外泌体于1983年由Pan和Johnstone[1]在研究绵羊网织红细胞体外成熟过程中被首次发现,他们发现网织红细胞能够分泌诸多包含多肽的囊泡,随后Johnstone等[2]将这种囊泡命名为“exosomes”,即为外泌体。最初研究者认为外泌体可能是清除细胞内退化蛋白质等代谢废物的载体[3],但2007年Valadi等[4]证明外泌体可作为细胞间交流的一种新的介质,且这一发现在随后的多数研究中得到证实[4-7]。外泌体的结构组成和功能研究成为新的研究热点。外泌体包含核酸、蛋白质和脂质等物质,核酸多为具有调控功能的小RNA,可以稳定存在并能够通过膜囊泡运输进入细胞[8-9],参与细胞的表观遗传调控[10]。外泌体上的一些蛋白质具有较高的细胞特异性,可以作为某些疾病的生物标记物[11-12]。近几年,外泌体在人类医学领域研究较多,其作为重要的生物标记物和信息传递介质,为包括癌症、HIV等疑难杂症的诊断[13-14]和靶向治疗[15-16]提供新的契机。外泌体在畜牧养殖中的研究目前相对较少。寄生虫病严重影响畜禽健康,是畜牧养殖中传播快、危害大的重要病害之一,目前主要依赖药用植物提取物和化学合成药物进行预防和治疗。寄生虫在宿主内的寄生机制复杂,研究表明寄生虫能够通过分泌外泌体与宿主发生信息交流,调控宿主的免疫反应[17],最终为寄生虫的寄生服务。寄生虫来源外泌体的深入研究对寄生虫病的早期诊断及靶向给药具有重要意义,外泌体可能成为寄生虫病诊疗的新切入点,促进畜禽养殖业的健康发展。
1 外泌体概述 1.1 外泌体的形成外泌体(exosomes)是一类由细胞分泌的囊泡,其广泛存在于血液、尿液、唾液和羊水等体液中,在体外培养的细胞及寄生虫上清液中也存在外泌体[18-19]。外泌体与微囊泡、凋亡小体、高密度脂蛋白和低密度脂蛋白都属于细胞外囊泡(extracellular vesicles),区别在于形态大小和形成方式的不同[20]。微囊泡直径200~2 000 nm,细胞受到刺激后细胞质膜的脂质重新分布导致微囊泡形成[21];凋亡小体直径500~2 000 nm,是细胞凋亡过程中细胞萎缩、碎裂形成的由膜包围的含有细胞核和细胞质碎片的小体[22];低密度脂蛋白直径21~27 nm,高密度脂蛋白直径7~13 nm[20];外泌体直径40~200 nm[20],外泌体的确切形成机制仍存在分歧。一般认为外泌体的形成与转运必需内体分选复合物(endosomal sorting complex required for transport,ESCRT)有关[23]。图 1展示了外泌体的形成过程与在细胞间传递的过程。细胞A细胞膜内陷形成包含信息物质的内吞小泡,内吞小泡相互融合形成早期核内体(early endsomes,EES),早期核内体在ESCRT系统的作用下形成晚期核内体(late endsomes,LES),即多泡体(multivesicular bodies,MVB),MVB与溶酶体融合则被降解,与细胞质膜融合则形成外泌体。外泌体形成后经胞吐方式释放,再经胞吞作用与细胞B的质膜融合将信息物质释放到细胞B中,发挥调控作用。
不同细胞来源的外泌体组分有所不同,但总体上都包括蛋白质、脂质和核酸。外泌体含有丰富的蛋白质且功能各异,包括内吞小泡形成相关蛋白[25](RAB27A、RAB11B和ARF6等)、多泡体形成相关蛋白[26](Alix和TSG101等)、热休克蛋白(HSP70和HSP90等)和跨膜蛋白家族[27](CD9、CD63、CD81和CD82)等,其中Alix、HSP70和跨膜蛋白家族是外泌体的标记蛋白,常用于外泌体鉴定。外泌体中的某些蛋白质还可作为疾病诊断中的生物标记[11-12]。外泌体中的脂质成分主要包含鞘磷脂、胆固醇、神经节苷脂GM3等[28],脂质具有维持外泌体的形态及参与细胞间信息交流的作用[29]。外泌体中的核酸成分包括mRNA和miRNA,其中主要为miRNA[4]。miRNA可促进肿瘤细胞的发生发展[10]和参与神经信号转导[30]等。此外,外泌体中还含有DNA[31]、tRNA和piRNA[32]等片段。
2 外泌体对寄生虫的生物学作用寄生虫病是危害畜禽健康的重要潜伏性疾病,可以通过饲料、饮水、粪便、物理接触以及吸血昆虫和垂直传播感染,且可通过动物源性食品危害人类的健康。寄生虫在宿主体内的寄生机制复杂,现有研究表明,毛滴虫、疟原虫、弓形虫和锥虫等多数寄生虫都可分泌外泌体,称为虫源外泌体[33]。外泌体是细胞间信息交流的重要途径之一,它在抗原提呈[34]、信号转导[30]等过程中都发挥着重要作用。虫源外泌体可以作为寄生虫与宿主之间联系的纽带,一方面:寄生虫在多种机制刺激下产生外泌体,外泌体包裹着大量的虫源性信息进入宿主体液及细胞内,进而调节宿主的免疫系统,抑制炎症反应,改善其寄生环境;另一方面,外泌体可直接携带有毒有害物质进入宿主循环系统。不同虫源外泌体的作用机制不尽相同[35-50](表 1)。
毛滴虫呈梨形,有4根前鞭毛,种类可分为阴道毛滴虫、禽毛滴虫、口腔毛滴虫和胎毛滴虫等[51]。毛滴虫对家禽和生猪危害严重,生产中常使用化学药物防治毛滴虫,但容易产生耐药性并存在抗生素残留的问题。目前,尚未见有关畜禽毛滴虫外泌体的报道,多数研究集中于人类阴道毛滴虫外泌体。Twu等[35]首次对阴道毛滴虫来源的外泌体进行了鉴定,并对其功能进行了深入研究。研究者通过透射电子显微镜、Western blotting以及纳米颗粒跟踪分析(Nanoparticle Tracking Analysis)鉴定阴道毛滴虫的囊泡形态、膜表面标志蛋白和粒径大小,并由此确定阴道毛滴虫可以分泌外泌体。随后发现在低黏附力的阴道毛滴虫和宿主细胞共孵育过程中添加高黏附力阴道毛滴虫外泌体时,低黏附力虫株对宿主细胞的黏附力显著增强,而添加低黏附力阴道毛滴虫外泌体时,黏附力没有显著增加,黏附力是阴道毛滴虫发挥致病性的重要因素[52],表明外泌体中某些内容物可能参与调控寄生虫致病力。且当虫源外泌体和宿主细胞共孵育后感染阴道毛滴虫,白细胞介素-8(IL-8)的表达量呈现下降趋势,进而影响嗜中性粒细胞向感染部位的迁移[53],嗜中性粒细胞通过凝集素途径或旁路途经激活补体而发挥杀虫作用,有氧环境下产生的活性氧中间物也可以破坏虫体的细胞膜[54],因此外泌体引起的IL-8的降低有利于寄生虫在宿主体内的存活。Olmos-Ortiz等[55]的研究也证明了阴道毛滴虫外泌体影响细胞因子的表达,利用近交系小鼠开展阴道毛滴虫攻毒试验,对照组正常攻毒,处理组在攻毒前阴道处涂抹一定浓度的阴道毛滴虫外泌体,结果显示,与对照组相比,处理组的细胞因子IL-10表达增加了15倍以上。IL-10作为一种可抑制单核巨噬细胞释放炎症介质的白细胞介素[56],其表达量大量增加有利于阴道毛滴虫的寄生。虫源外泌体可以增加阴道毛滴虫对宿主细胞的黏附力,增强致病力;并通过调节细胞因子影响宿主的免疫应答,但这些作用具体和外泌体哪些组分有关,作用机制如何,目前尚未见报道。
2.2 疟原虫疟原虫常寄生于红细胞内,致病力较强,可造成68%~75%的感染鸡死亡[57],对家禽生产危害严重。疟原虫感染的红细胞外泌体可以有效地活化巨噬细胞[37],巨噬细胞有助于CD8+ T细胞的局部增殖,最终导致感染疟原虫的小鼠死亡[58]。这种机制可能与MyD88和TLR4信号通路有关,当这两个信号通路被切断,外泌体诱导的巨噬细胞活化被完全消除[36]。除了在激活先天免疫方面的作用,外泌体还可以调控细胞因子的表达。Mantel等[37]发现恶性疟原虫外泌体促进细胞因子IL-6的表达。IL-6可以抑制肝细胞内疟原虫的生长发育[59],导致宿主的急性炎症反应,阻止疟原虫的感染。
外泌体不仅可以调控免疫反应,还能作为寄生虫和宿主之间或寄生虫之间信息交流的介质。Regev-Rudzki等[60]通过不同药物抗性的恶性疟原虫与红细胞共培养,发现外泌体可以通过在疟原虫之间转移核酸传递耐药性。此外,有研究发现疟原虫未裂解红细胞前,在其性成熟和分化期间,外泌体的释放量会明显增加,通过进一步的体外试验,纯化的外泌体刺激无性期的恶性疟原虫,会使其分化为配子,即有感染蚊子能力的虫体,表明外泌体在恶性疟原虫的分化中有其作用,改变外泌体分泌的水平会直接影响恶性疟原虫配子的数量[37]。
此外,外泌体还可以作为鉴定无症状带虫者可能的生物学方式。间日疟原虫起源于非洲地区[61],寄生虫在初次感染后的几年内可以在肝中潜伏,而不会引起任何症状,这种状态称为潜伏期。潜伏期的宿主疾病复发并将寄生虫传播给其他人称为潜伏期的再激活。目前没有潜伏期疟原虫感染的诊断方法。而Gualdrón-López等[62]利用被间日疟原虫感染的含有人肝细胞的“人源化”小鼠,从中分离的人源外泌体中含有17种与潜伏期再激活有关的蛋白质,这些结果表明,肝组织来源的外泌体可能是帮助我们识别潜伏期再激活感染的生物标志物。疟原虫外泌体可以传递耐药性,调节免疫反应,作为疟原虫病早期诊断的有效切入点,这些作用都暗示着外泌体或许是疟疾防治的新突破点。
2.3 弓形虫弓形虫病是一种由刚地弓形虫引起的人和动物的共患病,感染猪、牛、羊等家畜,造成严重的经济损失,且可通过动物源性食品危害人类健康,孕妇受到的危害极大,可导致流产、早产、畸胎或者死胎[63]。Pope和Lässer[43]通过微阵列法对比被弓形虫感染的宿主细胞及其外泌体mRNA和miRNA的表达情况,其中EEF1A1、TMSB4X、LLPH和Rab-13等4种mRNA在弓形虫感染组中高表达,且这4种mRNA调控与神经活动有关的蛋白质;miR-23b在感染组中特异表达,这种miRNA可以作为抗炎介质抑制宿主细胞因子IL-17的表达。由此推测弓形虫分泌的外泌体可能通过mRNA调控某些蛋白质进而影响宿主的免疫应答。此外,Li等[41]的研究也揭示了外泌体对免疫反应的调控作用,其结果表明弓形虫外泌体可以调节巨噬细胞的体外活化,激活体液和细胞免疫应答,同时对急性寄生虫感染有保护作用。进一步研究发现弓形虫外泌体可激活末端激酶通路,使IL-12和干扰素的表达量明显增加,即弓形虫外泌体可能通过激活末端激酶通路调控宿主免疫反应[42]。上述研究表明,弓形虫外泌体可以通过不同的途径影响宿主的免疫应答。然而Kim等[64]的研究发现,外泌体的作用并不局限于此,弓形虫的寄生可改变宿主细胞增殖,甚至可以引起邻近细胞的增殖变化,并且虫源外泌体的微阵列分析表明,外泌体miRNA对于调节细胞增殖相关的靶基因至关重要,虫源外泌体可以改变细胞周期并影响宿主细胞的增殖。由此,弓形虫外泌体不仅可以调控宿主免疫应答,还可能通过改变细胞周期影响宿主细胞的增殖,其在弓形虫寄生过程中的作用机制值得深入探究。
2.4 锥虫锥虫寄生于脊椎动物血液和组织引起锥虫病,在牛、马、驴和骆驼等畜种中感染严重。牛锥虫病发病率高、死亡率极高,不仅会致养殖者经济效益受到影响,且可通过动物性食品对食品安全造成较大的危害。目前关于布氏锥虫和克氏锥虫外泌体的研究较多,Szempruch等[65]揭示了布氏锥虫鞭毛膜出芽形成动态膜纳米管并由此形成外泌体。布氏锥虫外泌体通过鞭毛膜脂质双层融合的形式在寄生虫之间转移血清抗性相关蛋白。血清抗性相关蛋白的表达可抑制锥虫裂解因子的活性,使得布氏锥虫具有锥虫裂解因子耐受性,实现免疫逃逸。此外,布氏锥虫外泌体也可与宿主红细胞细胞膜高度融合,从而改变红细胞细胞膜的物理性质,造成红细胞被迅速清除,导致动物和人类的贫血。对克氏锥虫外泌体的研究显示,克氏锥虫外泌体帮助其实现免疫逃逸,有利于克氏锥虫的寄生[66-67]。此外,克氏锥虫外泌体在宿主细胞免疫应答的不同时期发挥着不同的调节作用,且可作为先天免疫的弱激活剂[68]。这些研究表明,外泌体导致了锥虫病的复杂性,它是一种可在寄生虫之间和寄生虫与宿主细胞之间传输非遗传性毒力因子、并帮助寄生虫实现免疫逃逸的细胞器。研究锥虫外泌体有助于探索锥虫病的发病机制,为该病的治疗提供新思路。
2.5 其他寄生虫血吸虫[48, 69-70]、利什曼原虫[44-45]、钩虫[47]及线虫[71-72]等多种寄生虫都能够释放外泌体。利什曼原虫外泌体可以转运到宿主巨噬细胞中,破坏宿主的内环境平衡,激活其表面糖蛋白GP63,GP63可影响外泌体对传递的内容物的选择并抑制宿主miRNA的加工[44],还有研究表明利什曼原虫外泌体可以释放毒力因子,抑制宿主巨噬细胞的活性,进而实现免疫逃逸[73]。研究发现,钩虫[47]和鼠鞭虫[64]分泌物中也存在外泌体,外泌体miRNA调节宿主的免疫应答,与Fromm等[74-75]的研究结果类似。Fromm等[74-75]发现肝片吸虫外泌体中含有55条miRNA,其中13条新发现的miRNA,且miR-10和let-7等家族成员可参与免疫调控。Samoil等[69]研究血吸虫外泌体的蛋白组学,结果显示外泌体中含有血吸虫与宿主相互作用的重要蛋白质,包括转移酶、钙蛋白酶等。Li等[46]研究了孢子虫外泌体,发现其可以迅速内化,将蛋白质传递给宿主细胞,并通过MAPK信号通路以TLR2依赖的方式调节宿主细胞的免疫应答。Buck等[76]对线虫的研究表明,虫源外泌体可以调控宿主的Dusp1基因,该基因是MAPK信号转导的关键调节因子并抑制Toll样受体的表达,从而调控免疫反应。这些寄生虫外泌体都参与调控宿主的免疫应答,但具体的作用机制尚不清楚,需要进一步探究。
3 展望寄生虫病是影响畜禽养殖的复杂疾病之一,大部分虫源外泌体都可以通过调节细胞因子的表达干扰宿主的免疫反应。在畜禽行业中,寄生虫源外泌体的研究尚不够深入,且多参考人类肿瘤等相关领域的外泌体研究。寄生虫源外泌体的研究面临诸多难点和挑战:其一,寄生虫与寄生部位的组织均能够分泌外泌体,虫源外泌体与宿主源外泌体难以区分;其二,组织外泌体的提取尚处于起步阶段;其三,虫源外泌体作用于宿主的机制仍不明确,其调控宿主细胞的生物活性物质还有待研究。虽然面对诸多技术难题,但虫源外泌体具有广阔的应用前景:应用于寄生虫病的快速诊断;通过筛选虫源外泌体特异表达的miRNA,研究其调控宿主免疫应答的机制,从分子水平阻断寄生虫的感染;应用于抗寄生虫病疫苗的研发等。Ofir-Birin等[77]研发的基于流式细胞成像技术的检测方法的建立为研究宿主细胞对寄生虫外泌体的摄取提供了可行的研究方法,且寄生虫外泌体的数据已经被收录到EuPathDB数据库(https://eupathdb.org/eupathdb/)中,这些新技术和新成果的应用将进一步推动寄生虫外泌体的研究。因此,对外泌体作用机制的深入研究将有助于寄生虫病的防治,促进畜禽养殖业的健康发展。
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