人体衰老的生物标志及特征

编译：王紫慧   

审校：李瑾

【摘要】衰老的特点是生理完整性的渐进性丧失，进而导致功能的损伤和死亡风险的增加。衰老是包括恶性肿瘤、糖尿病、心血管疾病和神经退行性疾病等人类主要疾病的首要危险因子。近年来，衰老研究取得了空前的进展，尤其是发现了进化保守的遗传途径和生化过程在一定程度上可以控制衰老的速度。本篇综述试图将不同生物体，尤其是哺乳动物的衰老归结为九项共性的特征标志。这些特征包括：基因组不稳定性（genomic instability），端粒缩短（telomere attrition），表观遗传学改变（epigenetic alterations），蛋白内稳态丧失（loss of proteostasis），营养素感应失调（deregulated nutrient sensing），线粒体功能异常（mitochondrial dysfunction），细胞衰老（cellular senescence），干细胞耗竭（stem cell exhaustion）和细胞间信息通讯改变（altered intercellular communication）。研究所面临的重大挑战是揭示上述特征之间的关联以及各自对衰老的贡献，最终目标为确定药物靶点，以提高人类衰老进程中的健康水平并最大程度地避免副作用。

【关键词】衰老；恶性肿瘤；DNA损伤；表观遗传；健康寿命；长寿；代谢；线粒体；端粒

广义地讲，衰老可被定义为大多数生物体均可发生的增龄性功能减退。在人类历史进程中衰老总是会激发人们的好奇和联想。30多年前（1983年），对秀丽隐杆线虫首个长寿品系的分离(Klass, 1983)，开启了衰老研究的新纪元。如今，基于对生命和疾病的分子及细胞基础的全面认识，衰老现象正经受着全面的科学审视。当前的衰老研究和数十年以来肿瘤方面的研究有着诸多相似之处。2000年一篇里程碑式的文献使肿瘤研究领域获得了强劲的动力，该文献归纳了肿瘤的六项特征（Hanahan and Weinberg, 2000），近期扩展为十项（Hanahan and Weinberg, 2011）。通过归纳，概念化地呈现了肿瘤的本质及其深层机制。

初看肿瘤和衰老似乎是对立的过程：肿瘤是细胞适应性异常的结果，而衰老则以细胞适应性丧失为特征。而深入来看肿瘤和衰老具有某些共同的根源。细胞损伤的增龄性蓄积是普遍认为的衰老原因（Gems and Partridge, 2013; Kirkwood, 2005; Vijg and Campisi, 2008），而细胞损伤又为特定细胞的癌变提供有利条件，并最终导致肿瘤的发生。因此，肿瘤和衰老可被看作是同一基础过程，即细胞损伤蓄积的两种不同表现。另外，诸如动脉粥样硬化、炎症等衰老相关病变，还涉及到细胞失控性过度生长或功能活跃（Blagosklonny, 2008）。基于上述概念框架，衰老领域应关注一系列关于衰老损伤生理性机制的关键性问题；如试图重建体内稳态的代偿性反应，不同损伤和代偿反应之间的相互联系，外源性干预延缓衰老的可能性。

本文试图对衰老的细胞和分子特征予以确认和归类。本文提出的九项特征已被普遍认为能够促进衰老的进展，并可共同决定衰老的表型（图1）。考虑到衰老特征的复杂性，本文重点关注针对哺乳动物衰老的最新认识，同时也会对低等模式生物的相关研究有所提及（Gems and Partridge，2013；Kenyon，2010）。各项衰老特征应严格符合以下标准：（1）其应在正常衰老过程中显现；（2）实验对其增强后应加速衰老；（3）实验对其削弱后应能够延缓正常衰老进程并由此增加健康寿命。本文提出的九项特征均不同程度的符合这套严格的标准，具体内容将在下文一一讨论。这套标准的最后一条是最难实现的，即使将其局限于衰老的某一方面亦是如此。因此，九项特征不是全部满足“通过干预成功延缓衰老”这一条件。上述各项衰老特征之间存在广泛关联，这意味着通过实验改变某一特征，可能会影响到其它特征。

注：本图展示了衰老的九大特征：基因组不稳定性、端粒损耗、表观遗传学改变、蛋白质稳态丧失、营养物感应失调、线粒体功能障碍、细胞衰老、干细胞耗竭和细胞间信息交换改变。

图1. 人体衰老标志物

一、基因组不稳定性

衰老的一个共同点是贯穿生命过程中的基因损伤累积（Moskalev et al., 2012）（图2A）。多种早老性疾病，如Werner综合征和Bloom综合征，均由DNA损伤增加所致（Burtner and Kennedy，2010），但是上述疾病及其他早老综合征与正常衰老之间的相关性尚未阐明，部分原因在于这些疾病仅能概括衰老的某些方面。外源性因素（如物理、化学和生物因子）和内源性因素，如DNA复制错误、自发性分解反应、活性氧（ROS）（Hoeijmakers，2009）等均可破坏DNA的完整性和稳定性。外源性或内源性损害造成的基因损伤类型各异，包括可由各种病毒或转座子共同作用所导致的点突变、易位、染色体获得或缺失、端粒缩短和基因断裂等。为尽可能减少上述损伤，机体DNA修复机制进化形成一个复杂网络，能够协同对抗针对细胞核DNA的大多数损害（Lord and Ashworth，2012）。基因组稳定系统还包括某些特殊机制，能够使端粒保持适当长度和功能（端粒与衰老的另一特征有关，见下文）以及确保线粒体DNA的完整性（Blackburn et al., 2006；Kazak et al., 2012）。除外DNA的直接损伤，细胞核结构的缺陷，即核纤层蛋白病，亦可引发基因组不稳定性并导致早老综合征（Worman，2012）。

1. 细胞核DNA：老年人和老年模式生物的细胞均会表现出体细胞突变的累积（Moskalev et al., 2012）。而其他类型的DNA损伤，如染色体非整倍体和拷贝数变异等现象亦被发现与衰老相关（Faggioli et al., 2012；Forsberg et al., 2012）。还有报道，大型染色体异常中的克隆镶嵌现象亦有所增加（Jacobs et al., 2012；Laurie et al., 2012）。上述各种类型的DNA改变均可影响到基本基因及其转录途径而导致细胞功能紊乱，若未通过凋亡或衰老而被清除，则会危及组织和机体的稳态。尤其当DNA损伤影响到干细胞的特定功能时，则会对组织再生造成影响（Jones and Rando，2011；Rossi et al., 2008）（见干细胞耗竭部分）。小鼠和人的研究发现生命过程中基因组损伤的增加与衰老呈现因果关系，表明DNA修复机制的缺陷会导致小鼠衰老加速，以及人类的多种早老综合征的发病，如Werner综合征、Bloom综合征、着色性干皮病、毛发硫营养不良、Cockayne综合征、Seckel综合征等（Gregg et al., 2012；Hoeijmakers，2009；Murga et al., 2009）。另外，在转基因小鼠中过表达BubR1（一种确保染色体精确分离的有丝分裂检查点组分），可增强对抗染色体非整倍性和恶性肿瘤的防御能力，并延长健康寿命（Baker et al., 2013）。这些发现提供了通过人为加强核DNA修复机制可以延缓衰老进程的实验证据。

2. 线粒体DNA：线粒体DNA（mtDNA）的突变和缺失也可促进衰老（Park and Larsson，2011），mtDNA被认为是衰老相关体细胞突变的主要靶点。原因在于线粒体的氧化微环境，mtDNA缺乏组蛋白的保护，而且与核DNA相比，mtDNA修复效率低下（Linnane et al., 1989）。由于线粒体基因组具有多重性，同一细胞中可并存突变型和野生型基因组，这一现象称为“异质性”。关于衰老mtDNA突变的因果关系的推测尚存争议。单细胞分析显示，尽管mtDNA突变的总体水平较低，但个体的衰老细胞突变负荷较显著，形成以突变基因组为主并达到同质性状态（Khrapko et al., 1999）。有趣的是与早期的预测相反，成年或老年细胞中多数mtDNA突变是由生命早期的复制错误所致，而非氧化损伤。这些突变会发生多克隆增殖，导致不同组织出现呼吸链功能障碍（Ameur et al., 2011）。HIV感染者经抗逆转录病毒药物（可干扰mtDNA复制）治疗后，会出现衰老加速，这支持了生命早期mtDNA突变的多克隆增殖加速衰老的观点（Payne et al., 2011）。

mtDNA损伤与衰老及增龄性疾病相关的最早证据来自mtDNA突变所致人类多系统疾病可模拟某些衰老表型（Wallace，2005）。更深入的因果关系证据来自于对mtDNA聚合酶γ缺陷小鼠的研究。这种突变小鼠有多种早老表现且寿命缩短，这与mtDNA随机点突变和缺失的累积有关（Kujoth et al., 2005；Trifunovic et al., 2004；Vermulst et al., 2008）。来源于此小鼠的细胞表现为线粒体功能受损，但出乎意料的是，并没有伴有ROS生成增加（Edgar et al., 2009；Hiona et al., 2010）。此外，这种早老症小鼠的干细胞对mtDNA突变的累积异常敏感（Ahlqvist et al., 2012）（见干细胞耗竭部分）。需要深入研究的是能否通过基因操作降低mtDNA突变而延长寿命。

3. 细胞核结构：除了涉及到细胞核及线粒体DNA的基因损伤外，核纤层缺陷也会导致基因组不稳定（Dechat et al., 2008）。核纤层蛋白是核纤层的主要成分，且可通过充当“脚手架”以牵曳染色质和蛋白复合物参与到基因组维护中（Gonzalez-Suarez et al., 2009；Liu et al., 2005）。核纤层中编码蛋白组分的基因突变及影响其成熟和动力学的因子可导致衰老综合征如Hutchinson-Gilford早老综合征和Néstor-Guillermo早老综合征（（HGPS 和NGPS）（Cabanillas et al., 2011；De Sandre-Giovannoli et al., 2003；Eriksson et al., 2003）），这一发现引起了衰老研究人员对核纤层的注意。在人类正常衰老过程中，亦可发现核纤层改变和早老素（突变核纤层蛋白前体A亚型）生成（Ragnauth et al., 2010；Scaffidi and Misteli，2006）。正常人成纤维细胞经体外长期培养，其端粒功能障碍亦可促进早老素生成。这提示在正常衰老过程中，端粒维护与早老素表达之间存在着密切关联（Cao et al., 2011）。不仅存在核纤层蛋白A的增龄性改变，核纤层蛋白B1水平亦随细胞衰老而降低，提示其可作为衰老过程中的生物标志物（Freund et al., 2012；Shimi et al., 2011）。

通过动物和细胞模型，已明确HGPS（早老症）特征性的核纤层异常可激活多条应激通路。这些通路包括p53激活（Varela et al., 2005）、生长轴失调（Mariño et al., 2010）以及成体干细胞损耗（Espada et al., 2008；Scaffidi and Misteli，2008）。观察发现，降低HGPS模型小鼠核纤层蛋白前体A或早老蛋白水平，可延缓早老症状的出现并延长寿命，这支持了核纤层异常与过早衰老之间的因果关系。而通过系统性注射反义寡核苷酸、法尼基转移酶抑制剂，或合用他汀类与氨基双膦酸盐（Osorio et al., 2011；Varela et al., 2008；Yang et al., 2006），也可取得上述效果。通过激素治疗恢复生长轴功能，或抑制NF-κB信号通路，亦可延长早老小鼠的寿命（Mariño et al., 2010; Osorio et al., 2012）。此外，对取自HGPS患者的诱导多能干细胞，采用同源重组策略可纠正其核纤层蛋白突变，这为今后开展细胞疗法开辟了道路（Liu et al., 2011b）。而能否通过强化核结构来延缓正常衰老，尚有待在今后研究中予以验证。

4. 小结：大量证据显示衰老过程伴随着基因组损伤，而人为诱导基因组损伤则会导致某些衰老加速。机体存在确保染色体准确分离的机制，遗传学证据显示，加强这种机制能够延长哺乳动物寿命（Baker et al., 2013）。一些特殊的早老症与核结构缺陷相关，而相关治疗可延缓此类早老症。寻找干预措施以加强细胞核和线粒体基因组的稳定性（如DNA修复）以及它们对正常衰老的影响，也是需要研究的方向（端粒问题较为特殊，以下单独讨论）。

二、端粒缩短

DNA损伤的增龄性累积对基因组的影响似乎是随机的，但在染色体的某些区域（如端粒）则特别容易发生增龄性损害（Blackburn et al., 2006）（图2A）。复制性DNA聚合酶不具备完全复制线性DNA分子末端的能力，而一种特异的DNA聚合酶（即端粒酶）则具备这种功能。然而，多数哺乳动物的体细胞不表达端粒酶，这导致了染色体末端端粒保护序列进行性和累积性的丧失。端粒缩短解释了某些类型的离体培养细胞增殖能力有限的原因，这种现象称为复制性衰老，或称为Hayflick极限（Hayflick and Moorhead，1961；Olovnikov, 1996）。事实上，通过异位表达端粒酶可有效实现普通细胞永生化，且不会发生致癌性转化（Bodnar et al., 1998）。重要的是端粒缩短在人类和小鼠的正常衰老过程中也可发现（Blasco，2007a）。

端粒缩短可以被视为躲避了DNA修复机制的DNA断裂（Palm and de Lange，2008）。端粒通过形成具有特异性保护作用的端粒蛋白复合体躲避DNA修复机制。端粒的这一特殊性使端粒不仅在没有端粒酶的情况下逐渐缩短，即使在端粒酶存在的情况下，由于端粒蛋白复合体的存在，外源性DNA损伤造成的端粒破坏也会逃避DNA修复机制。因此，端粒产生持久的DNA损伤会导致包括衰老和/或凋亡在内的有害的细胞效应（Fumagalli et al., 2012；Hewitt et al., 2012）。

人类端粒酶缺陷与某些疾病的过早发生相关，如肺纤维化、先天性角化不良、再生障碍性贫血等，这些疾病均涉及到不同组织再生能力的丧失（Armanios and Blackburn，2012）。而端粒蛋白复合体组分的缺陷也会导致严重的端粒脱帽现象（Palm and de Lange，2008）。端粒特异性保护蛋白复合物突变在某些再生障碍性贫血和先天性角化不良病例中亦有发现（Savage et al., 2008；Walne et al., 2008；Zhong et al., 2011）。多项模型研究显示，若端粒蛋白复合体各组分功能丧失，其组织再生能力下降且衰老加速，即使端粒仍处于正常长度这一现象依然存在（Martínez and Blasco，2010）。

通过基因修饰动物模型可在端粒丧失与细胞衰老、机体衰老之间建立因果联系。端粒缩短或延长的小鼠分别表现为寿命缩短或延长（Armanios et al., 2009；Blasco et al., 1997；Herrera et al., 1999；Rudolph et al., 1999；Tomás-Loba et al., 2008）。近期证据亦显示，通过激活端粒酶可逆转衰老，特别是针对端粒酶缺陷的小鼠。在其老年阶段采用基因手段重新激活端粒酶，则该小鼠的早老症状能够逆转（Jaskelioff et al., 2011）。此外，通过对成年野生型小鼠端粒酶采用药理激活或系统性病毒转导可延缓其正常生理性衰老，且肿瘤发病率未见增加（Bernardes de Jesus et al., 2012）。近期荟萃分析结果亦表明人类端粒缩短与死亡风险有强相关性，且在较年轻个体尤为显著（Boonekamp et al., 2013）。

小结：哺乳动物的正常衰老过程伴随着端粒缩短。并且，病理性端粒功能障碍会加速小鼠和人类的衰老。通过实验性刺激端粒酶则能延缓小鼠衰老。因此，端粒缩短完全符合衰老特征的认定标准。

三、表观遗传改变

各种表观遗传学改变会终生影响到所有的细胞和组织（（Talens et al., 2012）（图2B）。表观遗传学改变包括DNA甲基化模式改变、组蛋白翻译后修饰以及染色质重塑。而H4K16乙酰化、H4K20三甲基化和H3K4三甲基化程度增加，以及H3K9甲基化、H3K27三甲基化程度降低，构成了表观遗传学的增龄性标志（Fraga and Esteller，2007；Han and Brunet，2012）。多种酶类系统能够确保表观遗传模式的形成和维护，如DNA甲基转移酶、组蛋白乙酰化酶、去乙酰化酶、甲基化酶、去甲基化酶以及染色体重塑相关蛋白复合体。

1. 组蛋白修饰：在无脊椎动物中，组蛋白甲基化符合衰老特征的认定标准。组蛋白甲基化复合物组分的缺失可延长线虫和果蝇的寿命（Greer et al., 2010；Siebold et al., 2010）。另外，组蛋白去甲基化酶通过靶向关键长寿通路，如胰岛素/胰岛素样生长因子-1信号通路来调节寿命（Jin et al., 2011）。目前尚不明确的是组蛋白修饰酶调控影响衰老的表观遗传学机制是通过影响DNA修复和基因组稳定性，还是通过影响细胞核外代谢或信号通路转录。

它与NAD依赖型去乙酰化酶和ADP核糖基转移酶相关的sirtuin（沉默信息调节因子2相关酶）家族成员已被广泛认为是潜在的抗衰老因子。人们对sirtuin蛋白家族与衰老关系的兴趣源自于一系列针对酵母、果蝇和蠕虫的研究报告。报告显示，上述生物体中唯一的sirtuin基因Sir2具有显著的长寿活性（Guarente， 2011）。最初发现，酿酒酵母过表达Sir2，复制型酵母寿命会延长（Kaeberlein et al., 1999）。之后的报告提示分别过表达Sir2在蠕虫（sir-2.1）和果蝇（dSir2）的直系同源基因，亦可延长这两种无脊椎模式生物的寿命（Rogina and Helfand，2004；Tissenbaum and Guarente，2001）。不过，近期对上述研究又出现质疑，报告认为蠕虫和果蝇研究所见寿命延长，很大程度上是由于其复杂的遗传背景差异，而与过表达sir-2.1或dSir2无关（Burnett et al., 2011）。实际上，经过细致的再次评估发现，过表达sir-2.1只能中等程度延长线虫寿命（Viswanathan and Guarente，2011）。

多项研究显示，哺乳动物的7种sirtuin同源基因中有数种亦可延缓小鼠衰老的多项参数（Houtkooper et al., 2012；Sebastián et al., 2012）。具体来看，哺乳动物转基因过表达SIRT1（为最接近无脊椎动物Sir2的同源基因），可提高衰老过程中各方面的健康水平，但并不能延长寿命（Herranz et al., 2010）。然而，SIRT1良性效应的产生机制较为复杂且各机制间相互关联，包括从提高基因组稳定性（Oberdoerffer et al., 2008；Wang et al., 2008）到增强代谢效率的广泛细胞作用（Nogueiras et al., 2012）（见营养素感应失调）。而在哺乳动物中，支持sirtuin介导促长寿效应的更有力证据来自于对SIRT6的研究，其可通过组蛋白H3K9去乙酰化来调节基因组稳定性、NF-κB信号通路和血糖稳态（Kanfi et al., 2010；Kawahara et al., 2009；Zhong et al., 2010）。SIRT6缺陷的突变小鼠表现为衰老加速（Mostoslavsky et al., 2006），而过表达SIRT6的转基因雄性小鼠较对照组而言寿命更长，这与降低血清IGF-1以及IGF-1信号通路其他指标有关（Kanfi et al., 2012）。有趣的是，位于线粒体的sirtuin蛋白SIRT3亦可介导饮食限制延长寿命的某些良性效应，不过这些效应并非因为组蛋白修饰，而是因为线粒体蛋白的去乙酰化（Someya et al., 2010）。最新报道显示，衰老的造血干细胞过表达SIRT3可逆转其再生能力（Brown et al., 2013）。因此，哺乳动物sirtuin家族至少有三个成员，即SIRT1、SIRT3和SIRT6有利于健康老龄化。

注：（A）基因组不稳定性和端粒缩短。所示为单个染色体受到内源性或外源性因子损伤所导致的多种DNA损伤。这些损伤可通过多种机制予以修复。而过度DNA损伤及DNA修复不足可促进衰老进程。需注意的是，细胞核及线粒体DNA（图中未展示）均会发生增龄性基因组改变（Vijg，2007）。

（B）表观遗传学改变。DNA和组蛋白的甲基化、乙酰化及其它染色质相关蛋白的改变均可诱导表观遗传学改变，从而促进衰老进程。

图2. 基因组和表观遗传学改变

2. DNA甲基化：DNA甲基化与衰老之间的关系较为复杂。早期研究描述了衰老与总体低甲基化相关，但随后的分析发现，包括对应于多种肿瘤抑制基因和多梳蛋白靶基因的多个基因座，实际上会随着增龄而高甲基化（Maegawa et al., 2010）。早老综合征患者细胞及小鼠细胞所显示的DNA甲基化和组蛋白修饰模式，在很大程度上能够概括正常衰老细胞的表现（Osorio et al., 2010；Shumaker et al., 2006）。而所有增龄过程中出现的表观遗传学缺陷或突变会影响到干细胞的行为和功能（Pollina and Brunet，2011）（见干细胞耗竭部分）。然而，目前尚无直接实验证据显示通过改变DNA甲基化模式能够延长生物体寿命。

3. 染色质重塑：与DNA和组蛋白修饰酶相配合，染色体的关键蛋白（如异染色质蛋白1α，HP1α）和染色质重塑因子（如多梳蛋白家族或NuRD复合体）的水平，在正常衰老和病理性衰老的细胞中均降低（Pegoraro et al., 2009；Pollina and Brunet，2011）。这与前述的表观遗传修饰（即组蛋白和DNA甲基化）相伴随，表观遗传因子的改变可决定染色质结构的变化（如异染色质整体缺失和再分配），这也成为了衰老的特征性表现（Oberdoerffer and Sinclair，2007；Tsurumi and Li，2012）。染色质改变与衰老的因果关系在果蝇研究中已获支持。该研究发现，HP1α功能丧失的突变果蝇寿命缩短，而过表达这种异染色质蛋白则会延长其寿命，并延缓老年期肌肉功能衰退（Larson et al., 2012）。

支持染色质表观遗传学改变与衰老之间存在功能相关性的证据还表现在DNA重复区域异染色质形成与染色体稳定性显著相关。特别是，发生在近着丝粒区的异染色质组装需要组蛋白H3K9和H4K20三甲基化，并与HP1α结合，这对染色体稳定性相当重要（Schotta et al., 2004）。对于哺乳动物，经修饰的染色质还包含丰富的端粒重复序列，这提示染色体末端可组装进入异染色质区（Blasco，2007b；Gonzalo et al., 2006）。而在亚端粒区也会显示结构性异染色质的某些特征，如H3K9和H4K20三甲基化、HP1α结合以及DNA高甲基化。因此，表观遗传学改变可直接影响到端粒长度的调节（衰老的另一特征）。此外，在DNA损伤时SIRT1和其他染色质修饰蛋白会重新定位到DNA断裂位点，从而促进修复和基因组稳定性。SIRT1除了在染色质重塑和DNA修复中的作用外，还调节蛋白质稳态，线粒体功能，营养素感应通路和炎症（见下文），这说明了衰老标志物之间存在相互联系。

4. 转录改变：衰老与转录噪音增加（Bahar et al., 2006）、mRNA异常生成和成熟有关（Harries et al., 2011；Nicholas et al., 2010）。对取自不同物种的青年和老年组织进行芯片分析比较，明确了编码炎症、线粒体及溶酶体相关降解通路的关键组分基因发生增龄性转录改变（de Magalhães et al., 2009）。这种增龄性转录特征还会影响到非编码RNA，其中一组miRNA（gero-miR，老年miRNA）与衰老进程相关，它可通过长寿网络的某些靶向组分及调节干细胞行为影响寿命（Boulias and Horvitz，2012；Toledano et al., 2012；Ugalde et al., 2011）。功能获得和缺失研究证实黑腹果蝇和秀丽隐杆线虫体内存在多种具有调节寿命功能的miRNA （Liu et al., 2012；Shen et al., 2012；Smith-Vikos and Slack，2012）。

5. 表观遗传学改变的逆转：与DNA突变不同，表观遗传学改变至少在理论上是可以逆转的。这为新型抗衰老药物的设计提供了可能性（Freije and López-Otín，2012；Rando and Chang，2012）。小鼠应用组蛋白去乙酰化酶抑制剂可恢复生理性的H4乙酰化，从而避免增龄性记忆损害的出现（Peleg et al., 2010）。这提示表观遗传学改变的逆转可能具有神经保护作用。而组蛋白乙酰转移酶抑制剂亦可减轻早老症小鼠的早老表型并延长其寿命（Krishnan et al., 2011）。近期研究发现，秀丽隐杆线虫存在长寿表观遗传学的跨代遗传现象，提示亲代的特异性染色质修饰能够诱导其后代产生长寿的表观遗传学记忆（Greer et al., 2011）。与组蛋白乙酰转移酶抑制剂的理念类似，组蛋白去乙酰化酶激活剂亦应具有延长寿命作用。白藜芦醇在衰老中多方面的作用机制已被广泛的研究探讨，其中包括上调SIRT1活性以及其他与能量缺陷相关的效应（见线粒体功能障碍）。

6. 小结：目前已有多重证据提示，衰老伴随着表观遗传学改变，而表观遗传学紊乱则可引发模式生物出现早老综合征。SIRT6作为表观遗传学相关的典型酶类，其功能丧失可缩短小鼠寿命，而功能获得则可延长小鼠寿命（Kanfi et al., 2012；Mostoslavsky et al., 2006）。总之，研究表明了解并操控表观基因组有望改善增龄性病变并延长健康寿命。

四、蛋白质稳态丧失

衰老和增龄性疾病与蛋白质稳态丧失有关（Powers et al., 2009）（图3）。所有细胞都利用一系列的质控机制以保持其蛋白质组的稳定和功能。蛋白质稳态涉及稳定正确折叠蛋白质（最主要的是热休克蛋白家族蛋白）的机制及经由蛋白酶体或溶酶体实现蛋白质降解的机制（Hartl et al., 2011；Koga et al., 2011；Mizushima et al., 2008）。而增龄性蛋白毒性的调节因子可通过不同于分子伴侣和蛋白酶的其他途径起作用，如MOAG-4（van Ham et al., 2010）。所有的机制以系统功能协调工作，以恢复错误折叠多肽的结构或完全清除及降解它们，从而阻止损伤成分的累积并确保胞内蛋白的持续更新。多项研究显示衰老过程伴随着蛋白质稳态的改变（Koga et al., 2011）。未折叠蛋白、错误折叠蛋白或蛋白聚合体的缓慢表达会发生某些增龄性病变，如阿尔茨海默病、帕金森病、白内障等的进展（Powers et al，2009）。

注：内源性和外源性应激导致蛋白质未折叠或破坏蛋白质合成过程中的正确折叠。未折叠蛋白质通常经由热休克蛋白实现重新折叠或通过泛素-蛋白酶体途径、溶酶体（自噬）途径清除。自噬途径包括蛋白伴侣介导的自噬（蛋白伴侣Hsc70识别未折叠蛋白，并将其移入溶酶体）和巨自噬（自噬体隔离受损蛋白和细胞器，随后与溶酶体融合）。若无法重新折叠或降解未折叠蛋白质则会导致这些蛋白累积及聚集，进而诱发蛋白毒性效应。

图3. 蛋白质稳态丧失

1. 蛋白伴侣介导的蛋白质折叠和稳定：在衰老过程中，应激诱导的胞质和胞器特异性蛋白伴侣的合成受到损害（Calderwood et al., 2009）。多种动物模型证实蛋白伴侣减少与寿命之间存在因果关系。特别是转基因过表达蛋白伴侣可延长蠕虫和果蝇寿命（Morrow et al., 2004；Walker and Lithgow，2003）。热休克蛋白家族某种蛋白辅助伴侣发生突变的缺陷小鼠会加速其衰老，而长寿品系小鼠则表现会为某些热休克蛋白的显著上调（Min et al., 2008；Swindell et al., 2009）。进一步发现，线虫热休克反应主要调节因子——转录因子HSF-1的激活可延长其寿命并增强耐热性（Chiang et al., 2012；Hsu et al., 2003），而在其增龄过程中，淀粉样蛋白结合组分能够维持蛋白质稳态并延长寿命（Alavez et al., 2011）。在哺乳动物细胞中，SIRT1可使HSF-1去乙酰化，从而增强热休克基因的反式激活，如Hsp70；而下调SIRT1则可减轻热休克反应（Westerheide et al., 2009）。已有多种方法可激活伴侣介导的蛋白质折叠和稳定，以维护或加强蛋白质稳态。针对肌萎缩模型小鼠，通过药物诱导热休克蛋白Hsp72，可保持其肌肉功能，并延缓肌萎缩病变发展（Gehrig et al., 2012）。在模式生物中，小分子也可作为药物性蛋白伴侣，以确保受损蛋白的再次折叠并改善增龄性表型（Calamini et al., 2012）。

2. 蛋白分解系统：在蛋白质的质量控制方面主要存在两种蛋白分解系统，即自噬-溶酶体系统，和泛素-蛋白酶体系统。两大系统的活性随年龄增长而降低（Rubinsztein et al., 2011；Tomaru et al., 2012）可支持蛋白质稳态丧失为老年阶段共同特征。

就自噬系统来言，在LAMP2a（蛋白伴侣介导自噬的受体）额外拷贝的转基因小鼠身上未见自噬活性增龄性下降，且衰老过程中肝功能保持稳定（Zhang and Cuervo，2008）。在发现持续或间歇应用mTOR抑制剂雷帕霉素可延长中年小鼠寿命后，通过化学物质诱导巨自噬（不同于蛋白伴侣介导自噬的另一类型自噬）激发起研究者的强烈兴趣（Blagosklonny，2011；Harrison et al., 2009）。值得注意的是，雷帕霉素可延缓小鼠多方面的衰老表现（Wilkinson et al., 2012）。在酵母、线虫和果蝇中，雷帕霉素的寿命延长作用严格取决于自噬的诱导（Bjedov et al., 2010；Rubinsztein et al., 2011）。然而，雷帕霉素对哺乳动物衰老的影响尚无类似证据。而其它机制，如抑制核糖体S6蛋白激酶1（S6K1，与蛋白质合成有关）（Selman et al., 2009）可能有助于解释雷帕霉素的长寿作用（见营养素感应失调部分）。与雷帕霉素相反，另一种巨自噬诱导剂——亚精胺并无免疫抑制的副作用，也可通过诱导自噬延长酵母、果蝇和蠕虫的寿命（Eisenberg et al., 2009）。同样，小鼠用含亚精胺的多胺营养补充制剂或用产多胺的肠道菌群均能够延长其寿命（Matsumoto et al., 2011；Soda et al., 2009）。线虫喂饲ω-6不饱和脂肪酸也可通过激活自噬而延长寿命（O’Rourke et al., 2013）。关于蛋白酶体系，EGF信号通路的激活可通过增加泛素-蛋白酶体系统多种组分的表达来延长线虫寿命（Liu et al., 2011a）。与此类似，通过去泛素化酶抑制剂或蛋白酶体激活剂增强蛋白酶体活性可加速人培养细胞中毒性蛋白的清除（Lee et al., 2010）并延长复制型酵母寿命（Kruegel et al., 2011）。通过FOXO转录因子DAF-16增加蛋白酶体亚基RPN-6的表达，赋予秀丽隐杆线虫蛋白毒性应激抵抗并延长其寿命（Vilchez et al., 2012）。

3. 小结：已有证据显示衰老与蛋白质稳态失调有关。实验性蛋白质稳态失调可促进增龄性病变的产生。还有一些实验表明基因操控可改善哺乳动物蛋白质稳态并延缓衰老的可能（Zhang and Cuervo，2008）。

五、营养物感应失调 

在哺乳动物中，促生长轴由生长素（GH，由垂体前叶产生）和次级调节因子胰岛素样生长因子-1（IGF-1）组成。后者在多种类型细胞，特别是肝细胞中可对GH产生应答。细胞内IGF-1信号通路与胰岛素诱发的反应类似，均可使细胞感应到葡萄糖水平。因此，IGF-1和胰岛素信号通路又合称为胰岛素/IGF-1信号（IIS）通路。值得注意的是，IIS通路是进化过程中最为保守的衰老调控通路，转录因子FOXO家族和mTOR复合体在此通路的多个靶点中，它们也与衰老相关且进化保守（Barzilai et al., 2012；Fontana et al., 2010；Kenyon，2010）。在人类和模式生物中均发现降低GH、IGF-1受体、胰岛素受体及其下游效应因子功能的基因多态性或突变与长寿有关，如AKT、mTOR、FOXO；由此可进一步凸显营养和生物能量通路对寿命的重要影响（Barzilai et al., 2012；Fontana et al., 2010；Kenyon，2010）（图4A）。它与营养物感应失调为衰老特征之一的观点相一致，饮食限制（Dietary Restriction，DR）可延长寿命或健康寿命。已研究过的生物包括几种不同门的单细胞和多细胞生物的所有真核生物物种（包括非人灵长类）（Colman et al., 2009；Fontana et al., 2010；Mattison et al., 2012）。

1. 胰岛素和IGF-1信号通路：通过多项基因操作发现，在不同水平上减弱IIS通路的信号强度均可延长蠕虫、果蝇和小鼠的寿命（Fontana et al., 2010）。遗传分析提示，蠕虫和果蝇IIS通路可部分介导饮食限制（DR）的良性效应（Fontana et al., 2010）。与蠕虫和果蝇寿命最为相关的IIS下游效应因子是转录因子FOXO（Kenyon et al., 1993；Slack et al., 2011）。在小鼠中存在4个FOXO成员，但其过表达对寿命的影响及其在介导减弱IIS通路以延长寿命中的作用均未明确。小鼠中FOXO1为DR肿瘤抑制效应的必需因子（Yamaza et al., 2010），但尚不清楚该因子是否涉及到DR介导的寿命延长效应。小鼠过表达肿瘤抑制因子PETN基因能够总体下调IIS通路并增加能量消耗，这与线粒体氧化代谢增加和棕色脂肪组织活性增强有关（Garcia-Cao et al., 2012；Ortega-Molina et al., 2012）。与其他IIS活性降低的小鼠模型一致，过表达Pten蛋白的小鼠以及表达PI3K亚效等位基因的小鼠寿命延长（Foukas et al., 2013；Ortega-Molina et al., 2012）。

矛盾的是在正常衰老过程以及小鼠早老模型中GH和IGF-1水平均降低（Schumacher et al., 2008）。由此，IIS降低成为生理性衰老和加速衰老的共性特征，然而组成性降低IIS则可延长寿命。以上研究结果从表面上看相互矛盾，但可以通过统一的模型加以解释（Garinis et al., 2008）。即在遭受系统性损伤的情况下IIS下调可作为对抗性反应，以实现最低限度的细胞生长和代谢。据此观点，机体组成性降低IIS能够存活更长时间，因为其细胞生长和代谢速度降低，由此细胞损伤速率也会更低。与此类似，生理性衰老或病理性衰老的机体，也是试图通过降低IIS以延长寿命。然而，针对衰老的防御反应最终可能会产生衰老恶化并加剧的风险（观点以下章节会提及）。因此，极低程度的IIS信号通路无法适应生存所需，一个例子是，PI3K或AKT激酶无效突变的小鼠，均具有胚胎致死性（Renner and Carnero，2009）。同样许多例子显示，给IGF-1低水平的早老症小鼠补充IGF-1可减轻其早老症状（Mariño et al., 2010）。

2. 其他营养物感应系统：mTOR、AMPK和sirtuins。除外参与葡萄糖感应的IIS通路，另有三种相互关联的营养物感应系统正成为研究的重点，即mTOR感应高浓度氨基酸；AMPK通过检测高水平AMP以感应低能量状态；sirtuins通过发现高水平NAD+以感应低能量状态（Houtkooper et al., 2010）（图4A）。

mTOR激酶存在两种多蛋白复合体，即mTORC1和mTORC2，其可基础性调节合成代谢的各个方面（Laplante and Sabatini，2012）。在酵母、蠕虫和果蝇中下调mTORC1基因活性，可延长寿命，并可进一步削弱DR的延长寿命效应，提示抑制mTOR可模拟DR的效应（Johnson et al., 2013）。应用雷帕霉素亦可延长小鼠寿命，是延长哺乳动物寿命的最强效化学干预手段（Harrison et al., 2009）。通过基因修饰降低mTORC1活性，同时保持mTORC2正常水平也可延长寿命（Lamming et al., 2012）；另外，S6K1（mTORC1主要底物）缺陷小鼠亦长寿（Selman et al., 2009）。因此，下调mTORC1/S6K1可能为mTOR相关长寿效应中的关键调节因子。另外，小鼠衰老过程中其下丘脑神经元的mTOR活性增加，从而导致增龄性肥胖；而通过下丘脑内直接注射雷帕霉素可逆转该现象（Yang et al., 2012）。这些观察结果以及与IIS途径有关的观察结果表明，由IIS和mTORC1通路介导的营养和合成代谢的活性增强是衰老的主要促进因素。虽然抑制TOR活性会对衰老过程产生明确的良性效应，但也存在某些不良的副作用，如在小鼠可见伤口愈合不良、胰岛素抵抗、白内障及睾丸退化（Wilkinson et al., 2012）。因此，重要的是通过机制研究明确抑制TOR的利弊程度并将两者区分开来。

另外，还存在两种营养物感应器，即AMPK和sirtuins。与IIS和mTOR的作用相反，这两者针对营养匮乏和分解代谢产生效应，而非针对营养过盛和合成代谢。相应地，两者上调有益于健康老龄化。激活AMPK会对代谢产生多重效应，特别是关闭mTORC1（Alers et al., 2012）。有证据显示，蠕虫和小鼠应用二甲双胍后可通过激活AMPK使其寿命延长（Anisimov et al., 2011；Mair et al., 2011；Onken and Driscoll，2010）。关于sirtuin调节寿命的作用，上文已论述（见表观遗传学改变）。另外，SIRT1亦可去乙酰化并激活PPARγ共激活因子1α（PGC-1α）（Rodgers et al., 2005）。而PGC-1α可协调一系列代谢反应，如线粒体生成、增强抗氧化防御功能、提高脂肪酸氧化作用（Fernandez-Marcos and Auwerx，2011）。此外，SIRT1与AMPK可参与到一个正反馈环中，从而将这两种低能量状态感应器联结于统一反应之中（Price et al., 2012）。

3. 小结：总之，现有的证据强烈支持合成代谢信号会加速衰老，而降低营养物信号则可延长寿命（Fontana et al., 2010）。进一步通过药物操作，如雷帕霉素模拟营养素物获取不足状态可延长小鼠寿命（Harrison et al., 2009）。

六、线粒体功能障碍

伴随细胞和机体衰老，呼吸链效率趋于降低，由此电子泄漏增加而ATP生成减少（Green et al., 2011）（图4B）。长期以来，一直有关于线粒体功能障碍与衰老之间关系的猜测，然而阐明其细节仍然是衰老研究中的重大挑战。

1. 活性氧：衰老的线粒体自由基理论认为，衰老过程中线粒体渐进性功能障碍会增加ROS的生成，而其又会进一步导致线粒体功能恶化和细胞全局损害（Harman，1965）。大量数据支持ROS在衰老中的作用，本文聚焦于近5年来的进展，这些研究正促使研究者对衰老的线粒体自由基理论予以重新评价（Hekimi et al., 2011）。一些有特定影响的非预期发现表明，酵母和秀丽隐杆线虫ROS增加反可延长其寿命（Doonan et al., 2008；Mesquita et al., 2010；Van Raamsdonk and Hekimi，2009）；通过基因操作使小鼠线粒体ROS生成和氧化损伤增加并不会加速衰老（Van Remmen et al., 2003；Zhang et al., 2009），而抗氧化防御能力增强的小鼠，则未显示寿命延长（Pérez et al., 2009）。最后，通过基因操作仅损伤线粒体功能而不增加ROS生成可加速衰老（Edgar et al., 2009；Hiona et al., 2010；Kujoth et al., 2005；Trifunovic et al., 2004；Vermulst et al., 2008）。上述及相似资料为重新探讨ROS在衰老中的作用铺平了道路（Ristow and Schmeisser，2011）。实际上，平行且独立于ROS损伤效应的研究，在胞内信号转导领域已积累的有力证据证实，ROS可启动细胞增殖和生存反应，以应对生理信号和应激状态（Sena and Chandel，2012）。以上两方面的证据可统一描述为ROS作为应激诱导的生存信号，旨在代偿衰老相关的进行性衰退。伴随生物学年龄的增长，ROS水平增加以试图保持细胞生存；而一旦超过某一阈值，将会加剧而非减轻增龄性损伤（Hekimi et al., 2011）。由此这一概念框架应可容纳ROS对衰老“正面效应”、“负面效应”或“中性效应”等相互矛盾的证据。

2. 线粒体完整性及其生物合成：正如针对DNA聚合酶γ缺陷小鼠的研究所见，线粒体功能障碍可单独促进衰老（独立于ROS）（Edgar et al., 2009；Hiona et al., 2010）（见基因组不稳定性）。线粒体功能障碍发生机制涉及多个方面：如线粒体缺陷可致应激反应时膜渗漏性增加，从而影响细胞凋亡的信号转导或是启动炎性反应（Kroemer et al., 2007）；后者通过ROS介导和/或炎症小体促渗透性活化作用（Green et al., 2011）。另外，通过影响线粒体外膜与内质网之间的线粒体相关膜面，线粒体功能障碍可直接影响细胞信号转导和细胞器之间相互应答（Raffaello and Rizzuto，2011）。 

衰老过程中，线粒体生物能量的生成效率降低可能是由多重机制导致的，其中机制之一是线粒体的生物合成减少，如端粒酶缺陷小鼠的端粒损耗表现及继发的p53介导的PGC-1α和PGC-1β抑制（Sahin and DePinho，2012）。在野生型小鼠的生理性衰老过程中，亦可发现其线粒体衰减，而通过激活端粒酶则可部分逆转（Bernardes de Jesus et al., 2012）。而SIRT1通过影响与转录共激活因子PGC-1α（Rodgers et al., 2005）和自噬去除受损线粒体的过程来调节线粒体的生物合成（Lee et al., 2008）。SIRT3是主要的线粒体去乙酰化酶（Lombard et al., 2007），与能量代谢的多种酶发生作用，包括呼吸链、三羧酸循环、酮体生成和脂肪酸β-氧化途径的成分（Giralt and Villarroya，2012）。SIRT3也可通过去乙酰化锰超氧化物歧化酶（线粒体主要抗氧化酶）直接控制ROS的生成速率（Qiu et al., 2010；Tao et al., 2010）。上述结果可支持以下观点即sirtuins可作为代谢传感器控制线粒体功能，从而发挥对抗增龄性疾病的作用。其他导致线粒体生物能量不足的机制还包括mtDNA突变和缺失的累积、线粒体蛋白氧化、呼吸链（超级）复合体大分子组装失稳、线粒体膜脂质成分改变、分裂和融合失衡所致线粒体动力学改变、线粒体自噬（靶向缺陷线粒体、降解蛋白质的细胞器特异型巨自噬）质控缺陷等（Wang and Klionsky，2011）。线粒体生物合成及清除率减低会导致其损伤累积而更新降低，这些都可能共同促进衰老进程（图4B）。有趣的是耐力训练和隔日禁食可能通过避免线粒体衰退，从而改善健康寿命（Castello et al., 2011；Safdar et al., 2011）。推测上述良性效应的原因在于耐力训练和禁食可作为强有力的触发因素诱导自噬的发生，至少在部分程度上（Rubinsztein et al., 2011）。当然，自噬诱导可能并非健康生活方式延缓衰老的唯一机制，因为通过精细的DR方案，其他长寿通路亦可能被激活（Kenyon，2010）。

注：（A）营养物感应失调。本图展示了促生长轴包括生长激素（GH）和胰岛素/胰岛素生长因子1（IGF-1）信号通路，以及其与饮食限制和衰老的联系。图中促衰老分子标为橙色，抗衰老分子标为浅绿色。（B）线粒体功能障碍。线粒体功能障碍源于增龄性mtDNA突变、线粒体生成减少、电子传递链（ETC）复合物失稳、线粒体动力学改变及质量控制（通过线粒体自噬实现）缺陷等。应激信号和线粒体功能缺陷会产生ROS，若ROS低于特定阈值，则会诱发生存信号以恢复细胞稳态；若ROS高于特定阈值或持续存在，则会促进衰老。与此类似，线粒体轻度损伤会诱发微应激效应（称为线粒体微应激效应）并启动适应性代偿反应。

图4. 代谢改变

3. 线粒体微应激效应：衰老过程中发生的线粒体功能障碍也与线粒体微应激效应有关，近期已有多项衰老研究聚焦于这一概念（Calabrese et al., 2011）。根据这一观点，经少量毒物处理后会促发良性代偿反应，并强于其诱发损伤后的修复反应，这样与损伤开始前状态相比，最终的细胞适应性反而更优。因此，虽然严重的线粒体功能障碍是致病性的，但轻微的线粒体呼吸缺陷则可延长寿命，这可能是因为微应激效应（Haigis and Yankner，2010）。微应激效应可能启动线粒体应激反应，在秀丽隐杆线虫中，该反应既可见于有线粒体缺陷的同一组织，也可见于较远组织（Durieux et al., 2011）。有力的证据显示某些复合物（如二甲双胍、白藜芦醇）具有轻微的线粒体毒性，能够通过增加AMP水平和激活APMK而诱导低能量状态（Hawley et al., 2010）。重要的是二甲双胍通过诱导代偿性应激反应而延长秀丽隐杆线虫寿命，是经由AMPK和抗氧化主要调节因子NRF2介导的（Onken and Driscoll，2010）。近期研究还显示，二甲双胍延缓可通过破坏肠道微生物组叶酸和甲硫氨酸的代谢来延缓蠕虫衰老（Cabreiro et al., 2013）。而就哺乳动物来讲，自生命早期开始用二甲双胍干预，亦可延长小鼠寿命（Anisimov et al., 2011）。而对于白藜芦醇以及sirtuin激活剂SRT1720，有确切证据显示二者可通过依赖于PGC-1α的方式保护免受代谢损伤并改善线粒体呼吸（Baur et al., 2006；Feige et al., 2008； Lagouge et al., 2006；Minor et al., 2011），不过白藜芦醇在正常饮食情况下并不能延长小鼠寿命（Pearson et al., 2008；Strong et al., 2013）。有报道指出PGC-1α过表达延长果蝇寿命与提高线粒体活性有关，该研究进一步支持了PGC-1α在衰老中的作用（Rera et al., 2011）。最后，无论是基因过表达线粒体解偶联蛋白UCP1，还是应用化学解偶联剂2，4-二硝基苯酚所导致的线粒体解偶联均可延长果蝇和小鼠寿命（Caldeira da Silva et al., 2008；Fridell et al., 2009；Gates et al., 2007；Mookerjee et al., 2010）。

4. 小结：线粒体功能对衰老进程有着复杂的影响。线粒体功能障碍可加速哺乳动物衰老（Kujoth et al., 2005；Trifunovic et al., 2004；Vermulst et al., 2008），尽管已有建设性证据存在，但尚不明确是否可通过改善线粒体功能（如通过线粒体微应激效应）来延长哺乳动物寿命。

七、细胞衰老

细胞衰老可定义为稳定的细胞周期停滞，并伴随表型的固化（Campisi and d’Adda di Fagagna，2007；Collado et al., 2007；Kuilman et al., 2010）（图5A）。该现象最早由海弗利克（Hayflick）对人成纤维细胞进行连续传代培养时加以描述（Hayflick and Moorhead，1961）。现在已知海弗利克所观察到的细胞衰老现象是由端粒缩短所致（Bodnar et al., 1998），但是还有其他衰老相关刺激可诱发衰老，且独立于上述端粒过程。最值得注意的是，非端粒性DNA损伤和INK4/ARF位点脱抑制（两者均随年龄增长而渐进性发生）也都能够诱发衰老（Collado et al., 2007）。衰老组织中衰老细胞的累积程度通常可通过替代性指标，如DNA损伤来推断。某些研究直接采用衰老相关β-半乳糖苷酶（SABG）来判定组织衰老（Dimri et al., 1995）。值得注意的是，通过对小鼠肝脏SABG和DNA损伤进行精细的量化平行分析，所获取的比较数据显示，青年小鼠肝脏中衰老细胞所占比例约为8%，而在老年小鼠约为17% （Wang et al., 2009）。在小鼠的皮肤、肺脏和脾脏亦有类似结果，但在其心脏、骨骼肌和肾脏（Wang et al., 2009）中则未见类似改变。基于这些数据可知，对于老年生物体而言，细胞衰老并非所有组织的普遍特征。有证据表明，衰老的肿瘤细胞受到严格的免疫监视，并可通过细胞吞噬作用而被有效清除（Hoenicke and Zender，2012；Kang et al., 2011；Xue et al., 2007）。可以确定的是衰老过程中衰老细胞的累积能够反映出衰老细胞生成速率增加和/或其清除速度降低，例如免疫应答减弱。 

正是由于衰老细胞随机体衰老而增加，因此普遍认为细胞衰老促进机体衰老。但此观点低估了细胞衰老的初始意图，即通过免疫系统阻止受损细胞增殖并触发其死亡。因此，细胞衰老可能是一种良性代偿反应，有助于将受损和潜在致癌细胞从组织中清除。然而，这一细胞检查点机制的实现需要高效的细胞更新系统以恢复细胞数量，该系统涉及到衰老细胞的清除和前体细胞的动员。在老年生物体中，上述细胞更新系统可能会变得效率低下，也可能是前体细胞再生能力耗竭，最终会导致衰老细胞的累积，进而加剧损伤并促进衰老（图5A）。

近年来，人们已经认识到衰老细胞分泌蛋白质组发生了显著改变，尤其是富含促炎性细胞因子和基质金属蛋白酶，被称为“细胞衰老相关分泌表型”（Kuilman et al., 2010；Rodier and Campisi，2011）。而这种促炎性分泌蛋白质组也有可能会促进衰老（见胞间通讯部分）。

1. INK4a/ARF位点和p53：除DNA损伤外，过量的促有丝分裂信号是其它形式应激中与细胞衰老相关最为强烈的一种（Gorgoulis and Halazonetis，2010）。近期数据总结显示有超过50种的致癌性或促有丝分裂改变能够诱导细胞衰老。虽然有越来越多的发现通过细胞衰老以响应致癌性侵袭的机制，但其中最为重要的仍是最早报道的p16INK4a/Rb和p19ARF/p53通路（Serrano et al., 1997）。对小鼠和人类的各类组织进行分析均发现，p16INK4a水平（以及较低程度的p19ARF）与生物学年龄相关，这一发现使衰老通路之间的相关性变得更加显著（Krishnamurthy et al., 2004；Ressler et al., 2006）。目前在不同组织、不同物种中尚未发现有其他蛋白或基因的表达与生物学年龄有如此强烈的关联，其平均变化幅度在青年组织和老年组织中存在一个数量级的差异。p16INK4a和p19ARF由同一基因位点编码，即INK4a/ARF位点。近期一项对300多项全基因组关联研究（GWAS）的荟萃分析发现，在所有基因组位点中，增龄性病变，如多种类型的心血管疾病、糖尿病、青光眼、阿尔茨海默病（Jeck et al., 2012）等与INK4a/ARF位点存在遗传相关性的数量最多。

p16INK4a和p53的关键作用是诱导细胞衰老，这支持了两者通过诱导细胞衰老以促进病理性衰老的假说。据此，考虑到p16INK4a和p53抑制肿瘤的益处，二者促衰老的作用或许是可以承受的代价。支持上述观点的是因广泛且持续损伤而早老的突变小鼠其细胞衰老程度十分显著，而若清除其p16INK4a或p53，其早老表型则有所减轻。这种情况可见于BRCA1缺陷小鼠（Cao et al., 2003）、HGPS模型小鼠（Varela et al., 2005）以及BubR1突变所致染色体稳定性缺陷的小鼠（Baker et al., 2011）。然而，其他证据提示这其中的情形更为复杂。与其预期的促衰老作用相反，系统性轻微增加p16INK4a、p19ARF或p53等肿瘤抑制因子表达的小鼠寿命延长，且这不能由其较低的肿瘤发病率来解释（Matheu et al., 2007，2009）。另外，清除p53反而会加剧某些早老突变小鼠的衰老表型（Begus-Nahrmann et al., 2009；Murga et al., 2009；Ruzankina et al., 2009）。正如前述的细胞衰老具有两面性，p53和INK4a/ARF活化可看作是良性代偿反应，从而避免损伤细胞增殖及其造成的衰老和肿瘤的后果。然而，一旦出现广泛的细胞损伤，组织再生能力会被耗尽或饱和。在这种极端情况下，p53和INK4a/ARF的反应将具有危害性并加速衰老。

2. 小结：细胞衰老是应答损伤的良性代偿反应，但当组织再生能力耗竭，此时细胞衰老具有危害性并加速衰老。正是由于其复杂性，对细胞衰老是否符合衰老特征标准的第三条尚无法给出一个简单的回答。适当增强诱导细胞衰老的肿瘤抑制通路，则可延长寿命（Matheu et al., 2007，2009），同样，清除实验性早老模型的衰老细胞，亦可延缓增龄性病变的发生（Baker et al., 2011）。因此，尽管上述两种干预手段的理念相反，但均可延长健康寿命。

八、干细胞耗竭

组织再生潜力下降是衰老最明显的特征之一（图5B）。例如，衰老过程中造血干细胞的减少导致适应性免疫细胞生成减少（此过程称为免疫衰老），以及贫血和骨髓异常增生的发病率增加（Shaw et al., 2010）。相似的干细胞功能损耗可见于全部重要的成年干细胞区室，如小鼠前脑（Molofsky et al., 2006）、骨骼（Gruber et al., 2006）和肌纤维（Conboy and Rando，2012）。老年小鼠研究发现，其造血干细胞（HSCs）的细胞周期活性呈现整体性降低，且老年期HSCs的细胞分裂能力弱于青年期HSCs（Rossi et al., 2007）。而上述变化与DNA损伤累积（Rossi et al., 2007）和细胞周期抑制蛋白，如p16INK4a的过表达有关（Janzen et al., 2006）。实际上，相比于老年期野生型HSCs，老年期INK4a-/-HSCs则表现出更强的植入能力和更高的细胞周期活性（Janzen et al., 2006）。在多种组织中均发现，端粒缩短也为衰老过程中干细胞减少的重要原因（Flores et al., 2005；Sharpless and DePinho，2007）。作为衰老领域的一项例证，这提示干细胞减少是多重损害的综合结果。

注：（A）细胞衰老。在青年期机体内细胞衰老可阻止损伤细胞的增殖，从而防止肿瘤发生及促进组织稳态。而在老年期机体内，衰老细胞的广泛损伤、清除及补充不足，会导致衰老细胞累积从而对组织稳态产生毒性效应，进而促进衰老。（B）干细胞耗竭。图中所示为造血干细胞（HSCs）、间充质干细胞（MSCs）、卫星细胞和肠上皮干细胞（IESCs）耗竭所致后果。（C）胞间通讯改变。举例说明胞间通讯改变与衰老相关。

图5. 细胞衰老和干细胞耗竭以及胞间通讯改变

虽然干细胞和前体细胞增殖能力不足对机体的长期稳态明显不利，但二者过度增殖会加速干细胞微环境的耗竭，同样是有害的。干细胞休眠对其长期保持功能也具有重要作用，这已被果蝇小肠干细胞过度增殖会导致干细胞耗竭及过早衰老而证实（Rera et al., 2011）。同样情形亦见于p21缺失小鼠，其表现为HSCs和神经干细胞的过早耗竭（Cheng et al., 2000；Kippin et al., 2005）。如此看来，衰老过程中INK4a诱导（见细胞衰老部分）和血清IGF-1降低（见营养物感应失调部分）均可能反映出机体试图保持干细胞休眠状态。近期研究亦发现，老年期的肌肉干细胞微环境中成纤维细胞生长因子2（FGF2）信号通路增强可导致干细胞休眠减少，最终出现干细胞消耗及再生能力丧失。而抑制该通路则可挽救上述损害（Chakkalakal et al., 2012）。上述发现可望启发如下的设计思路，即通过抑制FGF2通路以减少衰老过程中的干细胞耗竭。这为通过抑制FGF2信号传导进而减少衰老过程中干细胞耗竭提供了可能性（Cerletti et al., 2012；Yilmaz et al., 2012）。

关于干细胞的一个重要议题是，其功能下降是与细胞-内源性通路还是细胞-外源性通路相关（Conboy and Rando，2012）。近期研究则强烈支持后者。特别是，DR可通过细胞-外源性通路增强小肠和肌肉干细胞功能。将青年小鼠的肌源性干细胞移植入早老小鼠后，后者寿命会延长且其退行性病变得到改善，即便是在未检测到供体细胞的组织中亦是如此，这提示上述有效治疗可能源于干细胞分泌因子的全身效应（Lavasani et al., 2012）。更有异种共生实验发现，来自青年期小鼠的系统性因子可逆转老年小鼠的神经和肌肉干细胞功能（Conboy et al., 2005；Villeda et al., 2011）。

而亦有研究探索采用药物干预来改善干细胞功能。特别是，雷帕霉素对mTORC1抑制，可通过提高蛋白质稳态（见蛋白质稳态丧失部分）和影响能量感应（见营养物感应失调部分）来延缓衰老，其还可改善上皮、造血系统和小肠的干细胞功能（Castilho et al., 2009；Chen et al., 2009；Yilmaz et al., 2012）。这些发现也表明，要阐明雷帕霉素的抗衰老机制还存在相当大的难度，也更凸显本文所讨论的衰老各项特征之间存在相互关联。另值得关注的是，老化造血干细胞中GTP酶CDC42活性增加，因此通过药物抑制CDC42，可使人体衰老细胞重新恢复活性（Florian et al., 2012）。

小结：干细胞耗竭是多种衰老相关损害的整体结果，也可能是组织和机体衰老的最终元凶之一。近期研究表明，干细胞再生可逆转机体水平的衰老表型（Rando and Chang，2012）。

九、细胞间信息通讯改变

除细胞自主性改变之外，衰老还涉及细胞间信息通讯改变，即内分泌、神经内分泌或神经方面的改变（Laplante and Sabatini，2012；Rando and Chang，2012；Russell and Kahn，2007；Zhang et al., 2013）（图5C）。因此，在衰老过程中随着炎症反应增强、对抗病原体和癌前细胞的免疫监视功能降低，以及细胞周围、细胞外环境组分的改变，易出现神经激素信号通路（如肾素-血管紧张素、肾上腺素、胰岛素-IGF-1信号通路）的失调，这影响了所有组织机械的和功能的特性。

1. 炎症：衰老相关的细胞间信息通讯的显著改变可被称为“炎性衰老”，即伴随哺乳动物衰老，表现为促炎表型的累积（Salminen et al., 2012）。炎性衰老的发生可能存在多重原因，如促炎组织损伤的累积、免疫系统功能失调导致无法有效清除病原体和功能障碍的宿主细胞；衰老细胞分泌促炎性因子增加（见细胞衰老部分）；NF-κB转录因子活性增强及缺陷性自噬反应发生（Salminen et al., 2012）。上述改变可增强NLRP3炎性小体及其他促炎通路的激活，最终导致IL-1β、肿瘤坏死因子和干扰素的生成增加（Green et al., 2011；Salminen et al., 2012）。炎症还参与到人群中肥胖和2型糖尿病的发生，这两种情况可促进或与衰老相关（Barzilai et al., 2012）。与此类似，炎性反应缺陷在动脉粥样硬化的发生中亦发挥重要作用（Tabas，2010）。近期研究发现，增龄性炎症可抑制上皮干细胞功能（Doles et al., 2012），这进一步表明，这些不同但均可促进衰老进程的特征之间存在错综复杂的联系。而适应性免疫系统的功能降低，亦与炎性衰老平行发生（Deeks，2011）。免疫衰老可在系统水平上加重衰老表型，这是因为免疫系统障碍导致其无法清除感染因子、感染细胞及癌前细胞。衰老细胞（见干细胞耗竭部分）和多倍体细胞可在衰老组织和癌前病变中累积，免疫系统的另一项功能就是识别和清除这两类细胞（Davoli and de Lange，2011；Senovilla et al., 2012）。

对衰老组织转录状态进行的整体研究进一步凸显了炎症通路与衰老的相关性（de Magalhães et al., 2009；Lee et al., 2012）。衰老转录特征之一表现为NF-κB通路的过度激活，而在转基因老年小鼠皮肤中条件性表达NF-κB抑制因子，会导致其皮肤组织表型及转录状态恢复至与青年期相对应的特征（Adler et al., 2007）。与之类似，在不同的衰老加速小鼠模型中，通过基因和药物抑制NF-κB信号通路可阻止其增龄性特征的出现（Osorio et al., 2012；Tilstra et al., 2012）。关于炎症与衰老关联的最新研究发现，炎症和应激反应可激活下丘脑NF-κB并诱导相关信号通路，导致神经元促性腺激素释放激素（GnRH）生成减少（Zhang et al., 2013）。而GnRH降低可导致多种增龄性改变，如骨骼脆性增加、肌无力、皮肤萎缩以及神经生成减少。相应地，小鼠采用GnRH治疗后，可阻止增龄性神经损伤及减慢衰老进展（Zhang et al., 2013）。以上结果提示，下丘脑可通过整合NF-κB驱动的炎症反应和GnRH介导的神经内分泌效应调节生物体整体衰老。进一步的体内研究证实，炎症与衰老的关联来自mRNA降解因子AUF1的作用，其通过介导细胞因子mRNA降解，参与到终止炎症反应中（Pont et al., 2012）。AUF1缺陷小鼠表现为明显的细胞衰老以及早老表型，而通过重新表达这种RNA结合因子而得以缓解。有趣的是，AUF1不仅可指导炎性细胞因子mRNA的降解，还可激活端粒酶催化亚基TERT（Pont et al., 2012），从而有助于端粒长度的保持，这再次说明单一因子可对多项衰老特征产生强烈影响。

类似的情形亦见于sirtuins，其也可影响衰老过程中的炎症反应。多项研究显示，SIRT1可通过去组蛋白去乙酰化及NF-κB等炎症信号通路的组分下调炎症相关基因表达（Xie et al., 2013）。与此发现相一致的是小鼠体内SIRT1水平降低与多种炎症性疾病的发生和进展相关，而通过药物激活SIRT1则可预防炎症反应（Gillum et al., 2011；Yao et al., 2012；Zhang et al., 2010）。且SIRT2和SIRT6亦可通过NF-κB亚基去乙酰化及其靶基因的转录抑制下调炎症反应（Kawahara et al., 2009；Rothgiesser et al., 2010）。

2. 其他类型的细胞间信息通讯：除炎症外，还有越来越多的证据显示，某一组织的衰老相关性改变可导致其他组织的衰老特异性衰退，这解释了衰老表型在不同器官之间存在协同性。还有其他例证表明，发生这种“传染性衰老”或旁观者效应的原因除炎症细胞因子外，衰老细胞通过间隙连接介导的细胞联系以及ROS相关过程亦可诱导邻近细胞衰老（Nelson et al., 2012）。在小鼠体内应用过继转移模型评估发现，其微环境可促进CD4 T细胞年龄相关性功能缺陷（Lefebvre et al., 2012）。同样，肾功能受损会增加人类患心脏病的风险。反过来，仅针对某一组织进行的延长寿命操作，亦可延缓其他组织的衰老进程（Durieux et al., 2011；Lavasani et al., 2012；Tomás-Loba et al., 2008）。

3. 恢复有缺陷的细胞间信息通讯：恢复衰老过程中有缺陷的细胞间信息通讯存在多种可能的手段，如遗传、营养或药物干预等可改善衰老过程中丧失的细胞间信息通讯特性（Freije and López-Otín，2012；Rando and Chang，2012）。这其中尤其令人感兴趣的是，通过DR方法（Piper et al., 2011；Sanchez-Roman et al., 2012）或再生策略（Conboy et al., 2005；Loffredo et al., 2013；Villeda et al., 2011）（应用异种共生研究中确定的血源性系统因子）可延长健康寿命。长期应用抗炎药物（如阿司匹林等）亦可延长小鼠寿命和人类健康寿命（Rothwell et al., 2011；Strong et al., 2008）。另外，鉴于肠道微生物组可塑造宿主免疫系统功能并产生系统性代谢效应，因此可通过操控复杂且动态的人体肠道细菌生态系统的组分和功能延长寿命（Claesson et al., 2012；Ottaviani et al., 2011）。

4. 小结：有令人信服的证据表明衰老并不仅仅属于细胞生物学现象，衰老与细胞间信息通讯的整体改变相关联，为在这一水平上调节衰老提供了可能性。令人兴奋的是，研究表明通过血液传播的因子来返老还童是可实现的（Conboy et al., 2005；Loffredo et al., 2013；Villeda et al., 2011）。

十、结论与展望

对本篇综述列举的衰老的九项特征性标志进行整体分析，可将其分为三种类型，即原发性特征、拮抗性特征和整合性特征（图6）。原发性特征的共同点为：它们均产生负面作用。其包括DNA损伤（包括染色体非整倍体）、线粒体DNA突变以及端粒缺失、表观遗传学改变和蛋白质稳态丧失。它与原发性特征相比，拮抗性特征则的效应根据其强度不同具有两面性。在较低水平可介导良性效应，而在较高水平则产生恶性效应。以细胞衰老为例，其可防御机体癌变，但一旦细胞衰老过度，则会促进衰老。它与此类似，ROS可介导细胞信号转导与存活，但慢性高水平的ROS则会导致细胞损伤；同样适当的营养物感应和合成代谢显然对细胞存活相当重要，但若过度则亦可导致病变。这三项特征可看做是机体为防御损伤或营养匮乏而设计的。而一旦这些特征加剧或慢性化，则其功效发生逆转，反而会造成更严重的损害。衰老特征的第三种类型为整合性特征，即干细胞耗竭和细胞间信息通讯改变，其可直接影响组织的稳态和功能。尽管上述特征相互关联，本文仍认为其存在一定程度的层次关系（图6）。原发性特征可作为启动性触发，随着时间延长，其损伤效应会进行性累积。原则上讲，拮抗性特征是有益的，但在部分因原发性特征而促进或加速的过程中则逐渐变为有害的。最后，当原发性特征和拮抗性特征无法通过组织稳态机制实现代偿时，整合性特征便会出现。由于衰老过程中这些特征同时发生又相互关联，因此，揭示这一精确的因果网络，将会是未来一项激动人心的挑战。

注：上述衰老的九项特征可被划分为三种类型：（上）这四项特征是导致细胞损伤的原发性因素。（中）这三项特征部分是针对损伤的代偿性反应或拮抗性反应。这种反应最初会缓解损伤，但若慢性化或加剧发展则最终转变为毒性反应。（下）这两项整合性特征是前两类特征的结局，最终导致增龄性功能下降。

图6. 衰老九项特征之间的功能相关性

阐明衰老的特征，既有助于为未来研究衰老的分子机制建立框架，又有助于提高人类健康寿命干预手段的设计（图7）。然而，理解衰老这一复杂的生物学过程，仍面临着大量的挑战biological process（Martin，2011；Miller，2012）。随着下一代测序技术的快速发展，可望对衰老研究产生特殊影响：针对衰老机体的个体细胞采用该技术，将更容易评价其遗传学和表观遗传学改变的特异性累积情况（de Magalhães et al., 2010；Gundry and Vijg，2012）。这项技术已应用于测定极度长寿个体的全基因组序列，比较长寿和短寿动物种系和品系之间的基因组差异，以及在最高分辨率下分析增龄性表观遗传学改变（Heyn et al., 2012；Kim et al., 2011；Sebastiani et al., 2011）。平行开展功能获得或缺失动物模型研究是必不可少的，这将跨越关联分析的层次并提供因果证据，以支持衰老过程中上述特征的含义。未来还需要采用系统生物学方法，其不仅可描述衰老的各项特征进行，还可解释导致衰老与伴随衰老的过程之间的机制性关联（Gems and Partridge，2013；Kirkwood，2008）。另外，在分子水平分析基因组与环境之间的交互调节衰老的作用，将有助于确定延长寿命的药物靶点（de Magalhães et al., 2012）。可预计的是，未来将有更为复杂的手段来最终解决正常、加速和延缓衰老的复杂性问题。相信这些手段的联合应用，将会更细致地理解衰老特征的潜在机制，从而促进未来研发出提高人类健康寿命及长寿的干预手段。

注：展示了衰老的九项标志及在小鼠体内已被研究证实的治疗策略

图7. 可能延长人类健康的干预措施

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