Exosomes that deliver specific miRNAs can regulate osteogenesis and promote angiogenesis

doi:10.12307/2022.152

Abstract

Abstract: BACKGROUND: Exosomes are nanoscale paracrine vesicles containing a variety of bioactive factors, such as miRNAs. Exosome miRNAs play an important role in intercellular communication. In recent years, more and more studies have focused on whether miRNAs in exosomes promote bone regeneration.
OBJECTIVE: To review research status of exosomal miRNAs promoting bone regeneration in the recent years so as to provide a theoretical basis for further research and application in the field of bone regeneration.
METHODS: Using “bone regeneration, bone repair, exosomes, miRNA” in Chinese and English as search terms, biomedical literature database, CNKI, and PubMed were retrieved for articles on exosomal miRNA and bone regeneration published from 2010 to 2021.
RESULTS AND CONCLUSION: Exosomes derived from different cells can effectively regulate osteogenesis and promote angiogenesis through the delivery of specific miRNAs, which has a broad prospect in bone tissue engineering.

Key words: cell, exosome, miRNA, bone regeneration, bone repair, review

Zhou Hongqin, Wu Dandan, Yang Kun, Liu Qi. Exosomes that deliver specific miRNAs can regulate osteogenesis and promote angiogenesis[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(7): 1107-1112.

Figures/Tables 1

2.1 miRNA调控骨再生 miRNAs是一类内源性小非编码RNA，通过与靶mRNA上的互补位点结合发挥转录后调控作用[10]。MiRNA在进化上是保守的，参与了各种生物学过程，包括细胞命运、细胞增殖、凋亡、分化和癌变[10-12]，许多miRNA参与细胞成骨分化。过表达miR-34a可显著提高人脂肪干细胞的碱性磷酸酶活性、矿化能力和成骨相关基因的表达，在脂肪干细胞中过表达miR-34a后植入裸鼠背部皮下也可以观察到体内异位骨形成增强。研究表明，miR-34a通过RBP2/NOTCH1/CYCLIN D1共调控网络促进脂肪干细胞的成骨分化[13]。MiR-21可通过PI3K/β-catenin通路促进骨髓间充质干细胞的成骨分化[14]，GENG等[15]将miR-21纳米胶囊加载到酸处理过的钛表面，将间充质干细胞接种在经过处理的钛表面，对比未加载miR-21纳米胶囊的钛表面，miR-21纳米胶囊处理过的钛表面更加显著促进间充质干细胞成骨和成血管，除此之外，还可提高间充质干细胞破骨活性；将此种钛材料植入兔股骨远端、胫骨，发现不仅能促进血管形成，还能促进成骨细胞生长，加速骨的重塑和成熟过程，从而提高骨与种植体的接触和结合强度。此外还有许多miRNA与成骨有关，例如miR-146a[16]、miR-31等[17]。 2.2 外泌体miRNA参与细胞间通讯 2007年，VALADI等[18]发现外泌体可在细胞间转移miRNA实现细胞间通讯，外泌体介导的miRNA转移可对受体细胞产生影响，如调节蛋白表达。外泌体相关miRNA可以在囊泡结构的保护下稳定在血液循环中而不被降解，然后转移到靶细胞中抑制某些基因的表达[19-20]。外泌体一旦被邻近或远端细胞吸收，外泌体miRNA在基因表达的转录后调控中起着重要作用。外泌体miRNA进入靶细胞，通过部分序列互补与靶基因mRNA结合。外泌体miRNA参与不同生理过程和病理发生发展，包括伤口愈合、炎症、骨折愈合和阿尔茨海默病[21-24]。miRNA的5’端有7个核苷酸组成种子区，这是mRNA识别的关键。miRNA与靶mRNA结合，通过形成miRISC调控蛋白质合成[25]。许多研究证明了细胞来源外泌体miRNA在细胞间通讯起重要作用，改变外泌体中miRNA可影响多种疾病进程，如癌症、阿尔茨海默症和心肌梗死等[26-28]。例如，氧化应激下心肌祖细胞分泌的外泌体miR-21通过靶向PDCD4抑制H9C2心肌细胞的凋亡[29]。 2.3 成骨诱导后外泌体miRNA改变在成骨诱导过程中，细胞内会发生一系列改变，外泌体内miRNA也会发生相应变化。不同细胞在成骨诱导分化过程中分泌的外泌体miRNA也会有所不同。间充质干细胞来源外泌体的应用为组织工程和再生医学提供一种新的策略。WANG等[30]发现来自不同成骨分化阶段的间充质干细胞外泌体可使同型细胞向成骨谱系分化，但只有来自成骨分化晚期的外泌体可诱导细胞外基质矿化。MiRNA谱分析发现一组与成骨分化后期差异表达的外泌体miRNA与成骨形成相关。靶标预测表明，这些miRNA富集通路参与了成骨分化的调控和外泌体发挥其功能的一般机制，如Wnt信号通路和内吞作用。最近，ZHAI等[4]提取成骨诱导10 d和15 d间充质干细胞的外泌体(EXO-D10、EXO-D15)，发现能促进间充质干细胞成骨分化，并将此外泌体加载到3D打印纯钛支架，然后植入大鼠骨缺损处，发现有哈弗斯管结构的新骨和血管形成；用下一代测序技术检测外泌体差异表达的miRNA，相对未成骨诱导间充质干细胞分泌的外泌体(EXO-D0)，EXO-D10、EXO-D15中分别有166和193种miRNA上调，139和150种miRNA下调；在EXO-D10、EXO-D15中，与成骨相关的miRNA如Hsa-miR-146a-5p、Hsa-miR-503-5p、Hsa-miR-483-3p、Hsa-miR-129-5上调，这些miRNA在EXO-D10、EXO-D15中的丰度高于其他外泌体，根据下一代测序技术测序结果分析，PI3K/Akt信号通路和MAPK信号通路可能在间充质干细胞成骨过程中起主导作用，miRNA可能与生长因子或受体如TARF6、FGF2、BMPR、BMP1和RUNX2相互作用，激活PI3K/Akt和MAPK信号通路，促进间充质干细胞的成骨。 XU等[31]使用安捷伦定制miRNA v3.5阵列绘制了人骨髓间充质干细胞成骨分化过程中外泌体内差异表达的miRNA，检测到79个miRNA。研究发现，在培养骨髓间充质干细胞0，0.5，1，1.5，2，2.5，3，3.5，4，4.5，7 d时，骨髓间充质干细胞培养上清中有14个外泌体miRNA的差异表达，其中0.5-7 d，5个miRNA (miR-199b，miR-218，miR-148a，miR-135b和miR-221)变化超过0 d变化最大值的2倍以上。通过DIANA-mirPath的生物信息分析表明，RNA降解、mRNA监视通路、Wnt信号通路、RNA转运是最显著的富集于与成骨分化相关的不同外泌体miRNA模式分位的通路。 YANG等[32]通过微阵列分析来自未分化和成骨分化脂肪间充质干细胞外泌体miRNA的表达谱，相对于未分化的脂肪间充质干细胞外泌体，成骨诱导分化的脂肪间充质干细胞外泌体有201个miRNA上调，33个miRNA下调。选取差异表达最明显的6个miRNA (miR-130a-3p、miR-513b-5p、miR-30b-5p、miR-34a-5p、miR-324-5p、miR-378f)，用qPCR验证微阵列分析，结果与微阵列获得的初步结论一致，与未分化脂肪间充质干细胞外泌体相比，成骨分化脂肪间充质干细胞外泌体中5个miRNA (miR-130a-3p、miR-30b-5p、miR-34a-5p、miR-324-5p和miR-378f)的表达显著增加。外泌体miRNA可以通过一系列生物学过程影响成骨分化，在这些过程中，它们首先靶向相关基因，从而调控MAPK信号通路和Wnt信号转导并修饰基因表达、细胞代谢等生物学功能促进成骨。MiRNA-mRNA网络分析显示一个miRNA可以同时识别多个靶标mRNA，一个基因也可能受多个miRNA的调控。更重要的是，大量miRNA被预测参与多种促进成骨分化的途径。例如，miR-130a-3p是差异表达最高的miRNA，预测其与SIRT7结合的可能性很高。既往研究发现，敲低SIRT7可增强骨髓间充质干细胞的成骨分化[33]。牙周膜干细胞是牙周膜来源的间充质干细胞，牙周膜干细胞较其他牙干细胞如牙髓干细胞和牙滤泡祖细胞具有更佳的再生能力和多能性[34]。牙周膜干细胞是一种比较容易获得的间充质干细胞，并已用于骨再生[35]。LIU等[36]发现成骨诱导可增强牙周膜干细胞来源外泌体的成骨能力，促进大鼠骨髓干细胞的成骨分化。通过RNA测序分析未分化和成骨分化牙周膜干细胞外泌体miRNA表达谱，结果显示，成骨诱导后72个miRNA表达上调(其中4个miRNA显著上调：miR-122-5p，miR-142-5p，miR-25-3p，miR-192-5p)，35个miRNA表达下调(其中3个miRNA显著下调：miR-125b-5p，let-7b-5p，miR-100-5p)。基因本体论分析和路径分析结果表明，外泌体miRNA差异表达的靶基因参与多种生物学过程，如催化活性、蛋白质绑定、代谢过程、细胞的发育和分化，并富含成骨分化相关途径，如MAPK信号传导、AMPK信号和胰岛素信号通路。诱导干细胞成骨分化后，干细胞分泌的外泌体miRNA表达与未分化的干细胞有所不同，许多变化的miRNA与成骨分化有关，但还未经过进一步的实验验证。 2.4 与成骨相关的外泌体miRNA 2.4.1 骨髓间充质干细胞外泌体miRNA促进骨再生 (1)miR-29a：miR-29a可调节血管生成[37]，LU等[38]发现骨髓间充质干细胞来源外泌体可被人脐静脉内皮细胞吸收，并促进人脐静脉内皮细胞增殖、迁移和管的形成。MiR-29a在骨髓间充质干细胞外泌体中表达较高，可通过VASH1依赖的方式转运到人脐静脉内皮细胞中调控血管生成。更重要的是，携带miR-29a的骨髓间充质干细胞来源外泌体具有促进小鼠血管生成和成骨的强大能力。 (2)miR-122-5p：LIAO等[39]用生物信息学方法推测 miR-122-5p可能通过调控SPRY2基因来影响股骨头坏死。用过表达miR-122-5p的骨髓间充质干细胞与成骨细胞共培养，发现骨髓间充质干细胞分泌外泌体能被成骨细胞摄取，并且使成骨细胞成骨、增殖能力增强和SPRY2表达下降。研究发现miR-122-5p通过RTK/Ras/MAPK信号通路抑制SPRY2的表达，激活RTK的活性来实现这一功能。体内实验证明miR-122-5p修饰的骨髓间充质干细胞来源外泌体促进兔股骨头坏死血管生成、骨修复和愈合，从而抑制股骨头坏死的发展。 (3)miR-31a-5p：GE等[40]发现骨髓间充质干细胞的miR-31a-5p在牙萌出过程中通过SATB2 信号通路调节成骨，而SATB2能够提高老龄人骨髓间充质干细胞的干性能力和成骨分化能力[41]。XU等[42]发现老年大鼠骨髓间充质干细胞成骨能力降低，伴随miR-31a-5p表达增加。将年轻大鼠骨髓间充质干细胞过表达miR-31a-5p后，骨髓间充质干细胞成骨能力降低；抑制老年大鼠骨髓间充质干细胞miR-31a-5p表达，则促进骨髓间充质干细胞成骨分化。研究显示，SATB2为miR-31a-5p靶基因。老年大鼠骨髓间充质干细胞来源外泌体中miR-31a-5p的表达明显高于年轻大鼠，抑制外泌体中的miR-31a-5p可以在翻译水平上提高RhoA的表达来减少破骨形成和骨吸收，体内实验显示抗agomiR-31a-5p处理的骨组织可防止老年大鼠的骨丢失和破骨细胞分化，说明外泌体包装的骨髓间充质干细胞来源miR-31a-5p不仅影响成骨分化，而且通过外泌体在细胞之间的穿梭影响破骨分化。 (4)miR-146a：锶(Sr)是人体内一种重要的微量元素，在元素周期表中与钙同族，具有相似的化学性质。既往研究表明锶元素可促进成骨[43-44]。锶代硅酸钙提取物促进骨髓间充质干细胞成骨和血管生成相关基因表达，锶代硅酸钙刺激后的骨髓间充质干细胞来源外泌体(BMSCs-derived exosomes after Sr-CS stimulation，Sr-CS-Exo)中miR-146a表达增加，并可促进脐静脉内皮细胞体外血管生成，外泌体转移的miR-146a参与了Sr-CS-Exo介导的血管生成，Sr-CS-Exo通过抑制Smad4和NF2上调miR-146a，促进脐静脉内皮细胞血管生成；Sr-CS-Exo促进斑马鱼发育血管生成，另外，Sr-CS-Exo促进体内SD大鼠血管生成和骨再生[45]。 (5)miR-375：CHEN等[46]研究发现miR-375在间充质干细胞成骨分化中具有正向调节作用，miR-375过表达显著增强了脂肪间充质干细胞的碱性磷酸酶活性和钙沉积，miR-375通过YAP1/DEPTOR/AKT调控网络促进脂肪间充质干细胞的成骨分化。CHEN等[47]继续研究发现miR-375可以通过在人脂肪间充质干细胞中过表达而在外泌体中富集，50 mg/L外泌体(miR-375)可促进骨髓间充质干细胞成骨分化。miR-375被骨髓间充质干细胞吸收后，胰岛素样生长因子结合蛋白3通过与其3’非编码端结合而被抑制，重组胰岛素样生长因子结合蛋白3降低了外泌体(miR-375)触发的成骨作用。与水凝胶结合后，外泌体(miR-375)表现出缓慢和可控的释放，进一步的体内分析表明，外泌体(miR-375)增强了大鼠颅骨缺损模型的骨再生能力。另外，有学者发现携带miR-375的神经母细胞瘤分泌外泌体可促进骨髓间充质干细胞的成骨分化[48]。 (6)miR-128-3p：间充质细胞的修复能力随着年龄的增长而下降。老年大鼠骨髓间充质干细胞来源外泌体的修复能力亦相对减弱。XU等[49]发现年轻大鼠间充质干细胞来源外泌体比老年大鼠间充质干细胞外泌体能更显著促进大鼠骨折愈合，通过miRNA微阵列分析，发现与年轻大鼠间充质干细胞来源外泌体相比，老年大鼠间充质干细胞外泌体中 miR-128-3p是表达上调最多的miRNA；并证实了miR-128-3p和Smad5之间的直接相互作用，体内实验表明miR-128-3p在体内具有靶向Smad5的抗成骨功能。外泌体miR-128-3p拮抗剂可能是一种很有前途的针对老年人骨折愈合的治疗策略。 (7)miR-186：绝经后骨质疏松症是老年妇女最常见的原发性骨质疏松症，行卵巢切除术SD大鼠骨髓间充质干细胞增殖及成骨能力均下降，人骨髓间充质干细胞外泌体可改善卵巢切除术大鼠骨髓间充质干细胞的成骨能力。LI等[50]通过miRNA-seq研究人骨髓间充质干细胞外泌体成骨作用的分子机制，结果显示，在外泌体+卵巢切除术组中有9个miRNA表达上调，5个miRNA表达下调，其中miR-186在外泌体+卵巢切除术组中的表达明显高于卵巢切除术组。体外实验也证实miR-186在外泌体+卵巢切除术大鼠的骨组织中上调。分子机制研究显示外泌体miR-186通过hippo信号通路调控卵巢切除大鼠成骨。 (8)miR-25：有研究发现miR-25通过上调Rac1的表达促进成骨细胞增殖、分化和迁移[51]。后来，JIANG等[52]发现外泌体miR-25介导的骨髓间充质干细胞外泌体促进MC3T3-E1C细胞的成骨分化、增殖和迁移，并且骨髓间充质干细胞外泌体可促进小鼠骨折愈合，但是抑制骨髓间充质干细胞外泌体内miR-25则无此种促进作用，进一步研究发现，SMURF1是miR-25下游基因，miR-25通过SMURF1促进MC3T3-E1C细胞的成骨分化、增殖和迁移。SMURF1通过促进Runx2泛素化降解抑制Runx2蛋白的表达。值得注意的是，骨髓间充质干细胞外泌体分泌的miR-25可通过SMURF1/Runx2轴加速成骨细胞的成骨分化、增殖和迁移，体外实验表明，骨髓间充质干细胞外泌体中的miR-25通过SMURF1调控Runx2的泛素降解，促进小鼠骨折愈合。 2.4.2 血源性干细胞外泌体miRNA促进骨再生 (1)miR-5106：巨噬细胞对骨折愈合的所有阶段都是至关重要的，是最早迁移到骨折部位的细胞。对于愈合过程尤为重要的是巨噬细胞表型从促炎性的M1亚型转变为抗炎性的M2亚型，而不愈合往往与延长的促炎性巨噬细胞反应相关[53]。XIONG等[54]发现M2亚型来源的外泌体(M2 macrophage-derived exosomes，M2D-Exos)能促进骨髓间充质干细胞成骨，而M1亚型来源的外泌体(M1 macrophage-derived exosomes，M1D-Exos)抑制骨髓间充质干细胞成骨；体内实验显示M2D-Exos加速小鼠股骨骨折愈合。微阵列鉴定M1D-Exos和M2D-Exos中差异表达的miRNA，发现M2D-Exos 中miR-709、miR-3112-3p、miR-3069-3p、miR-6406、miR-6389、miR-5106、miR-8103、miR-1934-3p、miR-22-3p、miR-505-5p上调，其中miR-5106上调最为显著并可从M2D-Exos传递至骨髓间充质干细胞。此外，用miR-5106抑制剂处理M2巨噬细胞并提取外泌体(ExosantagomiR- 5106)，发现ExosantagomiR-5106抑制骨髓间充质干细胞成骨并抑制大鼠骨折愈合，分子机制研究显示miR-5106直接靶向SIK2和SIK3参与骨髓间充质干细胞成骨分化。 (2)miR-26a：CD34+干细胞是一组血管祖细胞，具有非凡的血管生成特性[55]。CD34+干细胞来源外泌体(CD34+-Exos)移植可通过促进新生血管形成治疗缺血性疾病[56]，miR-26a是重要的成骨正向调节因子[57-58]。ZUO等[59]在CD34+-Exos中过表达miR-26a(miR-26a-CD34+-Exos)，然后通过体外迁移和管形成能力评估miR-26a-CD34+-Exos的血管生成能力，发现miR-26a-CD34+-Exos增强了人脐静脉内皮细胞的迁移和成管能力。此外，为了观察miR-26a-CD34+-Exos对骨髓基质细胞的成骨作用，采用茜素红染色、碱性磷酸酶活性测定和qPCR检测，发现miR-26a-CD34+-Exos可在体外糖皮质激素作用下增强骨髓间充质干细胞的成骨分化。最后，将miR-26a-CD34+-Exos注射到糖皮质激素诱导的股骨头坏死大鼠模型中，发现miR-26a-CD34+-Exos增加了股骨头坏死大鼠股骨头血管密度和骨小梁完整性，从而抑制了股骨头坏死的进展。 2.4.3 其他 (1)miR-27a-3p：越来越多的证据表明，骨骼和肌肉组织作为内分泌器官都能影响葡萄糖、能量和其他代谢。最近有研究表明，骨和肌肉也可以通过内分泌的方式相互调节[60-61]。C2C12成肌细胞源性外泌体可进入前成骨细胞MC3T3-E1，促进其成骨分化。具有成骨功能的miR-27a-3p在C2C12成肌细胞源性外泌体中富集，此外泌体miR-27a-3p可能通过靶向APC调控成骨细胞分化，从而激活对成骨有重要作用的β-catenin通路以促进成骨[62]。 (2)miR-181b-5p：机械张力可促进大鼠骨髓间充质干细胞成骨分化[63]。在炎症环境中，机械张力下MLO-Y4细胞分泌的外泌体不仅能促进牙周膜干细胞的增殖，还能促进成骨分化；GO富集分析表明，机械张力下MLO-Y4细胞来源外泌体miRNA在代谢过程中起关键作用。在差异表达的miRNA中，选择miR-181b-5p进行进一步研究，发现外泌体miR-181b-5p通过PTEN/AKT信号通路调控牙周膜干细胞增殖[64]。 (3)miR-130a-3p：通过成骨诱导可使细胞外泌体miRNA表达谱发生改变，部分增强细胞成骨分化。诱导成骨分化14 d 的脂肪间充质干细胞外泌体(Exos-D14)较未诱导成骨分化的脂肪间充质干细胞外泌体(Exos-D0)miR-130a-3p显著增加。过表达miR-130a-3p可增强脂肪间充质干细胞外泌体成骨分化，同时降低miR-130a-3p靶点SIRT7的蛋白和mRNA水平。进一步发现，过表达miR-130a-3p可导致SIRT7表达下调，Wnt信号通路相关蛋白上调。研究结果表明，外泌体miR-130a-3p可部分通过介导SIRT7/Wnt/β-catenin轴促进脂肪间充质干细胞外泌体的成骨分化，从而促进外泌体miRNA在骨再生领域的应用[65]。 2.5 外泌体miRNA促进成血管骨再生成功的关键在于是否有血管化发生，以便输送足够的营养物质、生长因子、矿物质和氧气用于组织修复[66]。多项研究发现携带特定miRNA的外泌体有成骨及成血管的双重作用。携带miR-29a、 miR-122-5p、miR-146a、miR-26a的外泌体都有这种成骨和成血管的双重作用，除此之外，外泌体miR-126也有此作用，与骨折愈合密切相关。有学者发现外泌体miR-126能同时促进成骨及成血管，JIA等[67]对SD大鼠行截骨术后进行牵张成骨，在骨缝隙内注射SD大鼠内皮祖细胞来源外泌体，与对照组比较，内皮祖细胞来源外泌体组能显著促进成骨和血管生成。后来，LIU等[9]发现低氧通过低氧诱导因子1α促进人脐带间充质干细胞外泌体释放并使其中miR-126上调，体内实验发现此种外泌体通过成血管促进小鼠骨折愈合，体外实验发现此种外泌体通过SPRED1/Ras/Erk途径促进人脐静脉内皮细胞增殖、血管生成和迁移，敲除外泌体miR-126可抑制人脐静脉内皮细胞的增殖、血管生成和迁移能力，体内注射敲除miR-126的外泌体可显著减少新生血管并抑制内皮细胞增殖，可见外泌体miR-126在成血管方面有重要作用，并可能因此促进骨折愈合。外泌体miR-126在成骨和成血管生成中有重要作用，未来有可能用于骨折的治疗。"

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