间充质干细胞早衰的原因及预防

doi:10.3969/j.issn.2095-4344.0632

中国组织工程研究 ›› 2018, Vol. 22 ›› Issue (29): 4721-4728.doi: 10.3969/j.issn.2095-4344.0632

• 干细胞综述 stem cell review • 上一篇下一篇

间充质干细胞早衰的原因及预防

周龙¹，何帆²，王磊¹

¹南京医科大学附属苏州医院(苏州科技城医院)骨科，江苏省苏州市 215153；²苏州大学骨科研究所，江苏省苏州市 215007

修回日期:2018-07-16 出版日期:2018-10-18 发布日期:2018-10-18
通讯作者: 王磊，副主任医师，南京医科大学附属苏州医院(苏州科技城医院)骨科，江苏省苏州市 215153
作者简介:周龙，男，1988年生，安徽省枞阳县人，汉族，2016年苏州大学毕业，硕士，医师，主要从事骨质疏松症的临床与相关基础研究。
基金资助:
国家自然科学基金(31570978)；国家自然科学基金青年科学基金(51203194)

Premature senescence of mesenchymal stem cells: causes and preventive measures

Zhou Long¹, He Fan², Wang Lei¹

¹Department of Orthopaedics, the Affiliated Suzhou Hospital of Nanjing Medical University (Suzhou Science and Technology Town Hospital), Suzhou 215153, Jiangsu Province, China; ²Orthopaedic Institute, Soochow University, Suzhou 215007, Jiangsu Province, China

Revised:2018-07-16 Online:2018-10-18 Published:2018-10-18
Contact: Wang Lei, Associate chief physician, Department of Orthopaedics, the Affiliated Suzhou Hospital of Nanjing Medical University (Suzhou Science and Technology Town Hospital), Suzhou 215153, Jiangsu Province, China
About author:Zhou Long, Master, Physician, Department of Orthopaedics, the Affiliated Suzhou Hospital of Nanjing Medical University (Suzhou Science and Technology Town Hospital), Suzhou 215153, Jiangsu Province, China
Supported by:
the National Natural Science Foundation of China, No. 31570978; the National Natural Science Foundation of China for the Youth, No. 51203194

摘要/Abstract

摘要：

文章快速阅读：

文题释义：
细胞衰老：是指DNA受到不可逆性损害，端粒缩短，细胞在生长过程中逐渐丧失自我更新能力，细胞周期停滞于DNA合成前期。间充质干细胞受到氧化应激刺激可提前进入衰老过程，即早衰。
预防间充质干细胞的衰老：间充质干细胞作为种子细胞可向成骨细胞、软骨细胞、脂肪细胞及神经细胞分化，预防及治疗其早衰为骨质疏松症、骨性关节炎等疾病的防治提供了一种新的思路。课题组先前的相关实验结果证实褪黑素及细胞外基质可显著提高干细胞的增殖及分化能力，有效预防骨髓间充质干细胞的早衰，在骨缺损方面有着重大的应用前景。

摘要
背景：间充质干细胞具有较强的自我更新和多向分化潜能，在组织工程和再生医学领域中已得到广泛应用，然而其在体外扩增过程中却不得不面临衰老的命运。
目的：阐明间充质干细胞早衰的特征及原因，总结近年来预防间充质干细胞衰老的措施，从而探索出安全而有效的方法对其早期干预，延缓衰老进程。
方法：用计算机检索1988至2017年PubMed数据库和和中国知网(CNKI)期刊全文数据库收录的相关文献。英文检索词为“mesenchymal stem cells；senescence；prevent；bone tissue engineering”，中文检索词为“间充质干细胞；衰老；预防；骨组织工程”，共纳入109篇文献。
结果与结论：端粒缩短及氧化应激使间充质干细胞发生衰老，调控生长因子、抗氧化剂、氧含量及细胞外基质可以明显促进间充质干细胞的增殖，延缓其衰老，但具体的作用机制还不明确，值得深入研究与探索。

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程
ORCID: 0000-0003-1847-985X(周龙)

关键词: 细胞衰老, 间充质干细胞, 骨组织工程, 细胞外基质, 氧化应激, 端粒, 干细胞, 国家自然科学基金

Abstract:

BACKGROUND: Mesenchymal stem cells (MSCs) have strong self-renewal and multi-directional differentiation potential, which have been widely applied in the tissue engineering and regenerative medicine. However, it is inevitable to face the aging of MSCs during in vitro amplification.
OBJECTIVE: To elucidate the characteristics and causes of premature senescence of MSCs, and to summarize the measures to prevent the aging of MSCs in recent years, so as to explore a safe and effective method for early interventions that can delay the aging process.
METHODS: A computer-based search of CNKI and PubMed was performed to retrieve relevant articles published from 1988 to 2017. The key words were “mesenchymal stem cells; senescence; prevent; bone tissue engineering” in Chinese and English, respectively. Totally 109 eligible were included in the result analysis.
RESULTS AND CONCLUSION: Telomere shortening and oxidative stress make MSCs senescent. Growth factors, antioxidants, oxygen content and the extracellular matrix can significantly promote the proliferation of MSCs, and delay the cell aging. However, the specific mechanism is still unclear, which is worth further investigations.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Mesenchymal Stem Cells, Aging, Tissue Engineering

中图分类号:

R394.2

周龙，何帆，王磊. 间充质干细胞早衰的原因及预防[J]. 中国组织工程研究, 2018, 22(29): 4721-4728.

Zhou Long, He Fan, Wang Lei. Premature senescence of mesenchymal stem cells: causes and preventive measures[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(29): 4721-4728.

图/表（结果） 2

2.1 MSCs早衰的特征 MSCs在经过多次复制传代进入生命晚期后具有一些特征，可与增殖期及静止期细胞区分。这些用以鉴别细胞衰老的特征包括细胞形态变得大而扁平，细胞周期停滞于G₁期，β-半乳糖苷酶表达增加^[11-12]。无论MSCs来源于何种组织，细胞衰老的特征都是相似的。传代晚期的MSCs的增殖能力下降，细胞周期停滞于G₁期，细胞内染色体组正常，无基因突变^[13-14]。衰老的MSCs扁平肥大，细胞核和细胞质颗粒固缩，细胞碎片累积。同时由于脂褐质累积，衰老的MSCs肌动蛋白过度表达，细胞黏附性降低，自体荧光水平增加。MSCs衰老时β-半乳糖苷酶染色阳性率接近80%，比正常细胞高出4倍^[15]。但值得注意的是，有部分研究显示传代晚期的MSCs的β-半乳糖苷酶染色阳性率不足40%，可能与细胞生长过度抑制有关，这也表明β-半乳糖苷酶染色并非是体外MSCs衰老的有效标记。

衰老的MSCs表达一些与细胞生物学广泛关联的基因，比如与细胞周期、DNA复制、DNA损伤修复、有丝分裂相关基因表达水平的下降^[16-17]。另一方面，细胞周期抑制剂(CDKN2b，p16^Ink4a)^[16]、炎性细胞因子(白细胞介素6)^[16]、抗纤维蛋白溶解物(PAI-1)以及促血管新生因子(转化生长因子β1)等基因则过度表达^[18]，这种改变在一些体细胞中也有类似现象^[19]。同时研究发现衰老MSCs内microRNAs表达量也发生改变，microRNAs是一种单股RNA分子，与基因表达的转录修饰密切相关^[20]。这些表观遗传变异以往被认为与DNA甲基化过程有关^[21]，而现在研究表明这种变化导致了细胞衰老^[22]。

有研究表明MSCs的成骨分化能力随着细胞寿命的延长逐渐减弱，这与体内年龄相关的骨形成效率降低和骨量丢失的理论一致^[23-24]。另一方面，一些研究显示传代后期的MSCs成骨分化潜能有所增加^[25]。这种成骨能力的保留与Runx2基因的表达增加有关^[26]，也有可能与衰老相关的细胞外基质钙沉积有关^[25]。无论MSCs对成骨和成脂分化的刺激反应如何，目前的观点倾向于MSCs经过连续传代后成骨和成脂分化能力下降^[23^，^25]。有趣的是，在含有Ⅰ型胶原的环境中培养骨髓MSCs，其成脂分化潜能增加^[27]。这种多层分化模式表明基于衰老MSCs的多向潜能变化是一个严格协调的过程，最初是骨-软骨-脂肪三层分化中脂肪分化潜能减弱，而后是骨-软骨两层分化中软骨分化潜能减弱。在这2个体系中，软骨分化潜能逐渐降低，而成骨潜能保持稳定^[28]。脂肪分化潜能的中止可能与微囊蛋白1生成增加和过氧化物酶体增殖物激活受体(PPAR?2)信号受累减弱有关^[29]。

2.2 MSCs早衰原因 细胞衰老是一个复杂的过程，同时受基因和环境的调控。成纤维细胞传代40-100次后细胞生长停滞，氧化应激诱导DNA链破坏导致端粒缩短，从而细胞出现衰老^[30-31]。上皮细胞出现复制停滞的时间更早，复制10-20次后，端粒缩短诱导细胞衰老^[32-33]。体外研究表明上皮细胞过早的生长停滞与较差的培养环境有关，培养在最佳的外在环境下，这种现象将会有所改善。在这种最佳的外在环境下，上皮细胞耗尽其寿命后最终以端粒缩短的方式进入衰老^[34]。

2.2.1 端粒的作用 端粒是细胞内染色体末端的DNA重复序列，长度相对恒定^[35]。传代早期MSCs内端粒长度与供者年龄有关，胚胎来源的MSCs端粒长度为10-11 kb，来源于产后组织的MSCs端粒长度则为7 kb^[36]。Baxter等^[37]发现来源于产后组织的MSCs端粒长度平均每年以17 bp的速度缩短。然而也有一些研究发现MSCs端粒长度可达11-13 kb，而且长度与供者年龄没有太大关系^[38]。

尽管关于MSCs端粒长度没有一致的结论，但MSCs连续多次传代后端粒长度逐渐缩短则是公认的。MSCs每传一次代，端粒将近缩短50-100 bp，与成纤维细胞类似^[39]。当MSCs端粒长度缩短至5.8-10.5 kb这个阈值时细胞便开始停止增殖^[37]，其他体细胞的这个阈值则低于MSCs^[40]，这表明一些端粒独立事件可能与体外MSCs的衰老有关。

体外细胞衰老时端粒缩短的一个直接证据便是在人端粒酶反转录酶作用下细胞寿命超过其预期的自然极限^[41]。端粒酶的主要作用是通过补充染色体末端的重复性序列TTAGGG来弥补端粒在连续复制传代后的缩短^[42]。然而大部分体细胞由于端粒酶转录水平的沉默而不具有活性^[43]。多数实验认为MSCs的端粒酶活性很低或是无法觉察到其活性^[44]。而且，一些研究者发现端粒酶活性具有年龄依赖性^[36]，另一些学者却认为两者没有关系^[38]。MSCs在培养过程中端粒酶的沉默可能与P53的功能和转化生长因子β的连续表达有关^[45]，它们也是人端粒酶反转录酶的较强抑制剂^[43]。

最近几年，大量研究证实MSCs通过基因转染的方式引进人端粒酶反转录酶后复制传代次数倍增(>260代)，染色体组型、端粒长度保持正常，衰老表型丧失，但体内外的分化潜能没有减弱^[46-48]。胚胎干细胞无限制的自我更新能力也强有力的说明了MSCs的衰老主要与由于端粒酶激活导致的端粒DNA逐渐磨损有关^[49]。

2.2.2 氧化应激的作用 氧化应激被认为是DNA损伤和衰老的主要原因^[50]。成纤维细胞在生命晚期时，活性氧生成增加，这是由于线粒体新陈代谢紊乱所致，包括细胞色素C氧化酶、烟酰胺腺嘌呤二核苷酸脱氢酶活性增加^[51]，线粒体膜电位降低，线粒体生物合成增加^[52]。氧化应激在细胞衰老中的作用已被研究证实，细胞培养在含氧过高的环境下加快了端粒介导的复制性衰老的进程^[53]，而经过氧化剂处理的细胞则以非端粒依赖的方式过早衰老^[54]。

相对于成纤维细胞和其他体细胞，氧化应激在MSCs生长和衰老中的作用则显得较为复杂。一些间接证据显示活性氧在骨髓和脂肪组织来源MSCs增殖中的作用，它们生长的速率和复制传代的寿命在抗氧化剂(抗坏血酸、白藜芦醇)存在的情况下明显提高^[55-56]。其他研究证实MSCs在较少的氧压力环境下寿命得以延长^[57]，可能与促进MSCs增殖的基因过表达有关，如血管内皮生长因子，肝素结合表皮生长因子等^[58]。

Lonergan等^[59]研究已发现线粒体在MSCs衰老中扮演重要角色。他们发现在MSCs传代晚期线粒体内三磷酸腺苷增加，耗氧量减少，由此表明细胞的衰老依赖于活性氧的积累。尽管有这些研究发现，氧化应激在MSCs衰老中的作用机制仍有许多未明确，尤其是活性氧积累导致的DNA损伤机制、线粒体功能障碍机制等。

2.3 预防MSCs早衰的措施

2.3.1 生长因子 生长因子可以维持并促进MSCs增殖及分化，但不能减缓端粒缩短的速率，增加端粒酶数量^[60-61]，见表1。5 μg/L成纤维细胞生长因子2可以在无论有无血清的培养环境下促进MSCs分裂增殖。Mizuta等^[62]在兔膝关节损伤模型中发现成纤维细胞生长因子2可以通过促进MSCs增殖提升软骨细胞的修复能力。Ito等^[13]也证实成纤维细胞生长因子2通过降低P21、P53、P16基因的表达水平，可以减缓人MSCs长期培养过程中出现的复制性衰老，也有可能是抑制转化生长因子β2 mRNA的表达来减少细胞停滞在G₁期的比例。100 μg/L胰岛素生长因子1可通过调节转化生长因子β2促进老化MSCs产生蛋白多糖。

2.3.2 抗氧化剂 N-乙酰半胱氨酸可以保护鼠MSCs免受肿瘤坏死因子诱导的死亡^[63]。褪黑素(N-acetyl-5-methoxytryptamine)，化学式为N-乙酰基-5-甲氧基色胺，是动物、植物、微生物中的一种天然化合物。在人体中，褪黑素主要由松果体分泌，是一种吲哚胺类激素，具有很强的抗氧化性，可以清除自由基，保护细胞核、线粒体、DNA免受氧化应激损伤^[64]。由于褪黑素对细胞有保护作用，因此具备抗衰老的潜能。褪黑素最近被发现在骨髓中也有高水平的分泌^[65]。许多研究表明，褪黑素调节各种生理功能，如促进睡眠、调节昼夜节律和神经内分泌活动^[66]。褪黑素有众所周知的抗氧化性能，包括清除过多的自由基和增加细胞内抗氧化酶的合成^[67]。研究表明，褪黑素通过减少活性氧生成和提高超氧化物歧化酶产生从而保护细胞免受促炎性细胞因子损害^[68]。此外，褪黑素已被证明可调控终末分化细胞(关节软骨细胞)的生物功能和MSCs的多向分化^[69-70]。然而，褪黑素对早衰MSCs的影响尚未阐明，对SIRT1介导的信号通路争论不一。Yu等^[71]发现褪黑素保护心肌缺血再灌注损伤通过以受体依赖的方式激活SIRT1。有令人信服的证据表明褪黑素在MCF-7癌细胞中通过增加P53的乙酰化作用促进细胞周期停滞^[72]。因此，褪黑素调控SIRT1及衰老MSCs细胞周期的潜在机制有待进一步研究阐明。褪黑素对MSCs和其他类型细胞遭受氧化应激的保护作用已被广泛研究。Wang等^[73]报道褪黑素预处理通过减少胞内活性氧产生及抑制caspase-3活性减弱H₂O₂诱导的MSCs凋亡。在鼠衰老模型中，褪黑素通过增加BCL-2_XL等抗凋亡蛋白的表达改善其寿命^[74]。外在的补充褪黑素可以通过减少活性氧的累积抵消促炎性细胞因子致凋亡的影响。目前的结果显示外在补充褪黑素成功保护了MSCs免受H₂O₂诱导的衰老，并具有浓度依赖性，因为褪黑素减少了β-半乳糖苷酶染色阳性率，增加了细胞增殖能力，下调了P16^INK4a的表达水平^[75]。一些抗氧化药物也同样具有抗衰老作用，例如内皮细胞外在补充白藜芦醇减弱了氧化应激诱导的衰老，促进细胞增殖^[76]。这些研究发现为利用基于MSCs抗氧化应激的细胞理论提供了实验策略。

2.3.3 低氧 21%的氧含量一直被认为是标准的细胞培养环境所需的含氧量，可以最优化的促进成纤维细胞生长，但其他类型的细胞却不适应这种环境。Moussavi-Harami等^[77]发现21%的氧环境明显减弱了MSCs的生长速率，这与氧化剂的产生过多有关。MSCs长期培养在低氧环境中，成软骨、成脂分化能力增强，成骨分化能力减弱^[78]。研究证实低氧阻止MSCs衰老是通过下调细胞外信号调节激酶表达，上调抑制P16基因表达等实现的^[79]。

2.3.4 细胞外基质 干细胞在一个特定的微环境中生长，微环境调控着干细胞如何参与组织再生，维持干细胞自我更新功能的平衡，防止其过度增殖耗尽寿命。细胞外基质作为干细胞微环境的重要组成部分，由各种胶原、蛋白聚糖、纤维蛋白组成，见图1^[80]。细胞外基质作为生长因子的储存库，为细胞分化过程的重塑提供了天然信号^[81-83]。由于细胞外基质在不同类型细胞中的成分保持相对恒定，来源于不同组织的细胞外基质，如心、肺、脑、肝、脂肪等都可以通过相似的简单生物化学方法获得^[84-86]。细胞外基质作为一种细胞支架，支持细胞增殖和分化，Urist^[87]研究证实去矿物质的骨基质能够诱导异位骨形成，这归功于骨形态发生蛋白的存在。

关于干细胞衰老的分子机制已有大量研究报道，普遍认为与干细胞从体内到体外所处的微环境改变有关。MSCs一旦离开体外特异的微环境，就逐渐失去其自我更新及多向分化的潜能。干细胞微环境的重要组成部分是细胞外基质，细胞外基质以其复杂的生物化学和物理信号维持着细胞功能。研究者们已经尝试重建细胞内天然的微环境，通过运用不同细胞外基质蛋白的组合调控MSCs的命运。Linsley等^[88]比较了纤连蛋白原、Ⅰ型胶原、纤连蛋白3种不同细胞外基质蛋白，发现纤连蛋白原在促进MSCs成骨分化方面起着最重要作用。然而，天然的细胞外基质不仅包含各种各样的蛋白质，如Ⅰ、Ⅱ、Ⅲ、Ⅲ、Ⅴ和Ⅵ型胶原，纤连蛋白和层黏连蛋白等黏附蛋白及大分子量蛋白聚糖，而且为细胞存在提供场所，加强MSCs对机械信号刺激的反应^[89]。在天然细胞外基质和有细胞外基质蛋白涂层的基质上培养的细胞显示出了不同特性^[90]，但天然细胞外基质调控MSCs功能的确切机制需要进一步的探索研究。

Li等^[10]研究显示MSCs来源的细胞外基质比软骨细胞来源的细胞外基质更能促进MSCs的增殖和软骨分化。人骨髓MSCs来源的细胞外基质为其自身提供了一种适宜的特异性微环境，促进骨髓MSCs软骨内骨形成^[91]。更为有趣的是Choi等^[92]发现衰老的人成纤维细胞在来源于年轻细胞的细胞外基质环境下培养能使其恢复到更为年轻的状态，可能延迟了端粒缩短。由此可见，细胞外基质内一些信号对细胞衰老有着深刻的影响，至少减缓了细胞衰老的进程。研究表明脱细胞外基质加强了细胞内褪黑素的抗氧化和骨诱导特性，而来源于干细胞的细胞外基质对MSCs早衰的作用及调控其早衰的潜在机制目前还没有完全阐明^[93]。

2.4 信号通路 MSCs在生长过程中不可避免发生衰老，具体的机制一直是研究的热点。研究已发现几种信号通路与体外MSCs的生长和衰老相关，但确切机制仍有待进一步深入探讨。

2.4.1 MSCs衰老有关的信号通路 分子水平的相关研究证实MSCs衰老主要与两种信号通路相关。复制性衰老具有端粒依赖性，受P53-P21信号通路调控，同时也包括DNA损伤应激反应机制^[94]。而非端粒依赖性的衰老被氧化应激诱导，与Erk-p38^MAPK信号通路有关^[95]。两种信号通路均与Rb有关，Rb即视网膜细胞瘤蛋白，调控细胞周期，介导端粒依赖性的细胞衰老。P53在调节细胞功能方面扮演者重要角色，调控细胞生长、DNA修复、凋亡、衰老^[96]。DNA损伤、端粒缩短时P53在G₁期激活，阻止正常的DNA进入S期，然后通过激活P21诱导Rb相关的衰老，这种衰老的过程随着后来P53的失活是可以逆转的^[97]。P53-P21信号通路也可以被Wnt/β-catenin通路激活，该通路可导致MSCs衰老^[98-99]。另外细胞衰老时p38^MAPK、P16激活，然后通过激活Rb，形成Erk-p38^MAPK-P16信号通路。该通路一旦被激活，衰老很难通过钝化Rb和沉默P16被逆转。P16基因的表达与人MSCs衰老密切相关，敲除P16基因后人MSCs的衰老能够被恢复。

总的来说，氧化应激导致的MSCs早衰主要是通过Erk-p38^MAPK信号通路，同时氧化应激也可引起端粒缩短激活P53-P21信号通路引起衰老，而复制性衰老的机制则主要受P53-P21信号通路介导，但复制性衰老过程中也伴随着内源性氧化应激的产生，激活Erk-p38^MAPK信号通路，所以无论复制性衰老还是氧化应激引起的早衰，Erk-p38^MAPK和P53-P21两种信号通路都参与其中。

2.4.2 SIRT1与MSCs早衰的关系 SIRTs是一类与酿酒酵母沉默信息调节因子2(Sir2)同源性的脱乙酰酶家族，具有NAD⁺依赖性。哺乳动物中有7个沉默调节蛋白，并且每一个包括一个恒定的中央核心脱乙酰酶结构域，通过变化两侧N端和C端的长度。有趣的是，SIRTs具有NAD⁺依赖性，这些脱乙酰酶对氧化应激和新陈代谢方面的环境变化反应迅速^[100]。SIRT1是SIRTs家族中研究最为广泛的成员，主要因为它可调控多种细胞功能及其治疗潜力。

氧化应激引起细胞早衰，SIRT1通过调节与DNA损伤和修复有关的分子如FOXOs、P53、P21等够保护细胞免受早衰^[101-103]。研究结果显示SIRT1通过调节FOXO3和衰老基因P53、P21的表达，避免氧化应激引起的肺细胞衰老^[104]。Langley等^[105]报道SIRT1的缺乏引起乙酰化P53的累积，这样增强了氧化应激诱导的衰老。在干细胞和祖细胞中，SIRT1也同样可以调节其衰老。组织蛋白酶可以减弱SIRT1在内皮祖细胞中的作用，导致氧化应激引起的早衰^[106]。SIRT1参与维持干细胞体内平衡^[107]。Homma等^[108]揭示SIRT1在调节人胚胎干细胞和多能干细胞功能方面的重要作用。SIRT1也可以通过增强人端粒酶反转录基因的表达阻止人脐带MSCs的复制性衰老^[109]。这些研究结果表明SIRT1对氧化应激环境下的干细胞复制性衰老和早衰调节的重要作用。

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间充质干细胞早衰的原因及预防

Premature senescence of mesenchymal stem cells: causes and preventive measures

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