,and the phenomenon of mitochondrial swelling crista disappeared.Immunofluorescence showed the clumps of mitochondrial marker VDAC and autophagic vesicle marker LC3,and the co-localization of them increased.Autophagy-related genes and gene expression increased significantly,and the protein expression level of genes downstream of mTOR pathway decreased.Conclusion Eight-week-old mdx mice showed degeneration and necrosis of muscle fibers in quadriceps femoris tissue,abnormal mitochondrial morphology,decreased mTOR activity and increased expression level of autophagy related proteins,and autophagy pathway was activated at this stage.