2. 天津农学院, 动物科学与动物医学学院, 天津 300384
2. College of Animal Science and Veterinary Medicine, Tianjin Agricultural University, Tianjin 300384, China
所有的细胞，原核生物和真核生物在正常或异常的生理状态均能释放细胞外囊泡[1]。根据大小及释放方式，可将细胞外囊泡分为外泌体、细胞微泡和凋亡小体[2]。表 1总结了这3种细胞外囊泡的主要特征。研究发现细胞外囊泡在细胞间通讯[3]、免疫应答[3-5]和传递外源性化合物到受体细胞均发挥重要的作用[6]。其中，外泌体是多囊泡体(multivesicular body，MVB)和质膜融合，通过胞吐作用向细胞间隙中分泌的直径最小(30~150 nm)的胞外囊泡[7]，且外泌体几乎存在所有的体液中，包括人类和动物的乳汁[8-11]。
在哺乳动物的饮食来源中，牛乳是消耗最多的饮品之一，且含有丰富的外泌体。外泌体可通过与质膜融合或内吞作用被细胞摄取[12]。根据不同的受体介导，内吞方式可分为：网格蛋白介导的内吞作用、小窝蛋白介导的内吞作用、脂筏介导的内吞作用、巨胞饮作用和吞噬作用。此外，外泌体的摄取还取决于细胞类型、生理状态以及外泌体表面的配体是否可识别细胞表面的受体[13]。研究表明，牛乳外泌体在哺乳动物出生后参与合成代谢，促进生长和完善免疫系统；同样有报道指出，牛乳外泌体所携带的部分内容物可引起一些慢性疾病的发展，如肥胖、2型糖尿病、骨质疏松症、常见癌症(前列腺癌、乳腺癌、肝癌)和帕金森病[14]。因此，本文综述了外泌体的组成及分选机制，并比较了外泌体的分离方法、在体内的分布和生物学功能，以期为国内牛乳外泌体的研究与开发提供参考。
1 外泌体的主要成分及分选机制 1.1 蛋白质外泌体中的蛋白质仅来源于细胞溶质、内吞小泡或细胞膜，没有内质网、高尔基体和细胞核相关的蛋白质[20]。有研究发现，泛素化在蛋白质翻译后修饰中发挥重要作用，其主要调控Fas配体进入MVB[21]，这一过程由转运必需核内体复合物(endosomal complex required for transport，ESCRT)介导，对维持外泌体中蛋白质的水平和功能具有重要意义。外泌体中的蛋白质与外泌体的形成和功能密切相关，例如，ESCRT中含有凋亡转接基因2互作蛋白X(Alix)、肿瘤易感基因101蛋白(TSG101)；小GTP结合蛋白家族(Rab27a、Rab11b和ARF6)参与外泌体的形成和释放；此外，外泌体中含有四跨膜蛋白(CD63、CD81和CD9)、参与信号转导的蛋白[表皮生长因子受体(EGFR)]、抗原呈递[组织相容性复合体(MHC)Ⅰ和MHCⅡ)]和其他跨膜蛋白[溶酶体关联膜蛋白1(LAMP1)和转铁蛋白(TFR)][13]。乳外泌体中的蛋白质不仅可作为营养物质，还在调节免疫系统方面发挥重要作用[22]。Samuel等[23]从初牛乳和成熟牛乳的外泌体中分别鉴定出8 124和4 443个蛋白质，发现初牛乳外泌体中的蛋白质与免疫反应和细胞生长息息相关。
1.2 脂质脂质对外泌体发挥生理功能有重要作用，但其相关研究较少。目前为止，ExoCarta数据库显示，1 116种脂类在外泌体中被发现。虽然不同细胞分泌的外泌体在脂质组成上存在差异，但其双分子层主要包含磷脂酰胆碱(PC)、磷脂酰丝氨酸(PS)、磷脂酰乙醇胺(PE)、磷脂酰肌醇(PIs)、磷脂酸(PA)、胆固醇、神经酰胺、鞘磷脂、鞘糖脂和其他低浓度的脂类[24-25]。外泌体中的神经酰胺、胆固醇和鞘脂等构成的脂质筏在传递免疫信号过程中发挥较大作用[24]。除牛乳以外，其他来源的外泌体中的胆固醇和鞘磷脂组成不依赖于供体细胞，而饱和脂肪酸中的PC和PE的含量则与供体细胞有关[26]。薄层色谱(TLC)、液相色谱(LC)、气相色谱(GC)和质谱(MS)是最常用的脂质分析技术，对脂质进行全面研究有助于阐明外泌体的生物学功能。
1.3 核酸在外泌体中发现了多种遗传物质。在少数情况下，外泌体含有DNA，包括单链DNA、双链DNA、基因组DNA、线粒体DNA，甚至是反转录互补DNA[27-28]。但总体而言，外泌体中含有丰富的非编码RNA，主要包括miRNAs、tRNAs、rRNAs、snRNAs、piRNAs、snoRNAs和RNA碎片[29-30]。外泌体中的RNA与细胞中的RNA具有相似的形态，但外泌体中的RNA种类更加固定[30]。其中，miRNAs在转录后水平上调控基因表达和蛋白质合成，参与细胞增殖、分化、凋亡和免疫系统发育，是外泌体中含量丰富且重要的一种RNA[31-32]。Quan等[33]在牛乳外泌体中共鉴定出273种miRNAs，且大部分与免疫相关。然而，外泌体如何分选miRNAs尚无定论，目前存在4种假说：第一，神经鞘磷脂酶2(neural sphingomyelinase 2, nSMase2)依赖途径。nSMase2是第1个被报道与外泌体分选miRNAs有关的蛋白质。Kosaka等[34]发现过表达nSMase2可增加外泌体miRNAs的数量，而抑制nSMase2表达则减少了外泌体miRNAs的数量。第二，miRNA基序和核糖核蛋白(heterogeneous nuclear ribonucleoproteins, hnRNPs)依赖途径。Villarroya-Beltri等[35]发现，单糖基化的hnRNPA2B1可以识别miRNAs序列中3’端的特定的基序(GGAG)，并使其进入外泌体。此外，hnRNPA1和hnRNPC也是hnRNPs家族蛋白，似乎也参与外泌体分选miRNAs，但具体机制尚不清楚[35]。第三，依赖于miRNA的3’端序列。Koppers-Lalic等[36]发现尿苷化的3’端有利于直接将miRNAs分拣到外泌体中，而3’端腺苷酸化miRNAs主要存在细胞中。第四，miRNAs诱导沉默复合体(miRNA induced silencing complex, miRISC)相关途径。成熟的miRNAs可结合真核翻译起始因子2C2(AGO2)形成miRISC，从而调控miRNAs是否加载到外泌体中。Guduric-Fuchs等[37]发现，敲除AGO2可降低某些miRNAs的种类和丰度。总之，miRNAs中的特定序列，某些酶和蛋白质都可能控制外泌体分选miRNAs。
2 外泌体的分离方法外泌体的分离方法可在一定程度上决定外泌体的质量。根据外泌体的特定特性，如密度、形状、大小和表面蛋白质，其分离方法可分为：差速超速离心、密度梯度离心、超滤、排阻色谱、聚合物沉淀、微流体分离和等电沉淀。表 2呈现了每种分离技术的优缺点。
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表 2 外泌体分离技术及其优缺点 Table 2 Various exosome isolation techniques and their advantages and disadvantages |
了解外泌体在体内的具体分布可更有针对性地去研究其生物学功能。Munagala等[45]用细胞膜近红外荧光探针(DiR)标记的牛乳外泌体灌胃裸鼠发现，外泌体主要分布在肝脏、脾脏、胰腺和肾脏中，少量存在结肠、肺和大脑中。Manca等[46]用不同浓度的外泌体灌胃小鼠发现，牛乳外泌体主要聚集在肠黏膜、肝脏和大肠中，而在其他组织中的浓度很低；与雌性小鼠相比，雄性小鼠器官中的外泌体浓度更低，表明性别会影响外泌体在体内的分布。当外泌体表面的蛋白质被胰蛋白酶处理后，外泌体在肝脏中的含量较少；而用氯膦酸钠清除小鼠体内的巨噬细胞后，灌胃的外泌体几乎只聚集在肝脏中；这表明牛乳中外泌体在机体内的分布与外泌体表面的蛋白质和机体中的巨噬细胞密切相关[46-47]。
外泌体中的RNA与外泌体的分布不同，其主要分布在脾脏和大脑中[46]。据推测，miRNAs可将外源乳外泌体转移到内源性外泌体并转运到大脑，但这一理论尚未被证实。miRNA-34a与空间认知功能、海马神经发生和大脑衰老有关，将合成的miRNA-34a转移到牛乳外泌体后发现其主要在大脑中积累[48-49]。此外，miRNAs的分布不仅取决于组织中互补mRNAs的丰富度[31]，还取决于miRNAs的序列，它可以结合核糖核酸蛋白，促进miRNAs分选到外泌体中[50]。
4 外泌体在体内的利用 4.1 肠道细胞外泌体因其独特而稳定的脂质双层膜，可携带其内容物躲避胃肠道的消化，直接被部分细胞吸收或进入大肠。Izumi等[51]发现，乳中外泌体可被人巨噬细胞吸收，进而将功能性RNA传递到人类机体中，但是其对巨噬细胞的影响还尚未可知。体外试验发现乳中外泌体可被人肠细胞、原代大鼠小肠细胞和人脐带血管内皮细胞吸收，并促进增殖[52-53]。此外，乳中外泌体还可促进大肠杆菌和植物乳杆菌WCFS1等细菌的生长并改变基因的表达，可能有助于维持肠道稳态和改善胃肠道功能[54]。
4.2 肠道微生物约75%的牛乳外泌体可逃避细胞的吸收直接进入大肠，是肠道微生物和机体发生互作的介质[46]。一方面，外泌体可直接影响肠道菌群结构进而影响机体的健康。研究发现，在门水平上，牛乳外泌体对小鼠肠道中厚壁菌门和拟杆菌门的相对丰度无现在影响，在科水平上，增加了梭菌科、瘤胃球菌科和毛螺旋菌科的相对丰度，对其他菌群无显著影响[55-56]。其中，瘤胃球菌科和毛螺旋菌科可维持肠道健康和改善小鼠营养不良[57]；此外，瘤胃球菌科与动脉粥样硬化密切相关[58]，而毛螺旋菌科可预防艰难梭菌感染和肥胖[59-60]。另一方面，外泌体可调控肠道菌群的代谢产物进而发挥作用。肠道内容物中的短链脂肪酸(SCFAs)是目前研究较多的代谢产物，可维持肠道黏膜的完整性，改善糖脂代谢，控制能量消耗，调节免疫系统和炎症反应[61]。Tong等[55]发现口服牛乳中的外泌体可提高小鼠粪便中乙酸、丙酸和丁酸的浓度，但对总挥发性脂肪酸和其他短链脂肪酸的浓度没有显著影响；然而，外泌体是否会影响菌群的其他代谢产物仍有待评估。
5 乳中外泌体的生物学作用及资源开发 5.1 有益功能及资源开发外泌体中含有丰富的miRNAs，可在转录和转录后水平上调控基因的表达，从而调节动物的生理过程[62]。此外，某些外泌体中的miRNAs与免疫应答相关，尤其是let-7家族[63]。Tong等[55]报道，牛乳外泌体可增加小鼠肠道中的吸收细胞，增加肠绒毛高度和隐窝深度，促进肠道中黏糖蛋白2、胰岛再生源蛋白(RegⅢγ)、髓样分化因子和转录因子的基因表达，提高肠中免疫球蛋白A(IgA)和分泌型免疫球蛋白A(sIgA)的水平，进而增强肠道免疫。此外，有研究发现，牛乳外泌体中的miRNA-148a和miRNA-21可靶向Rho相关卷曲螺旋形成的蛋白激酶1和抑制DNA甲基转移酶的活性，从而改善肠道屏障功能和缓解溃疡性结肠炎，这表明乳中外泌体可在炎症性肠病患者的肠内营养配方中作为一种营养素[64-65]。Yun等[66]发现牛乳中外泌体可提高骨质疏松模型小鼠的骨密度和恢复肠道菌群结构，进而用于预防骨质疏松，改善骨骼的健康。Arntz等[67]报道乳中外泌体可通过直接或间接调节肠道中的T细胞的分化治疗自身免疫性关节炎。Wang等[68]发现，牛乳外泌体可减轻肠隐窝上皮细胞在氧化应激下的嘌呤核苷酸代谢，改善细胞能量状态，对氧化应激具有保护作用。Gao等[69]发现，牛乳外泌体中的miRNAs可显著提高大鼠肠上皮细胞(IEC-6)中脯氨酸羟化酶的表达，降低低氧诱导因子-α及其下游的血管内皮生长因子的表达，进而减轻缺氧对肠道的损伤。此外，牛乳外泌体中的ncRNAs可能在免疫调节、表观遗传调节、代谢疾病和靶向治疗中发挥生物学作用，然而，关于牛奶外泌体中ncRNAs的来源、摄取和生理作用等的研究较少，还要很多问题亟待解决[70]。
牛乳源性外泌体的脂质双分子层不仅可提高药物的溶解性，还可保护其化学成分不被降解。Munagala等[45]首次证明了牛乳源性外泌体可携带药物并实现肿瘤靶向性治疗。Agrawal等[71]发现口服牛乳源性外泌体给药与腹腔注射给药具有相似的治疗效果。因此，牛乳源性外泌体作为亲水和亲脂药物(包括化疗药物)的载体具有巨大的潜力。
5.2 潜在风险然而，目前对牛乳源性外泌体的生物学功能研究并不全面，仍存在很多争议。流行病学家指出，牛乳源性外泌体可能会引入病原体从而引发一系列的疾病，还可能激活与衰老相关的哺乳动物雷帕霉素靶蛋白1(mTORC1)的活性，降低人的寿命，增加死亡率[14, 72]。首先，牛乳源性外泌体可增加巨大胎儿的发生率，有研究发现，怀孕期间牛奶摄入量与胎盘和出生体重变化呈正相关[73]。miRNA-21是牛乳中外泌体的标志miRNAs[74-75]，可到达胎盘并促进胎盘滋养层细胞的生长。此外，周期蛋白依赖性激酶抑制剂(CDKN1C、p57kip2)的缺失突变可导致5%~10%的婴儿出现过度生长综合征、巨舌、腹壁缺损等现象[76-77]；而miRNA-21是直接靶向CDKN1C的因子之一[78]，进一步提高巨大胎儿的发生率。其次，牛乳源性外泌体可诱发肥胖。外泌体中的miRNAs在间充质干细胞形成的脂肪细胞中发挥重要的促进作用[79-80]。Wnt1和Wnt10b可抑制脂肪细胞形成，而外泌体中的miRNA-148a可抑制其表达，此外，miRNA-148a还可抑制DNA甲基化转移酶家族(DNMT)的活性而促进脂肪细胞分化[81-83]。外泌体中的miRNA-21和miRNA-29b均可促进脂肪细胞增殖分化[84-85]；miRNA-155可通过减弱棕色脂肪组织的分化和产热，促进白色脂肪组织的储存，进一步促进肥胖[86]。此外，长期暴露于miRNA-148a和miRNA-21可能会诱发动脉粥样硬化，从而可能增加心血管发病率和死亡率[14]。外泌体中miRNA-155是侵入大脑的主要免疫调节miRNAs之一[46]，在α-突触核蛋白(α-syn)相关的神经退行性疾病中发挥着核心作用[87]，可能是帕金森病发生的关键因子。最后，外泌体中的一些miRNAs在前列腺癌、乳腺癌和肝细胞癌等一系列疾病中也发挥作用[14]。
6 小结外泌体作为一种功能性的微型囊泡，不仅可介导细胞间通讯，还在机体生理病理过程中发挥重要作用。已有研究表明，牛乳源性外泌体可调节肠道的菌群结构，有利于免疫调控，以及缓解肠炎和骨质疏松等疾病，表明牛乳源性外泌体可被机体有效利用，有望成为一种新型的功能性食品。此外，牛乳源性外泌体特殊的生物学结构可使其成为实用的药物载体，传输药物作为疾病的靶向治疗。但是，牛乳源性外泌体可能会传递某些有害的物质，从而诱发疾病。因此，有必要全方位了解牛乳外泌体的生物学功能，从而为开发相关产品提供理论依据。
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