猪卫星细胞原代培养的自噬调节

A. Vincent, I. Louveau, F. Dessauge
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引用次数: 0

摘要

自噬是一种溶酶体降解途径,通过自我消化在细胞成分的转换中发挥作用。在过去的十年里,它被认为是维持细胞和能量稳态的一个重要过程。然而,农场动物很少注意到这一过程。在猪中,自噬在骨骼肌稳态中的作用,特别是在多核肌纤维形成中的作用需要确定。卫星细胞(常驻肌肉干细胞)的原代培养是研究主要自噬过程——大自噬(以下简称自噬)的合适模型。本研究的目的是评估在该细胞模型中监测自噬的工具,并明确自噬在细胞分化中的作用。从3至4日龄仔猪身上采集最长肌样本。分离后,将卫星细胞接种在生长培养基中,使其增殖至80%汇合,然后放置在适当的培养基中分化为肌管。从分化的第0天至第3天对细胞进行探索。通过蛋白质印迹检测自噬相关蛋白和细胞能量的主要传感器腺苷单磷酸激活蛋白激酶(AMPK)。与自噬相关基因的表达也通过qPCR进行定量。微管相关蛋白1轻链3β形成率在细胞分化过程中增加,而磷脂酰肌醇3-激酶和螯合体1蛋白显著降低。线粒体蛋白表达也随着卫星细胞分化而显著降低。然后,在我们的卫星细胞培养模型中,用自噬流量抑制剂巴菲霉素A1处理细胞,证实在成肌细胞转化为肌管的过程中,自噬被激活,AMPK也被激活。总之,我们为猪在组织或细胞中的自噬研究提供了工具,并证明了基础自噬和能量代谢在体外猪肌肉发生过程中同时受到调节。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Autophagy modulation in primary culture of porcine satellite cells

Autophagy is a lysosomal degradation pathway with a role in the turnover of cell components via self-digestion. Over the past decade, it has been recognised as an essential process to maintain cellular and energy homeostasis. Nevertheless, little attention has been paid to this process in farm animals. In pigs, the role of autophagy in skeletal muscle homeostasis and more specifically on the formation of multinucleated muscle fibres needs to be determined. Primary culture of satellite cells, the resident muscle stem cells, is an appropriate model to investigate macroautophagy (hereafter autophagy), the main autophagy process. The objective of the current study was to evaluate tools to monitor autophagy in this cell model and to specify the role of autophagy on cell differentiation. Samples of longissimus muscle were collected from 3- to 4-day-old piglets. After isolation, satellite cells were plated in growth medium, allowed to proliferate up to 80% confluence and then placed in an appropriate culture medium to differentiate into myotubes. Cells were explored from day 0 to day 3 of differentiation. Autophagy-related proteins and Adenosine Mono Phosphate-activated protein kinase (AMPK), a major sensor for cell energy, were detected by Western blotting. Expression of genes related to autophagy were also quantified by qPCR. The Microtubule-associated protein 1 light-chain 3β forms ratio increased during cell differentiation whereas phosphatidylinositol 3-kinase and sequestosome 1 proteins decreased significantly. Mitochondrial protein expression also decreased significantly with satellite cell differentiation. Then, cell treatment with an inhibitor of autophagy flux, Bafilomycin A1, confirmed that autophagy was activated during the conversion of myoblasts into myotubes along with AMPK activation in our satellite cell culture model. In conclusion, we provided tools for porcine autophagy investigation in tissues or cells and demonstrated that basal autophagy and energy metabolism are concomitantly modulated during porcine myogenesis in vitro.

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