mTOR激活在低氧刺激所致C2C12细胞脂质沉积中的作用

发布时间：2018-06-07 19:11

  本文选题：低氧 + C2C12成肌细胞　； 参考：《皖南医学院》2017年硕士论文

【摘要】：目的:机械通气是急慢性呼吸衰竭的主要治疗手段,机械通气过程中可能由于多种因素引起膈肌收缩力下降并进行性加重,临床主要表现为脱机困难,即呼吸机相关膈肌功能障碍(ventilator-induced diaphragmatic dysfunction,VIDD)。研究表明机械通气所致脂质代谢异常及其触发的线粒体损伤是导致VIDD的重要因素,但其启动调控机制尚不明确。雷帕霉素靶蛋白(mTOR)激活在动脉粥样硬化脂质代谢中起重要调控作用,但其在VIDD中的作用尚不清楚。机械通气导致膈肌氧供减少所引起的低氧环境可能是膈肌纤维脂质沉积的刺激因素,从而导致膈肌氧化应激增强及膈肌功能障碍。推测机械通气过程中脂质代谢紊乱引起mTOR信号通路的激活,导致膈肌脂质沉积的加重,加速机械通气早期膈肌功能障碍的发生发展。本研究拟通过建立C2C12小鼠成肌细胞低氧环境模型,从细胞水平观察低氧环境下,mTOR通路激活对细胞脂质沉积的作用,揭示机械通气条件下膈肌萎缩的启动和调节机制,为临床VIDD的预防和治疗提供理论基础。方法:C2C12小鼠成肌细胞在10%胎牛血清DMEM高糖培养基培养传代后,在2%马血清高糖DMEM高糖培养基诱导分化成具有肌管后,显微镜拍照观察细胞形态变化,并分为四个实验组:1)对照组:常氧(37℃、5%CO2)二氧化碳培养箱中的分化成具有肌管的C2C12成肌细胞。2)低氧组:在低氧条件(5%二氧化碳、1%氧气、94%氮气)下分别处理分化成肌管的C2C12成肌细胞12小时,24小时。3)雷帕霉素组:在分化成肌管的C2C12成肌细胞培养基中加入终浓度为10ng/ml雷帕霉素分别处理12小时,24小时。4)雷帕霉素加低氧组:终浓度为10ng/ml雷帕霉素的分化成肌管的C2C12成肌细胞在低氧条件下分别处理12小时,24小时。油红染色用显微镜拍照观察各组各时间点细胞脂质沉积情况,另收集细胞,提取蛋白行Western blotting检测各组各时间点p-mTOR、p-4EBP1、p-S6K1、SCAP、SREBP-2、LDLR等蛋白表达水平,并用细胞实时荧光法检测mTOR、4EBP1、S6K1、SCAP、SREBP-2、LDLR mRNA表达情况。结果:(1)用2%马血清高糖培养基C2C12成肌细胞,7天左右细胞分化成具有肌管形态。(2)油红染色,结果显示各低氧组细胞有脂质沉积,雷帕霉素可以减轻细胞内脂质沉积。(3)低氧环境下,细胞内mTOR信号通路和脂质代谢通路相关因子蛋白和mRNA表达增加,并伴随者细胞内脂质的沉积。(4)随着低氧刺激时间的延长,mTOR信号通路和脂质代谢通路相关因子蛋白和mRNA表达增加,雷帕霉素能够减轻细胞mTOR信号通路和脂质代谢通路蛋白和mRNA表达增加,并且减轻脂质沉积。结论:(1)脂质代谢异常可能是mTOR信号通路激活的刺激因素(2)mTOR信号通路激活加重细胞脂质的沉积,可能是调控细胞脂质代谢的一个靶点
[Abstract]:Objective: mechanical ventilation is the main treatment for acute and chronic respiratory failure. That is ventilator-induced diaphragmatic dysfunction VIDD. The results showed that the abnormal lipid metabolism induced by mechanical ventilation and mitochondrial damage triggered by mechanical ventilation were important factors leading to VIDD, but the mechanism of its priming regulation was not clear. The activation of rapamycin target protein mTORplays an important role in the lipid metabolism of atherosclerosis, but its role in VIDD is unclear. The hypoxic environment induced by mechanical ventilation may be a stimulating factor for the deposition of lipid in the diaphragm, which may lead to the increase of oxidative stress and the dysfunction of the diaphragm. It is speculated that lipid metabolism disorder during mechanical ventilation causes activation of mTOR signaling pathway, exacerbates lipid deposition of diaphragm, and accelerates the occurrence and development of diaphragm dysfunction in the early stage of mechanical ventilation. The aim of this study was to investigate the effects of activation of mTOR pathway on lipid deposition in C2C12 mouse myoblast under hypoxic environment, and to reveal the mechanism of initiation and regulation of diaphragmatic atrophy under mechanical ventilation. To provide a theoretical basis for the prevention and treatment of clinical VIDD. Methods the myoblasts were cultured in 10% fetal bovine serum DMEM high sugar medium, and then differentiated into myotubes on 2% equine serum high glucose DMEM medium. The morphologic changes of the cells were observed by microscope. And was divided into four experimental groups: control group: normal oxygen 37 鈩，

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