电刺激对肌卫星细胞发育及分化影响的研究

发布时间：2018-05-30 06:18

  本文选题：功能性电刺激 + 肌卫星细胞　； 参考：《南方医科大学》2014年硕士论文

【摘要】：研究背景及意义： 功能性电刺激原理产生的各种起搏器正逐渐应用于临床,而肌肉的起搏器如麻痹的环杓后肌喉起搏器、麻痹的膀胱逼尿肌起搏器和正常神经支配的吞咽肌起搏器等已进入临床实验并在临床中使用。目前,起搏器带来的电刺激对肌肉包括麻痹肌肉和正常肌肉的影响还不明确。肌肉起搏器在补偿肌肉功能的同时也导致肌肉本身的变化,肌卫星干细胞作为肌纤维细胞的主要来源,在电刺激的影响下对肌肉的再生影响尚不十分明确。近年来,研究发现肌卫星干细胞是肌肉再生的主要来源,而电刺激对肌卫星干细胞的研究也非常少。 头颈肌是发声、呼吸、吞咽及头颈部运动的重要肌肉。支配头颈肌的神经发生不可逆性损伤后,肌肉发生不可逆性萎缩。卫星干细胞作为肌肉细胞的主要来源,是肌肉再生和修复的关键。因此,我们以电刺激头颈肌卫星干细胞为研究立足点展开工作。本研究拟通过电刺激体外培养卫星干细胞,了解其活性、增殖和分化的变化。以期为卫星干细胞增殖提供有利的刺激手段,并为卫星干细胞的增殖和分化机制研究提供一定的基础,更希望为临床肌肉起搏器的进一步研发提供实验室基础。 目的： 利用小鼠肢体来源的C2C12细胞系及比格犬的颏舌肌卫星干细胞体外培养作为研究对象,使用固定电压、固定刺激时间、不同脉宽和不同频率的脉冲电培养细胞,了解不同模式电刺激对卫星干细胞的增殖和分化能力的影响。 方法： 1.取体外培养的肌源性C2C12细胞株,采用固定电压(5V)、固定刺激模式、不同脉宽(Oms, lms,2ms,4ms,8ms,20ms六组)的串刺激(5s,3s-on,2s-off)细胞系,以及固定电压(5V)、固定刺激模式、不同频率(Opps、2pps、10pps、20pps、40pps、80pps六组)使用MTT比色法、流式细胞周期检测、qRT-PCR观察细胞的活性、增殖及分化。 2.取体外培养的比格犬颏舌肌卫星干细胞体外培养,采用固定电压(5V)、固定脉宽、不同频率(Opps、2pps、10pps、40pps、80pps、160六组)的串刺激(5s,3s-on,2s-off)刺激细胞,使用MTT比色法、流式细胞周期检测和Western blot观察细胞的活性、增殖及分化。 3.用SPSS19.0统计软件进行分析。采用Spearman相关分析了解刺激脉宽及频率的增加同细胞活性、增殖和分化状态的相关性。采用Kruskal-Wallis H非参数检验方法比较组间差异。以P0.05为差异有统计学意义。 结果： 1.随着脉冲电刺激的脉宽增大会导致肌源性C2C12细胞的活性降低(r=-0.724,P0.01),分化能力有提高,在脉宽20ms时影响较大(p=0.044)；随着电刺激的频率加快,肌源性C2C12细胞的增殖(r=0.643,P=0.004)和分化能力提高(r=0.637,P=0.004；r=0.660,P=0.003),分化的肌细胞亚型有从快型向慢型转变的趋势,在80hz时较明显(P0.05)。 2.随着电刺激的频率加快,颏舌肌卫星干细胞活性和增殖没有变化,分化能力明显提高,肌细胞亚型从快型向慢型转变,在频率为160hz变化明显(P0.01)。 结论：体外实验中,随着脉冲电刺激的脉宽增大会导致卫星干细胞的活性降低,分化能力加强,肌细胞亚型从快型向慢型转变。随着电刺激的频率加快,卫星干细胞的分化能力提高,肌细胞亚型从快型向慢型转变。
[Abstract]:Research background and significance:
Various pacemakers produced by the principle of functional electrical stimulation are gradually applied to the clinic, while the muscle pacemaker such as paralytic posterior dipper pacemaker, paralyzed bladder detrusor pacemaker and normal denervation pacemaker have entered clinical trials and used in clinical practice. At present, the electrical stimulation of pacemaker brings to muscle package. It is not clear that the effects of paralytic muscles and normal muscles are not clear. Muscle pacemakers also cause muscle changes while compensating muscle function. Muscle satellite stem cells are the main source of muscle fiber cells. The effect of electrical stimulation on muscle regeneration is not very clear. In recent years, the study found that muscle satellite stem cells are muscles. The main source of regeneration is the electrical stimulation of muscle satellite stem cells.
The head and neck muscles are important muscles of the vocal, respiratory, swallowing, and head and neck movement. After the irreversible damage to the neurogenesis of the head and neck muscles, the muscle is irreversible. As the main source of the muscle cells, the satellite stem cells are the key to the muscle regeneration and repair. Therefore, we use electric stimulation of the head and neck muscle satellite stem cells as the research foothold. The purpose of this study is to stimulate the activity, proliferation and differentiation of satellite stem cells by electrical stimulation in vitro, in order to provide a favorable stimulus for the proliferation and differentiation of satellite stem cells, and to provide a basis for the study of the mechanism of proliferation and differentiation of satellite stem cells, and hope to provide a further research and development of the clinical muscle pacemaker. Laboratory basis.
Objective:
The effects of different modes of electrical stimulation on the proliferation and differentiation of satellite stem cells were investigated by using the C2C12 cell lines from the limbs of the mice and the culture of the genial muscle satellite stem cells of the Beagle in vitro.
Method锛，

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