·综 述·
1983年,Pan等[1]在体外培养绵羊网织红细胞时发现一种从未发现的细胞外囊泡,1987年被命名为外泌体[2]。作为一种传递信号的介质,外泌体在细胞与细胞间、细胞与组织间起到了多种调控作用,并在心血管疾病中扮演着重要的角色。
在心血管疾病如冠状动脉粥样硬化型心脏病(coronary atherosclerotic heart disease, CAD)过程中,外泌体的介导作用是一把双刃剑,既可导致心肌细胞损伤[3-7],也可产生心肌保护作用,这与外泌体来源细胞的种类和诱导因素相关。如血管平滑肌细胞外泌体会介导miR-155破坏血管内皮,而单核巨噬细胞受尼古丁诱导后产生的外泌体会促进血管平滑肌细胞迁移及增殖,延缓冠脉狭窄的进展[8-9]。
根据《中国心血管健康与疾病报告2019》,CAD是我国城乡居民疾病死亡的首要原因,其中急性心肌梗死(acute myocardial infarction, AMI)的死亡率呈快速上升趋势[10]。由于冠状动脉粥样斑块破裂形成的血栓堵塞或冠脉痉挛等原因,冠状动脉发生急性、持续性闭塞,从而导致心肌血供不足,心肌细胞缺血缺氧,因而AMI有着起病急、进展快、预后差的特点。目前AMI的治疗手段以溶栓和介入治疗为主,即使及时接受了治疗,患者仍可能遭受心脏破裂、心律失常、心力衰竭或心源性休克等并发症的折磨,心源性猝死风险大大提升[11-13]。因而研究外泌体介导AMI的作用机制,有助于了解AMI病理生理过程,以利开发新的诊疗方式。
已有众多报道表明外泌体在AMI后心脏具有修复作用[14-16],外泌体在临床应用中具有巨大潜力,有望为防治心脏疾病开辟一条蹊径。
外泌体形态呈杯状,直径为50~150 nm,具有双层脂质膜结构[17]。外泌体通过旁分泌途径传递信息,其起源于多囊体(multivesicular bodys, MVBs)产生的腔内囊泡,在与质膜融合后向胞外分泌[18],释放受哺乳动物雷帕霉素靶蛋白复合体1(mechanistic target of rapamycin complex 1, mTORC1)开关调节[19]。
靶细胞通过内吞途径摄取外泌体。目前已经发现的外泌体内吞途径包括网格蛋白依赖的内吞作用、小窝蛋白依赖性内吞作用、大胞饮作用、吞噬作用以及脂筏介导的内吞作用等[20-23]。在对大鼠乳鼠来源的心肌成纤维细胞、心肌细胞和小鼠骨髓来源树突状细胞的通讯观察中发现,靶细胞对外泌体的吞噬具有选择性,外泌体的内吞作用通过受体介导的调节机制实现[7,24]。研究外泌体释放和摄取的机制有助于解析其在细胞间的作用机制,为外泌体介导和干预AMI发病机制指明新方向。
外泌体通过转运内含物传递细胞间信号,从而参与体内多种生理及病理过程,如介导细胞死亡、调节炎症过程、促进病毒传播等[25-29]。目前已发现的内含物种类包含核酸、蛋白质、糖类、脂质等,其中含RNA超过10 000种,RNA是外泌体中最主要的信号传递分子[30]。
不同来源的外泌体在不同的机体环境下所产生的作用也不尽相同[7]。对外泌体内含物参与的信号通路的解密,有助于揭露外泌体介导AMI过程的相关机制。
外泌体可以将囊泡内的RNA转运到靶细胞中,其RNA种类十分丰富,包括miRNA、lncRNA、cRNA、siRNA等,但目前研究主要集中于miRNA和lncRNA。外泌体同样运载DNA,但鲜有报道其生物学意义。
1.2.1 外泌体miRNA在AMI中的作用 AMI发生后心肌细胞因缺血缺氧受到损伤,虽然适时溶栓或介入治疗降低了AMI死亡率,但无法逆转梗死区心肌细胞的受损及纤维化的发展,而外泌体miRNA介导AMI后修复方式是近5年研究热点之一。AMI后再灌注过程中,心脏成纤维细胞通过miR-423-3p抑制RAP2C,增强心肌细胞对复氧的耐受,同时心肌球细胞外泌体中的miR-181b可通过miRNA与蛋白质的协同作用抑制促凋亡蛋白PKCδ,阻止心肌细胞发生凋亡[31-32]。AMI后血清中的循环外泌体可以通过富集miR-1956激活脂肪间充质干细胞诱导血管内皮生长因子的释放,同时下调miR-939-5p表达从而增强iNOS-NO活性,促进血管新生[33-34]。此外,胚胎干细胞分泌的外泌体中miR-294靶向修复受损心肌细胞,增强梗死后心脏的功能[35]。但是AMI后巨噬细胞外泌体通过传递促炎信号加重心肌损伤,这些外泌体miRNA(如miR-155或miR-182)表达下降能够靶向抑制巨噬细胞,逆转AMI后心脏重构[4,36-37]。由此可见不同细胞外泌体miRNA对AMI后修复作用不尽相同。
1.2.2 外泌体lncRNA在AMI中的作用 目前已知的外泌体lncRNA对心脏的保护作用大多建立在干细胞相关研究中,需与miRNA协同发生。如人骨髓间充质干细胞外泌体中的lncRNA KLF3-AS1通过miR-138-5P/SIRT1轴抑制AMI过程中心肌细胞的焦亡[26]。在动物实验中,大鼠间充质干细胞的lncRNA-UCA1通过靶向miR-873,解除对抗凋亡因子XIAP的抑制作用后同时增加抗凋亡蛋白BCL2的表达,从而维持细胞生存[38]。阿托伐他汀作为降胆固醇药物,在AMI治疗中起到关键作用,阿托伐他汀预处理后的大鼠间充质干细胞会以lncRNA H19为媒介促使miR-675表达,激活血管内皮生长因子和细胞黏附分子-1(cell adhesion molecule-1, CAM-1),从而促进血管新生,同时抑制心肌细胞凋亡[39]。
研究表明,外泌体内装载的蛋白有心脏保护作用。从成年雄性大鼠和健康的人类男性志愿者的血液中纯化出外泌体,外泌体携带的热休克蛋白70(heat shock protein 70, HSP70)通过刺激Toll样受体4(Toll-like receptors 4, TLR4)信号通路激活HSP27,从而保护受损心肌[40]。
向AMI小鼠心肌内注射富含白细胞介素-10(interleukin-10,IL-10)的小鼠心脏内皮细胞外泌体,显著改善了左心室心功能,抑制了细胞凋亡,减小了MI瘢痕大小,促进了MI后新血管生成,而注射敲除了IL-10的小鼠心脏内皮细胞外泌体显示出了完全相反的治疗效果[41]。
临床上用于确诊AMI的血清学指标主要是肌钙蛋白(troponin, Tn)I或T。其中TNI在AMI起病3~4 h后方才升高明显,因此以TNI为诊断指标可能会错过AMI治疗的最佳时机[11-12]。此外,其他非AMI因素心肌损伤也可导致TNI升高,对临床诊断工作会造成一定干扰。因此迫切需要更多AMI早期诊断标志物用于临床诊断。众多研究证据表明,外泌体中的miRNA和lncRNA可为AMI早期诊断提供新的依据。
对AMI患者血清和血浆进行检测,会发现心肌细胞来源的miR-1、miR-133、miR-208和miR-499表达量升高[42-44]。而在AMI小鼠模型中验证确定外泌体miR-1a、miR-208a和miR-499-5p在血液循环中升高,而血浆中的非外泌体部分可检出升高的miR-133a[42]。此外,AMI患者循环外泌体中还存在miR-126、miR-183和PTEN基因的高表达,lncRNA-UCA1、lncRNA-NEAT1和lncRNA MMP-9等lncRNA含量也较常人更丰富,而miR-21、miR-204的表达量有所下调[38,45-47],其中,miR-126、miR-183的高表达与患者的心肌缺血程度呈正相关[45-46]。因此,外泌体中的miRNA和lncRNA具有预测心肌缺血损伤程度和诊断AMI的潜力。AMI后1年内发作心力衰竭的患者血清中检测出了高表达的miR-192,患者血清外泌体中的miR-194及miR-34a的表达与之协同上调,这3种miRNA具有预测AMI后1年内发生心力衰竭的潜力[48]。
AMI往往导致心肌长时间缺血缺氧,致使心肌细胞形成不可逆的损伤和坏死[49],因此对外泌体的利用主要聚焦于对再灌注后受损心肌的修复,治疗思路为阻断损伤性外泌体的摄取、增强治疗性外泌体的传递[50-51]。
在相关的研究中,往往将外泌体与基因工程相结合,借助外泌体对基因的运载和释放功能,从基因层面实现对AMI的靶向治疗[52-53]。例如过表达低氧诱导因子-1(hypoxia inducible factor-1, HIF-1)可上调心脏内皮细胞分泌的外泌体中的miR-126和miR-210表达量,其中miR-126上调Vegfa和Fgf2基因的表达,促进血管新生,而miR-210降低线粒体代谢,从而提升被移植的心脏祖细胞的存活率[54]。
在此基础上,以外泌体为载体,将外泌体与利于心肌修复的信号分子整合为治疗用药,可以减少信号分子在体内的代谢消耗,定向、定量、靶向AMI区域局部给药[50-52]。Song等[53]通过向梗死区心肌注射外泌体,将装载的外源性miR-21导入AMI小鼠心肌细胞和内皮细胞,从而大大抑制了细胞凋亡,使心功能得到明显改善。北卡罗来纳州立大学陈柯团队[55]直接向AMI大鼠梗死心肌区域内注射人正常心脏组织来源的外泌体,发现其能够阻止小鼠左心室射血分数下降,减轻AMI后心脏重塑。
在大型动物模型中,局部外泌体注射治疗的疗效也得到了验证。美国西奈山医学院Eduardo Marban研究组将外泌体对心脏的保护作用在猪AMI模型上进行了验证。Eduardo Marban研究组[32]构建了大鼠和猪的AMI模型后,向两种模型的心肌梗死区均注射了人心肌球衍生细胞源外泌体,结果显示梗死区域瘢痕减小,减轻不利的重塑,且改善梗死后左心室射血分数。美国阿拉巴马大学伯明翰分校张建一教授和国内同济大学附属东方医院高崚团队合作[50],同样构建了猪AMI模型,报道了由人诱导多能干细胞(hiPSCs)衍生的心脏细胞外泌体于心脏梗死区域注射治疗后,可改善梗死猪心脏的细胞能量代谢,改善了猪AMI模型的心脏功能并限制了不良重塑,并且不会增加心律失常并发症的发生率,为心肌损伤提供了无细胞治疗的新选择。
两方团队都尝试冠脉内注射予药,然而疗效则无,这提示外泌体治疗的予药需集中在梗死局部,减少体内循环代谢对外泌体的消耗。对此,亦有研究提出通过利用磁性吸附的原理,局部募集外泌体,使之在梗死区域释放的可能性,并在不同动物AMI模型中予以了尝试[56]。
干细胞移植是AMI后心肌修复治疗的前沿研究方向[57],在动物实验中已证明干细胞能够帮助AMI后梗死区心肌组织再生和修复,但是移植后细胞存活率不高,严重制约了干细胞移植的临床应用[50-54]。
心肌内注射人胚胎干细胞来源的心脏祖细胞外泌体或小鼠胚胎干细胞外泌体同样可以改善小鼠AMI后心功能[13,35],既能收获与干细胞移植同等的效益,又可以规避干细胞移植存活率低的局面。
外泌体还可以用于抑制干细胞移植治疗后发生的免疫反应,减轻移植后排斥反应。人脐带间充质干细胞通过miR-24/Bim途径降低了移植后排斥反应,从而提高移植细胞生存率[58]。
目前外泌体在AMI等心血管疾病中的研究尚处在起步阶段,集中于外泌体在细胞和组织水平上通讯载体功能,主要围绕着AMI过程中的炎症反应、梗死面积的加重等损伤过程,抑或是减少心肌细胞死亡、刺激血管新生等的保护机制,具有诊断和治疗AMI的潜力。
由于外泌体粒径单位只有纳米级别,在电荷、粒径大小上都很难与其他细胞囊泡或者成分分离,因而如何分离和纯化外泌体一直是研究中的一大难题。依托于科学技术的进步,外泌体的检测方法也得到发展[56,59-60],如特异性配体受体结合或者磁珠捕捉法、纳米流式分析系统、原子力显微红外光谱仪等,从而简化外泌体的定性定量流程[60-63]。微流控外泌体捕捉芯片,因其对外泌体样品需求量小、检测灵敏度高,具有成为健康检测和疾病诊断工具的潜能[64-65]。有研究团队以仿生外泌体粒子为灵感,通过技术手段人为建造外泌体模拟纳米囊泡,提升了其可控性和可修饰性,在外泌体的生产技术领域实现革命性突破[66-68]。
随着外泌体检验技术的发展,其检验手段已经初步具备临床检验的能力,因此外泌体具有良好的临床应用前景,值得受到更多研究者的关注。
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