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Biologia

肌肉干细胞中自噬的原位免疫荧光染色

Published: June 12, 2017
doi:
10.3791/55908
, , ,
1Department of Medicine, Institute of Translational Pharmacology,Italian National Research Council, 2Epigenetics and Regenerative Medicine,IRCCS Fondazione Santa Lucia, 3Biological and Environmental Sciences and Engineering Division,King Abdullah University of Science and Technology (KAUST), 4Department of Life Sciences,University of Modena and Reggio Emilia

Summary

活动性自噬与生产性肌肉再生有关，肌肉再生对肌肉干细胞（MuSC）的激活至关重要。在这里，我们提供了原位检测LC3的方案，这是来自对照组和受伤小鼠的肌肉组织切片的MyoD阳性MuSC中的自噬标记。

Abstract

越来越多的证据表明自噬是维持组织稳态的重要调节过程。已知自噬涉及骨骼肌发育和再生，并且自噬过程已经在几种肌肉病理学和年龄相关的肌肉疾病中描述。与肌肉修复期间卫星细胞的功能衰竭相关的自噬过程的最近描述的块支持活性自噬与生产性肌肉再生相结合的概念。这些数据揭示了在正常和病理状态（如肌营养不良）的肌肉再生过程中自噬在卫星细胞活化中的关键作用。在这里，我们提供了在肌肉再生条件下监测成年肌肉干细胞（MuSC）隔室中的自噬过程的方案。该协议描述了LC3的原位免疫荧光成像的设置方法，autophagy标记和MyoD，一种肌原性谱系标记，来自对照组和受损小鼠的肌肉组织切片。报告的方法允许监测一个特定细胞室的自噬过程，MuSC隔室在调节肌肉再生中起着核心作用。

Introduction

骨骼肌再生是成体干细胞（肌肉卫星细胞，MuSCs）与参与再生过程的其他细胞类型之间相互作用的结果。肌肉的体内平衡和功能由肌肉利基和全身线索1,2所组合的信号保持。在整个一生中，已经报告了MuSC功能，肌肉利基和系统线索的变化，导致老年人的功能能力下降3 。 MuSCs位于基底层下方的一个利基，并且在肌肉损伤时被激活以修复损伤的肌肉4,5 。为了确保有效的再生反应，至关重要的是，MuSC协调退出静止，自我更新和增殖扩张阶段所需的不同过程通过肌原性分化6 。在老年人和肌肉慢性疾病中，所有这些功能受到损害，导致肌肉功能变化2,3,6,7,8,9,10,11,12,13。

Macroautophagy（以下称为自噬）正在成为维持组织稳态至关重要的生物过程14 。自噬过程包括贩运机制，其中细胞质，细胞器和蛋白质的部分被吞入囊泡，其最终通过溶酶体途径降解，促进有毒分子的去除和大分子的再循环cules。这提供了能量丰富的化合物，以支持在应激或其他不利条件下的细胞和组织适应15,16 。与其细胞存活活动一起，自噬也可以作为细胞死亡诱导剂，取决于细胞组织背景（ 例如，正常与癌组织）和应激刺激的类型17,18 。

最近的证据表明，自噬需要维持肌肉质量和肌纤维完整性19,20 ，据报道在不同的肌营养不良21,22,23 中受损，包括Duchenne肌营养不良症（DMD）24,25,26,27 </su同样，在失去肌肉量（称为肌肉减少症）后，31,33,34,35岁的老年人中观察到自噬过程的逐渐减少32,33,34 ， 35,36,37和肌纤维存活38 。

热带实验室的一项研究预计自噬和骨骼肌再生潜能之间有着密切的关系，这表明热量限制增强了MuSC的可用性和活性。这不行最近观察到Foxo3-Notch轴激活了自我更新期间的自噬过程40以及从静止到增殖状态的MuSC转变。这些数据符合青年时期老年人和老年人的基础性自噬的逐渐减少，以及老年期间MuSCs的数值和功能下降42 。

在最近的一篇论文中，我们展示了自噬和补偿性肌肉再生之间的密切关系，区分了DMD进展的早期阶段。因此，当肌肉再生受损和纤维化组织沉积发生时，我们观察到疾病进展后期阶段的自噬通量减少。有趣的是，我们表明，在再生条件下，自噬在MuSCs中被激活，并且调节自噬过程影响MuSC激活和fu功能30

总而言之，这些数据突显了在正常和病理状况下以及整个寿命期间，在肌肉再生过程中探索MuSCs自噬过程的紧迫性。在这里，我们提供了一个协议，通过对微管相关蛋白1A / 1B-轻链3（LC3） 进行原位免疫染色监测肌肉再生条件下的自噬过程，LC3是自噬标志物43 ，MyoD是一种标记肌原纤维谱系，在对照组和受损小鼠的肌肉组织切片中。报告的方法允许监测一个特定细胞室的自噬过程，MuSC在编排肌肉再生中起关键作用。

Protocol

根据标准动物设施程序培育和维持小鼠，所有实验方案均经过动物福利保证和意大利卫生部内部动物研究伦理委员会批准，并遵守“NIH护理和使用指南”实验动物。 肌肉损伤和自噬通量的体内阻滞 肌肉受伤。 为了诱导急性骨骼肌损伤，将20μL的10μM心脏毒素（CTX）直接注射到体重约为20g的2个月大的C57BL / 6J小鼠的左胫骨前部（TA）肌肉中。使?…

Representative Results

该方案描述了在肌肉再生过程中检测MuSCs中的自噬的有效的原位方法。 CTX 体内治疗： 使用CTX诱导TA肌肉的肌肉损伤，并使用不受干扰的肌肉作为对照。由于自噬是高度动态的，通过执行CLQ的IP注射来阻断自噬通量（ 图1 ）。 CLQ治疗对于评估自噬通…

Discussion

该协议描述了如何在代偿性肌肉再生期间监测骨骼肌干细胞中的自噬。尝试了几种用于LC3和MyoD共染色的抗体，在这里列出了在小鼠组织切片中工作并产生成功结果的抗体 （参见材料表 ）。强烈推荐使用甲醇渗透（见步骤3.2.2）以进行成功染色。

该方案的局限性与小鼠的内在变异性相关，强迫使用至少3只小鼠/实验组。该方法反映了在骨骼肌再生…

Declarações

The authors have nothing to disclose.

Acknowledgements

这项工作得到了NIAMS AR064873，Epigen Project PB的支持。 P01.001.019 / Progetto Bandiera Epigenomica IFT to LL

Materials

C57BL/6J The Jackson Laboratory 000664 WT mice
Cardiotoxin 1 Latoxan L8102
Millex-VV Merck Millipore SLVV033RS Syringe Filter Unit, 0.1 µm, PVDF, 33 mm, gamma sterilized
Chloroquine diphosphate salt Sigma-Aldrich C6628 Caution:
Harmful if swallowed
BD Micro-Fine + 0,5 mL BD 324825
Tissue-Tek O.C.T. compound Sakura Finetek 25608-930
Tissue-Tek Cryomold Intermediate Sakura Finetek 4566
2-Methylbutane Sigma-Aldrich 277258
Hematoxylin Solution, Harris Modified Sigma-Aldrich HHS32
Eosin Y solution, alcoholic Sigma-Aldrich HT110132
o-Xylene Sigma-Aldrich X1040 Caution:
Flammable liquid and vapour; May be fatal if swallowed and enters airways; Harmful in contact with skin; May cause respiratory irritation; Causes serious eye irritation
Paraformaldehyde Sigma-Aldrich P6148 Caution:
Flammable solid; Harmful if swallowed; Causes skin irritation; May cause an allergic skin reaction; Causes serious eye damage; May cause respiratory irritation; Suspected of causing cancer
DPBS, no calcium, no magnesium Thermo Fisher Scientific 14190-094
Bovine Serum Albumin Sigma-Aldrich A7030
Glycerol Sigma-Aldrich G5516
Eukitt – Quick-hardening mounting medium Sigma-Aldrich 3989
AffiniPure Fab Fragment Goat Anti-Mouse IgG (H+L) Jackson ImmunoResearch 115-007-003
LC3B Antibody Cell signaling Technology 2775
Monoclonal mouse anti-MyoD
(concentrated) clone 5.8A		 DAKO – Agilent Pathology Solutions M3512
Laminin-2 (α-2-chain) monoclonal antibody Enzo Life Sciences 4H8-2
Alexa Fluor 488 Goat Anti-Rabbit IgG (H+L) Life technologies A11008
Alexa Fluor 594 Goat Anti-Mouse IgG (H+L) Life technologies A11005
Alexa Fluor Goat Anti-Rat IgM Antibody Life technologies A21248
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) Thermo Fisher Scientific D1306
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Tags

AutophagyMuscle Satellite CellsSkeletal Muscle RegenerationImmunofluorescent StainingCardiotoxinChloroquineLC3Cryosection
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Castagnetti, F., Fiacco, E., Imbriano, C., Latella, L. In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells. J. Vis. Exp. (124), e55908, doi:10.3791/55908 (2017).
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