中华急诊医学杂志  2019, Vol. 28 Issue (5): 652-654   DOI: 10.3760/cma.j.issn.1671-0282.2019.05.028
外泌体的体内代谢进展
朱亚婕 , 王杨 , 唐嘉隆 , 潘曙明     
上海交通大学医学院附属新华医院急诊科 200092

外泌体(exosome)是一种由细胞主动分泌的具有脂质双分子层膜结构的小囊泡,直径约30~100 nm,表面表达CD63、CD9、CD81、MHC-1等标志物,内含蛋白质、核酸等物质[1-2]。外泌体是一种重要的细胞间通讯工具,多种细胞均都可分泌外泌体,将蛋白质、mRNA、miRNA等物质送达受体细胞,调控细胞行为,在肿瘤转移、免疫识别、炎症反应等众多生理病理过程中扮演重要角色[3-4]。近年来外泌体的治疗作用逐渐得到重视,间充质干细胞(mesenchymal stem cells,MSC)来源外泌体可以治疗急性肺损伤、急性肾衰竭、脑梗死等多种疾病,并已开始相关临床试验[5-7]。药物在体内的代谢动力学是其发挥疗效的基础[8], 故认识外泌体在体内的代谢特点十分重要。

1 外泌体的生成与释放

外泌体最初被误认为细胞用来排泄废物的小泡。随后研究发现外泌体的生成和释放是受细胞精密调控的。外泌体在细胞内的多泡体(multivesicular bodies,MVBs)中产生,由ESCRT、TM4SF、RAB、SNARE等一系列蛋白参与其形成、运输、转移与释放[9-11]。不同细胞来源的外泌体其大小、表面受体和内容物往往具有不同特征[12]。外泌体释放后可广泛分布于血清、尿液、唾液、母乳、精液等体液中,并通过血液循环到全身。

2 外泌体的摄取机制

细胞可通过多种方式摄取外泌体,包括直接融合、网格蛋白/小窝蛋白依赖的胞吞途径、巨胞饮、吞噬及脂筏介导的内吞途径[11, 13-14]。细胞对外泌体的摄取行为是主动耗能的,摄取量大小取决于受体细胞本身[15]。此外,不同细胞摄取外泌体的方式有所侧重,往往依赖于上述某一种途径,故可通过抑制其关键蛋白如小窝蛋白、网格蛋白来抑制相应的摄取行为。同时,环境的酸碱度也是影响因素之一,酸性环境可以增强外泌体被细胞摄取,主要因酸性环境能促进外泌体表面小窝蛋白-1表达增加[16-17]

3 外泌体在体内代谢研究的基础

由于外泌体大小不一,每种外泌体所含蛋白不同,不能实现完全定量,目前多用总蛋白定量结合描述外泌体的直径范围与来源细胞来衡量试验中外泌体的量。同时需要注意提取外泌体的方式,不同的离心条件可导致分离出不同的外泌体亚群[18-19]。目前公认的最佳标准是通过差速离心法分离出直径约在100 nm左右的外泌体用于实验[20-21]。收获外泌体后,用荧光染料标记、化学荧光标记和同位素示踪法来研究外泌体在体内的代谢过程。其中荧光染料标记是最常用的方法,多使用碘化物DiR、DiD染料共同孵育,但特异性较差。Takahashi等[18]用Gaussia荧光素酶(gLuc)与外泌体特有蛋白乳凝集素(lactadherin)连接,前者遇到其催化底物时产生化学荧光从而实现外泌体的体内追踪,特异性较好。也有少数实验使用同位素示踪法[22-23]

4 外泌体在体内代谢特点 4.1 静脉注射

外泌体的颗粒大小可能是决定其代谢分布的重要决定因素之一。纳米/微米颗粒(nano/micro particles,NMPs)是通过纳米制造技术制成的微球,可用于研究药物的颗粒大小与流体力学特点。研究发现,向血管中注射外泌体大小类似的NMPs,只有少量NMPs能贴近血管壁与白细胞、内皮细胞直接作用。大部分NMPs与红细胞一起循环,并被肝、脾摄取清除[24]。人的肝窦的内皮细胞窗大小约为107 nm,直径小于此值的颗粒可以通过内皮细胞窗进入肝窦周隙,被肝实质细胞捕获,而大于内皮细胞窗直径的颗粒则会被阻挡在肝外,由脾负责清除[25]。研究显示当NMPs直径在100 nm左右时,肝和脾共同的清除作用最小,NMPs在血液循环中存留的时间最长。而其他器官对于NMPs的摄取量则主要取决于NMPs在血液循环中的浓度和循环时间[26]。外泌体进入血液循环后迅速从血管中消除,进入实质器官,血浆半衰期仅为2~4 min。从小鼠尾静脉注入荧光标记的外泌体,通过活体成像观察到其主要被肝、脾、肺、肾、骨髓等器官捕获,其中以肝、脾、肺的荧光最强[19]; 静脉注射用125I标记的外泌体4 h后,在肝、肺、脾中分别检测到28%、7%、1.6%的放射活性。进一步研究发现,外泌体在肝和脾中主要被巨噬细胞吞噬摄取。抑制巨噬细胞的活性,外泌体的血浆半衰期明显延长[27]。这一过程可能由于外泌体可以表达磷脂酰丝氨酸,作为一种凋亡信号被巨噬细胞识别并吞噬[28-29]

4.2 其他给药方式

腹腔注射和皮下注射可以避免外泌体直接进入血液循环,极大降低肝、脾的清除作用,但会出现外泌体在胰腺和消化道的明显聚集[30]。外泌体还可以通过口服进入体内,外泌体的膜结构可以抵抗胃酸和RNA酶的作用,将其携带的内容物安全送达肠道,被巨噬细胞及其他肠道细胞摄取从而发挥作用[31]。研究表明口服牛乳来源的外泌体,可以改善免疫调理功能,减轻小鼠的关节炎[32]此外,可以通过局部给药,提高外泌体对靶器官的作用浓度和作用时间。对比静脉注射,直接在肿瘤内注射或通过经鼻颅内给药等方式,可以防止外泌体进入体循环被肝、脾大量清除[22, 33]

4.3 生理情况下的代谢特点

在生理情况下,静脉注射进入血液循环的外泌体被固有免疫非特异性地清除,这一过程尽管有补体蛋白参与其中,但与特异性的抗原抗体反应无关无论是外泌体表面表达的蛋白还是其特殊的脂质成分对清除速度都没有明显影响[19, 34]

4.4 病理情况下的代谢特点

外泌体在损伤、炎症反应等病变的组织中具有聚集性。在大鼠脑出血模型中,从尾静脉注入外泌体,可见其主要分布在出血灶、肺、肝和脾中[35]。在对兔、小鼠脑缺血—卒中模型的研究中发现,脑缺血灶对外泌体有强烈的摄取行为[33, 36],同时外泌体会在缺血损伤区域持续存在(> 24 h)、浓度基本不变,而正常脑组织中外泌体浓度则不断地下降[38]。向急性脊髓损伤的小鼠注射MSC来源的外泌体,发现它们在脊髓损伤处聚集,且主要是被损伤处的M2型巨噬细胞所吞噬[37]。在急性肾损伤小鼠模型中观察到,在损伤的肾组织中出现了外泌体的富集,而对照组无此现象[38]。外泌体在病理改变的组织中具有聚集性的机制尚不清楚,考虑可能与外泌体表面表达特异性的受体被炎症细胞识别有关,也可能因炎症和受损组织通透性增高所致。

此外,外泌体对肿瘤组织没有特殊的亲和性,机体是否患有肿瘤也几乎不影响外泌体在体内的清除过程[39],但恶性肿瘤来源的外泌体可增加肺血管内皮通透性,从而实现在肺间质中的聚集[40]

5 外泌体的毒性

外泌体的治疗作用在一定范围内可呈剂量依赖性,但正如其他药物一样,过大的剂量也有毒性[40]。目前对外泌体的毒性研究极少,仅有报道对小鼠注射超过200 μg的外泌体时可能出现副作用。Smyth等[22]发现,向Balb/c小鼠静脉注射400 μg 4T1细胞来源外泌体后,观察到小鼠迅速出现呼吸困难,并于3 min后死亡,成像显示外泌体主要在小鼠的肺中富集。这可能由于外泌体静脉注射后汇聚到肺组织中,或在体内凝集成栓子栓塞肺血管导致,其产生毒性的机制仍待明确。

6 展望

外泌体在体内的代谢动力学研究是其他研究的基础,是科研成果转化的重要基石,有助于我们集中精力早日实现外泌体的治疗作用。外泌体本身大小不一,成分复杂,发挥作用的机理很多还不明确,需要进一步地去探索。

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