Factors regulating the adipo-osteogenic differentiation of bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2017.17.022

Abstract

Abstract:

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) with multiple differentiation potential can be induced into osteogenic or adipogenic differentiation under certain conditions.
OBJECTIVE: To review the related factors regulating the adipo-osteogenic differentiation of BMSCs.
METHODS: A computer-based search of CNKI and PubMed databases was performed for literature concerning the related factors regulating the adipo-osteogenic differentiation of BMSCs published from January 2006 to August 2016. The search terms were “bone marrow mesenchymal stem cells, osteogenic differentiation, adipocyte differentiation” in Chinese and English, respectively.
RESULTS AND CONCLUSION: Signaling pathways, transcription factors, and small molecule compounds that are interacted are key factors in the regulation of BMSCs differentiation, so the techniques to intervene BMSCs differentiation based on these key molecules may correct bone or fat abnormality and can be applied to tissue engineering and regenerative medicine in the future. Additionally, the biological clock is also one of the most important factors for adipo-osteogenic differentiation of BMSCs by regulating signaling pathways or transcription factors.

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: Stem Cells, Mesenchymal Stem Cells, Adipogenesis, Tissue Engineering

CLC Number:

R318

Zhou Xin, Zhao Jia-jia, Chen Li-li. Factors regulating the adipo-osteogenic differentiation of bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(17): 2759-2765.

Figures/Tables 1

2.1 调控BMSCs成骨-成脂分化相关的信号通路近几十年来的研究表明，一些关键的信号通路参与调控细胞谱系的功能，包括Wnt通路、TGFβ/BMPs通路、Notch通路、FGFs通路、Hedgehogs通路等。这些信号通路也参与调节BMSCs成骨和/或成脂分化。 2.1.1 Wnt信号通路 Wnt通路参与了许多重要的生物过程，如Wnt复合受体低密度脂蛋白受体相关蛋白5(LRP5)缺失可导致骨质疏松-假性神经胶质瘤综合征，这是一种罕见的常染色体隐性遗传疾病，表现有先天性或幼年失明，严重的幼年型骨质疏松[5]。骨质疏松-假性神经胶质瘤综合征的发现，引起了众多科学家和临床医生对于Wnt信号通路在骨生物和骨疾病中的作用的重视。越来越多的证据表明，激活Wnt信号可促进成骨分化[6]，抑制成脂分化[7]。本课题组于2015年发表于《Stem Cells》上的文章中报道：对细胞增殖和多向分化有重要调控作用的Wnt/β-catenin信号通路，在CD31-/CD34+/ CD146+细胞中相较于CD31-/CD34+/CD146-的脂肪前体细胞有更强的活性，因而CD31-/CD34+/CD146+干细胞亚群相较于CD31-/CD34+/CD146-干细胞亚群的成脂分化能力较弱，而成骨分化能力较强[8]。动物实验证明，过表达Wnt10b可激活Wnt信号通路，进而增加骨小梁厚度[9]，敲除/敲低Wnt10b可导致骨密度降低[10]，年龄相关的脂肪细胞增加也被认为与Wnt10b的减少有关。这些研究强有力地证明Wnt信号通路参与调节间充质干细胞成骨成脂分化平衡。 Wnt信号通路受到多因素的调控，生物钟基因可通过Wnt通路调节成骨成脂分化。生物钟是生命对地球光照及温度等环境因子周期变化长期适应而演化的内在自主计时机制。地球自转而导致光照等环境因子以大约24 h为周期的循环变化，塑造了生命过程以大约24 h为周期的近昼夜节律。生物钟赋予生命预测时间和环境变化的能力，以协调体内的生命过程，如代谢、生理和行为等[11]。部分研究报道生物钟基因正是通过影响Wnt通路来调控骨稳态，Bmal1可通过作用于Wnt信号通路抑制3T3-L1及C3H10T1/2细胞的成脂分化，提示生物钟基因在胚胎发育及成体干细胞分化过程中发挥了重要的作用[12]。He等[13]证明生物钟基因Bmal1和Wnt信号通路在某个特定阶段对促进BMSCs成骨分化具有协同作用。 2.1.2 TGF-β/BMPs信号通路 TGF-β家族包括30多名成员，广泛参与调节细胞增殖、细胞分化及胚胎发育[14]。TGF-β超家族分为3个亚型：TGF-β1、TGF-β2、TGF-β3，BMPs属于TGF-β1家族[15]。Tsuji等[16]发现，BMP-2对骨折的创伤愈合极为重要，不能产生BMP-2的小鼠四肢易产生自发性骨折，且不能自然愈合。用其他的成骨刺激因子也不能弥补因BMP-2缺失导致的骨形成异常，提示BMP-2对骨形成及骨折创伤愈合的重要性。在BMSCs分化过程中不同成员发挥不同功能，TGF-β/BMPs信号途径已被普遍认为在调节成脂和成骨分化中起到双重作用[17]。例如，BMP2与罗格列酮相互作用可促进间充质干细胞成脂分化[18]。TGF-β/BMPs信号通路可以激活经典的Smad通路和非经典的p38 MAPK通路[19]，进而通过调控Runx2和PPARγ来影响成骨-成脂分化[15，17]。 2.1.3 Notch信号通路 Notch信号通路在成骨成脂分化过程中也发挥重要的作用。有研究报道，阻断Notch信号可促进自噬介导的间充质干细胞成脂分化[20]。除了对成脂分化的作用外，Notch信号可以通过抑制Wnt/β-catenin信号而抑制成骨分化[21]。这也反映了信号通路之间不是单一发挥作用的，而是可以相互作用。 2.1.4 Hedgehog信号通路 Hedgehog信号通路参与多种细胞的增殖和分化活动，对骨的发育和骨量的维持非常重要。研究发现，氧化应激通过抑制Hedgehog信号通路从而抑制BMSCs成骨分化[22]。BMSCs成脂分化过程中，由于Gli蛋白表达减少导致Hedgehog信号通路下调[23]。这些研究表明，Hedgehog信号通路起到了促进BMSCs成骨分化，抑制成脂分化的作用。与此同时，Hedgehog信号和BMP信号之间可相互作用，通过调节smad共同促进成骨分化[24]。 2.1.5 交感信号成骨细胞表达多种神经肽受体，这表明他们确实可以整合多个神经元的信号[25]。例如谷氨酸受体阻滞剂被报道降低DNA结合活性和成骨细胞Runx2的表达，从而抑制成骨[26]。Fu等[27]在2005年《Cell》中报道敲除生物钟基因Per1、Per2的小鼠骨量增加，这是由于Leptin失调，可通过交感神经信号系统调节生物钟系统，最后导致骨量增加。这一研究不仅提示交感信号在骨形成中的重要作用，同时也表明生物钟基因作为一种新颖的骨稳态调节因子，能从多方面调节骨形成。 2.1.6 其他与成骨成脂分化相关的信号通路除此之外，还有一些信号通路(如FGFs、PDGF、EGF、IGF等)也与BMSCs成骨成脂分化相关，例如FGFs受体激活后可启动下游ERK1/2、p38 MAPK、PKC、SAPK/JNK和PI3K信号，进而调控成骨成脂分化[28-29]。通过以上论述发现，与BMSCs分化相关的信号通路它们都不是单独发挥作用的，而是相互作用形成信号通路网，在特定的微环境里，信号通路网将被激活，共同调控BMSCs的分化方向。 2.2 调控BMSCs成骨-成脂分化相关的转录因子许多研究表明，转录因子可影响BMSCs向不同成熟细胞类型的分化。通过激活转录因子，上调相关基因的表达，诱导特定细胞类型的分化和发展。在下面的章节中将讨论各种转录因子影响BMSCs成骨成脂分化的特点和功能。 2.2.1 转录因子Runx2 Runx2是BMSCs成骨分化及骨形成过程中的必要的转录因子，能将BMSCs导向分化为前成骨细胞，并且抑制其向脂肪细胞和软骨细胞的分化[30]。许多可影响成骨作用的激素、细胞因子及信号通路(包括Wnt，BMP和Notch信号通路)均可调控Runx2的表达[31-32]。例如，HOXB7可通过上调Runx2增强成骨细胞的分化[33]。BMP9促进smad1，5，8的激活，Smad与runx2相互作用，促进BMSCs成骨分化，将Runx2在C-末端诱导突变可破坏Runx2-Smad蛋白转录活性，进而抑制成骨分化[34]。Runx2可与许多成骨特异基因的启动子结合，并促进其表达。例如，Runx2可与骨钙素基因的启动子区成骨细胞特异性的顺式作用元件Osteoblast-specific cis-acting element 2(OSE2)结合，促进成骨细胞标志物如Ⅰ型胶原蛋白和骨钙素的表达。此外Runx2被认为是整合各种信号影响成骨细胞分化的交汇点，能直接刺激成骨分化过程中骨钙素、Ⅰ型胶原、骨桥蛋白和胶原酶Ш等基因的转录[35-36]。Runx2的mRNA在有丝分裂后对称分离到子代细胞中仍能维持成骨细胞的表型[37]，表明Runx2在成骨细胞的分化和成熟过程中不但起着关键作用，而且是必需基因。研究表明，Runx2在骨组织里呈生物节律性表达[38]，实际上，超过1/4小鼠颅骨基因表达都具有生物节律性[39]，研究报道与BMSCs的分化和矿化沉积相关的基因直接受到BMAL1：CLOCK异二聚体的调控[40-42]。因此，与成骨分化相关的基因基本是受生物钟的调控；当生物钟紊乱后，BMSCs成骨分化能力会发生异常。He等[43]发现过表达生物钟基因Rev-erbα可促进BMSCs的增殖和成骨分化。Samsa等[44]研究指出，敲除生物钟基因Bmal1小鼠骨量明显减少且BMSCs成骨分化的能力降低。本课题组近期的研究也发现，Bmal1敲除小鼠发育会明显迟缓，股骨及下颌骨的骨量明显减少，具体表现在小鼠体质量增长曲线较正常老鼠低，下颌骨长度、质量均较小，另外Mic-CT结果显示股骨、下颌骨的骨密度、骨体积分数、骨小梁厚度等相对较小，骨小梁间隙等相对较大。 2.2.2 转录因子Osterix Osterix(Osx)是在小鼠体内发现的由成骨细胞表达的转录因子，人类Osx基因编码产物称为特异蛋白7(Specific protein 7，Sp7)。研究表明，Sp7/Osx是成骨细胞分化过程中的关键转录因子，为骨形成所必需。Nakashima等[45]报道敲除Osterix后，骨皮质和骨小梁都没有发生，同时Runx2/Cbfa1敲基因小鼠的Osterix不表达，提示Osterix是Runx2的下游分子。Cheng的研究小组发现Msx2对成脂分化和成骨分化具有双向调节能力，也是“成脂-成骨”分化平衡的一个调节因子，Msx2可通过上调细胞内Osx的表达，增强碱性磷酸酶活性及细胞外基质钙化以促进成骨分化，同时通过直接与C/EBPa蛋白质相互作用抑制PPARγ启动子区的活化，进而抑制成脂分化[46]。 2.2.3 转录因子PPARγ PPARγ是已被确定在BMSCs成脂分化中起关键调节作用且被研究最广泛的转录因子。PPARγ在成脂分化过程中表达上调，若抑制PPARγ的表达，则成脂分化也会抑制[47]。本课题组前期研究发现，脂肪组织来源血管基质成分SVF中的CD31-/CD34+/CD146-细胞具有更强的成脂分化能力，PPARγ-Axin2-Wnt信号通路在其中起着非常关键的作用, PPARγ能通过促进Axin2的表达来下调Wnt信号通路，从而促进脂肪干细胞的体外成脂分化[8]。Yu等[48]报道PPARγ2和PPARγ1在促进BMSCs分化为脂肪细胞中起着至关重要的作用。有趣的是，敲除C/EBPα后，抑制PPARγ2但不抑制PPARγ1，表明PPARγ2对成脂分化的作用更显著。生物钟也能通过调控成脂相关转录因子影响成脂分化，例如Nocturnin (NOC)是一个有自主生物节律的基因，可通过刺激PPARγ核转运，促进3T3-L1细胞成脂分化[49]。这也提示生物钟基因在调节BMSCs成脂分化中可能起到相关作用。 2.2.4 转录因子C/EBPα 除了PPARγ，C/EBPα是成脂分化过程中另一个重要的转录因子，C/EBPs因具有激活特定基因DNA增强子CCAAT重复序列的功能而得名，该家族包括C/EBPα、C/EBPβ和C/EBPδ等成员[50]。而前脂肪细胞表达的C/EBPα是脂肪细胞决定性调控因子之一。C/EBPα在大多数特异性脂肪细胞基因转录之前就已表达，此外许多特异性脂肪细胞基因的最近端启动子上都含有C/EBPα结合点，C/EBPα可直接作用于PPAR-γ启动区激活其转录，引发细胞成脂分化[51]。 2.2.5 其他与成骨成脂分化相关的转录因子研究报道TNF-α、DLX5、YAP蛋白等转录因子参与调节间充质干细胞的成骨分化[52-54]；FoxA1、HOXC8、SOX2、OCT4等转录因子参与调节间充质干细胞的成脂分化 [55-57]。对PPAR-γ和Runx2的表达及活性具有直接或间接调节作用的因子，也可能具有调节BMSCs成骨成脂分化平衡的能力。如TAZ被报道可通过分别激活Runx2的转录活性和抑制PPAR-γ的转录活性，促进BMSCs的成骨分化并抑制成脂分化，TAZ也因此被认为是“成骨-成脂”分化平衡的调节因子[58]。此外，缺氧逐渐增加骨髓间充质干细胞CBF1α表达，增强BMSCs分化为成骨细胞的潜力[59]。而EBF-1是在细胞功能和分化中起重要作用的一员，在促进BMSCs分化为脂肪细胞、骨细胞中也起着关键的作用[60]。 2.3 调控BMSCs成骨-成脂分化相关的小分子化合物 2.3.1 microRNAs 虽然已发现了多种分子机制调节干细胞的分化，但一类新的表观遗传调控者”micro- RNAs”被报道在干细胞的分化中发挥关键的作用。microRNAs (miRNAs)是一类在真核生物中内源性表达的非蛋白编码小分子RNA，长约22 nt。miRNAs广泛存在于哺乳动物各种组织内，人类基因组上大部分编码基因3'UTR上都有miRNAs的结合位点[61]。miRNAs可通过与靶基因mRNA结合抑制靶基因蛋白转录或使mRNA降解，从而在转录后水平对靶基因表达进行调控。鉴于miRNAs的广泛存在及对靶基因的调控作用，近年来研究发现，一些miRNAs在“成脂-成骨”分化平衡中发挥调控作用。如miR-204/211、miR-30、miR-320等可通过抑制其靶基因Runx2表达，进而抑制成骨分化并促进成脂分化[62]，miR-637可通过抑制成骨分化关键调节因子Osx进而抑制成骨分化并促进成脂分化[63]。这提示miRNA可通过改变内源性靶基因的表达来激活或抑制靶基因的功能作用，调控成骨成脂的平衡，从而改变骨的结构。Li等[64]发现miR-2861能通过抑制其靶基因HDAC5促进成骨分化，将miR-2861拮抗剂经鼠尾静脉注射大鼠后，可加速骨量丧失，同时，青少年原发性骨质疏松症临床病例测序结果也发现pre-miR-2861发生突变，此研究证明miR-2861在成骨分化方面具有重要的生理功能。另外有研究发现敲除了miR-188基因小鼠年龄相关的骨丧失降低，骨髓脂肪组织的堆积也减少，而向骨髓腔内注射BMSCs靶向适配体拮抗miR-188能刺激骨形成，降低小鼠骨髓腔脂肪的堆积。这些研究证明了miRNAs在“成脂-成骨”分化平衡中发挥的调控作用，并为治疗骨丢失提供了新的治疗靶点[65]。 2.3.2 外泌体Exosomes 外泌体Exosomes(Exos)是直径40-100 nm的膜性微囊结构，可由多种类型细胞产生，其内包含有多种功能性蛋白、脂质、mRNA、miRNA等。有研究表明，其在细胞–细胞沟通中起着至关重要的作用，一个细胞分泌的exosomes可以跟另一个细胞的胞膜融合，将其含有的RNA及蛋白质等直接释放到靶细胞的胞浆内，也可以通过胞吞的方式，整体进入到靶细胞中，从而实现细胞与细胞之间的信号转导[66]。Exosomes是近几年来发现并引起广泛关注的细胞间信号通讯的重要途径，是干细胞重要的旁分泌形式[67]。目前最新的研究发现，hiPSC-MSC-Exos能促进骨髓间充质干细胞的增殖和成骨细胞分化，调节成骨相关的基因和蛋白表达，并且体内实验验证，在颅骨缺损模型中，hiPSC-MSC-Exos能促进骨缺损处新骨再生[68]。提示Exosomes有望成为临床上治疗骨缺损的有效方法，这些作用的潜在机制可能是通过Exosomes内所包含的多种功能性蛋白、脂质、mRNA、miRNA释放作用于目的细胞所介导的。 2.3.3 其他与成骨成脂分化相关的小分子化合物除此之外，还有其他的小分子化合物调节干细胞的分化。例如长链非编码RNA(long noncoding RNA，lncRNA)过去被认为是转录过程中的杂音，没有实际作用。但近些年来，随着研究的深入，科学家们发现其在表观遗传、转录及转录后基因表达中起着重要的调控作用[69]。Wang等[70]对比了骨髓间充质干细胞成骨分化前后的lncRNA变化，发现有1 206个lncRNA在分化过程中产生了变化。lncRNA H19可通过TGF-β1/Smad3/HDAC信号通路促进BMSCs成骨分化[71]。同时，lncRNA H19抑制BMSCs成脂分化[72]。整合素家族(Integrins)是介导细胞–基质与细胞–细胞间相互作用的跨膜受体，配体结合后使其激活，从而导致FAK磷酸化，随后活化一系列信号蛋白包括PI3K、MAPK ERK1/2、PKC等[73]。FAK介导的ERK1/2和p38的激活可以磷酸化并激活Runx2，致使MC3T3-E1成骨分化的增加[74]。激活α(5)β(1) integrin 可以促进BMSCs成骨分化，利于骨形成和骨修复[75]。纤维粘结蛋白Fibronectin与细胞膜上的Integrins受体结合，也可抑制成脂分化[76]。"

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