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Biologie

技术诱导和量化细胞衰老

Published: May 01, 2017
doi:
10.3791/55533
,
1Laboratory of Epidemiology and Population Science,National Institute on Aging, National Institutes of Health

Summary

细胞衰老，细胞周期停滞的不可逆状态，可通过各种细胞应激来诱导。在这里，我们描述的协议来诱导细胞衰老和方法来评估衰老的标志。

Abstract

响应于细胞应激或损害，增殖细胞可诱导特定的程序发起的长期细胞周期停滞的状态，称为细胞衰老。衰老细胞积累的有机体衰老和通过体外培养持续发生。衰老细胞中影响许多生物过程，包括胚胎发育，组织修复和再生，肿瘤抑制和老化。衰老细胞的标志包括，但不限于，增加的衰老相关β半乳糖苷酶的活性（SA-β-GAL）; P16 INK4A和p53和p21的水平;更高水平的DNA损伤，包括γ-H2AX;衰老相关异染色质灶（SAHF）的形成;和获取一个衰老相关分泌表型（SASP），这种现象的特征在于数促炎细胞因子和信号分子的分泌的。在这里，我们描述协议都复制和DNA损伤诱导的衰老中培养的细胞。另外，我们强调技术使用几种衰老相关的标志物，包括SA-β-GAL，γ-H2AX和SAHF染色监测衰老表型，并量化细胞周期调控和SASP因子蛋白和mRNA水平。这些方法可以适用于衰老的各种型号和组织的评估。

Introduction

半个多世纪前，海弗利克和他的同事描述了细胞是如何在未转化增殖的文化，但只是时间1有限的时期。人成纤维细胞的长期培养导致细胞增殖停止;然而，他们是代谢活跃，这被称为细胞衰老。衰老可用于抑制肿瘤发生是有利的，但它也可能是有害的，因为它被认为有助于其与衰老2,3发生的再生能力的损失。衰老细胞已被证明在组织中积累作为人类4年龄和在许多生物过程，包括胚胎发育，伤口愈合，组织修复，和年龄相关的炎症2的有牵连。

在培养细胞的持续传代引起复制性衰老，这已链接以端粒耗损和基因组不稳定性。各种细胞的应力，包括DNA损伤和癌基因，也可引起衰老3。由除端粒耗损等因素衰老经常被称为应力诱导或早衰和通常取决于P16 INK4A / Rb途径5。虽然增殖，未转化的细胞通常出现主轴在形状，衰老细胞可被识别为具有特定的特征，包括一平的，大的形态和提高衰老相关β半乳糖苷酶的活性（SA-β-GAL）（ 图1和2）。衰老细胞也积累的DNA损伤标记物，包括γ-H2AX（ 图3）6，以及潜在的衰老相关异灶（SAHF）（ 图4）7。衰老细胞中具有较高水平的细胞周期调节的，包含p 16（P16 INK4A）和/或p21蛋白和p53（ 图5）8,9。此外，最近的数据已经表明，衰老细胞可通过分泌许多促炎性细胞因子和趋化因子具有非自主效应称为衰老相关分泌表型（SASP）10。虽然这种现象SASP可以从细胞类型而有所不同，以细胞类型，一般地，它是通过增加白细胞介素-6（IL-6），IL-8，粒细胞 – 巨噬细胞集落刺激因子（GM-CSF），生长 – 证明调节的原癌基因α（GRO-α），和GRO-β，等等（ 图6）。诱导衰老的特定应力或损坏还可能影响分泌表型11，12，13。 SASP可以通过测量使用的ELISA或细胞因子/蛋白质阵列分泌的蛋白质的水平来检测S = “外部参照”> 10，14。尽管转录后的机制可以调节SASP蛋白水平11，15，16，17，在mRNA水平的变化也可以在许多情况下检测。这些变化通常更敏感，更容易比蛋白质水平的测量来量化。其它衰老标记物也可以被评估，包括持久性DNA损伤核灶，与染色质的改变称为DNA片段增强衰老（DNA-SCARS）18，以及各种其它的标记3，19，20。

在这里，我们将介绍常见的技术在培养细胞诱导的衰老，也为测量衰老的几个标志物，包括SA-β-Gal的，γ-H2AX，SAHF和衰老的蛋白和mRNANCE相关分子。

Protocol

1.诱导复制性衰老解冻低传代的人二倍体成纤维细胞（ 例如，WI-38和IMR-90）或其他细胞系。 注意：在此，人二倍体成纤维细胞使用，但这些协议可以用来评估在其它细胞类型，例如内皮细胞，上皮细胞，或间充质干细胞衰老。培养条件可对用于由于不同的生长速率或生长条件的不同的细胞类型进行优化。 注：细胞应是增殖和具有主轴的形态（参见图1的示意图?…

Representative Results

图2 -图6示出从SA-β-gal染色的代表性结果;染色γ-H2AX和SAHF; P16 INK4A，P21和p53的蛋白水平的评估;和mRNA和衰老相关分子的蛋白水平。增加SA-β-gal染色以复制和DNA损伤诱导的衰老发生。此外，观察与衰老发生形态变化。相比增殖的成纤维细胞的主轴外观细胞成为扩大且平坦。对于此实验范例，细胞染色和RNA /蛋白质被暴露于IR辐射之后，分离7天。更健壮的结?…

Discussion

在这里，我们已经描述了使用人二倍体成纤维细胞用于复制和DNA损伤诱导的衰老的方法。此外，用于定量蛋白质和多种衰老相关蛋白的mRNA水平的技术包括，以及染色SA-β-gal和对DNA损伤标记物γ-H2AX。这些协议可广泛用于评估在体外和体内表型衰老，虽然用于体内 20表征衰老存在许多警告。其他细胞可表达衰老标记，即使它们不衰老细胞。例如，细胞如破骨细?…

Divulgations

The authors have nothing to disclose.

Acknowledgements

这项研究是由美国国立卫生研究院老龄研究所院内研究计划的支持。作者要感谢迈娅姆·戈罗斯佩和寇柏Abdelmohsen约衰老和寇柏Abdelmohsen的批判也读手稿许多有益的讨论。我们也感谢我们的实验室成员，特别是道格拉斯·德卢岑批判地阅读手稿。

Materials

16% Tris-glycine gels Invitrogen XP00160BOX
Acid-Phenol ChCl3 Ambion AM9720
Alexa-Fluor 568 goat anti-mouse antibody Invitrogen A11031 1:300 dilution
Cell lifters Corning Inc. 3008 Cell scraper
ECL anti-mouse HRP linked antibody Amersham NA931V
ECL Plus Western Blotting Substrate Pierce 32132 ECL
DAPI Molecular Probes MP01306 stock 5 mg/ml in dH2O
GAPDH antibody Santa Cruz sc-32233 1:1,000-5,000 dilution
GlycoBlue Ambion AM9515
Histone H3 dimethyl K9 monoclonal antibody Abcam 1220 1:500 dilution
Human IL-6 Quantikine ELISA assay R&D systems D6050
Human IL-8 Quantikine ELISA assay R&D systems D8000C
Human GROa Quantikine ELISA assay R&D systems DRG00
N-N-dimethylformamide  Sigma D4551 DMF
p16 monoclonal antibody BD Biosciences 51-1325gr 1:500 dilution
p21 monoclonal antibody Millipore 05-345 1:750 dilution
p53 monoclonal antibody Santa Cruz sc-126 1:500 dilution clone DO-1
phospho-H2AX (Ser139) FITC conjugate antibody Cell Signaling 9719 1:2000 dilution
POWER SYBR-green PCR master mix  Applied Biosystems 4367659
Pre-stained molecular weight markers Biorad 161-0374
ProLong Gold Antifade  Invitrogen P36930
PVDF membrane  Thermo Scientific 88518
Senescence b-Galactosidase Staining Kit Cell Signaling 9860
TRIzol Ambion/Life Tech 10296028
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Tags

Cellular SenescenceSenescence InductionSenescence QuantificationGamma IrradiationSenescence-associated Beta-galactosidase AssayCell CultureMicroscopyImage Analysis
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Noren Hooten, N., Evans, M. K. Techniques to Induce and Quantify Cellular Senescence. J. Vis. Exp. (123), e55533, doi:10.3791/55533 (2017).
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