修回日期: 2016-06-13
接受日期: 2016-06-21
在线出版日期: 2016-12-18
外泌体是细胞分泌的一种纳米级囊泡结构, 在血液、唾液、尿液等多种体液中均有分布. 作为一类重要的细胞间通信分子, 外泌体含有核酸和蛋白质等多种具有生物活性的成分, 可通过多种方式在人体中发挥调节作用. 在多种类型的细胞中均发现外泌体的存在, 肿瘤来源或肿瘤相关的外泌体是调控肿瘤发生发展的重要机制, 对肿瘤外泌体的分析和检测可以辅助肿瘤的早期诊断、疗效评价和预后分析, 已成为肿瘤领域的研究热点. 本文将就近年来外泌体在消化系统肿瘤诊断中的作用进行简要综述.
核心提要: 外泌体中蛋白质和核酸等内容物可以近似真实地反映其源细胞的特征和状态, 因此可用于肿瘤辅助诊断、疗效检测以及预后预测, 靶向肿瘤的外泌体的标志物可为疾病的治疗提供新的指标和途径.
引文著录: 崔琮, 徐建明, 王友亮. 外泌体在消化系统肿瘤诊断中的应用. 世界华人消化杂志 2016; 24(35): 4644-4651
Revised: June 13, 2016
Accepted: June 21, 2016
Published online: December 18, 2016
Exosomes are nanovesicles that are secreted by their host cells and distributed in the blood, saliva, urine, and other body fluids. Exosomes have emerged as a novel important mediator in facilitating intercellular communication by virtue of regulatory molecules in its cargo (nucleic acids and proteins) and inducing physiological and genetic changes in targeted cells. Exosomes can be released in many and perhaps all biological fluids, and tumor-derived or -associated exosomes are emerging as key players in intercellular communication between cancer cells and their microenvironment through horizontal transfer of information via their cargo. Exosomes could be serving as a novel means for tumor genetic detection and potential biomarkers for cancer diagnostics and prognostics. This article reviews recent progress in the understanding of the role of exosomes in diagnosis of digestive system cancers.
- Citation: Cui C, Xu JM, Wang YL. Role of exosomes in diagnosis of digestive system cancers. Shijie Huaren Xiaohua Zazhi 2016; 24(35): 4644-4651
- URL: https://www.wjgnet.com/1009-3079/full/v24/i35/4644.htm
- DOI: https://dx.doi.org/10.11569/wcjd.v24.i35.4644
"外泌体"这一术语第一次出现在公众视野中是在1981年, 由Trams等[1]在研究细胞核酸外切酶活性时发现并加以描述的. Johnstone等[2]发现外泌体是细胞分泌的一种双层膜的囊状小体, 几乎所有的真核细胞均分泌这种纳米级的双层膜囊泡[3]. 外泌体的大小不一, 一般认为是一种30-150 nm的囊泡, 密度为1.13-1.21 ng/mL, 成球形或杯状[4,5]. 外泌体广泛存在于各种体液, 如血液、淋巴液、唾液、尿液、精液、脑脊液、乳汁、腹水、羊水、眼泪、鼻腔黏液、精液和支气管肺泡灌洗液等[6]. 外泌体携带有细胞来源相关的多种蛋白质、脂类和多种核酸, 参与细胞间通讯、细胞增殖、细胞迁移、细胞分化、血管新生和免疫调节等过程. 在糖尿病、心血管疾病、艾滋病、慢性炎症疾病以及肿瘤中都发现细胞外囊泡水平的升高, 很有可能成为这类疾病的诊断标志物[7]. 在肿瘤中, 由于肿瘤细胞与周围支持细胞间需要大量的信号交流, 随着需求的增加, 外泌体的分泌量明显提高, 参与肿瘤微环境的调解、肿瘤的转移, 并可能在其中发挥着关键性的作用, 对肿瘤外泌体的分析和检测可以辅助肿瘤的早期诊断、疗效评价和预后分析[8].
外泌体起源于多泡体, 以细胞管腔内囊泡的形式存在. 细胞的胞吞形成带有外源性抗体的内体小泡, 在高尔基体等细胞器的作用下形成早期核内体, 早期核内体的囊膜内陷、突入形成多个小囊泡, 并选择性的接受细胞内的蛋白质、核酸、脂类等成分, 最终形成晚期核内体, 晚期核内体与细胞膜融合, 并将外泌体排出胞外[9]. 这是一个连续而又复杂的过程, 从内体小泡到成熟外泌体, 需要在各细胞器间传递并包装, 包括: TSG101、Alix蛋白、CD63、CD81、神经酰胺、胆固醇等[10]. 外泌体的排出跟其他胞内小泡大致相同, 受到Rab家族蛋白的调控, 敲除细胞的Rab 27a和Rab 27b蛋白后, 外泌体不能排出, 且从高尔基体到晚期核内体的囊泡运输不受影响[11].
外泌体中含有的生物大分子丰富而复杂, 主要为蛋白质、酯类和核酸, 据统计, 已经确定有27000种蛋白质、1100种脂质、19000种mRNA和2838种微小RNA(microRNA, miRNA)存在于不同组织和细胞来源的外泌体中. 外泌体所含生物大分子与其来源细胞、组织有着一致性, 如: 抗原提呈细胞来源的外泌体中富含MCHⅡ, T淋巴细胞来源的外泌体富含CD3[9]. 外泌体中携带许多细胞内特异性的标志性蛋白, 如: 与细胞膜转运和融合相关的GTP酶家族蛋白Rab27、Rab11和Rab35; 调节膜动态性的膜联蛋白(Ⅰ、Ⅱ、Ⅲ、Ⅳ)以及跨膜蛋白(CD63、CD81和CD9)[3,12]. 同时也携带许多细胞内特定的胞质辅助蛋白, 如: 微管蛋白、膜联蛋白、MCH Ⅰ/Ⅱ和热休克蛋白(HSP70和HSP90)[10]. 尽管起源于细胞核, 但外泌体通常不含有内质网、线粒体和细胞核的蛋白. 外泌体也富含核酸(包括: DNA、miRNA、mRNA和lnRNA)[8], 外泌体中的mRNA可以参与细胞周期、血管生成及组蛋白修饰等进程[13]. 外泌体中的miRNA可参与干细胞分化、器官形成、血细胞生成和肿瘤的发生及转移[14]. 外泌体也含有大量的双链DNA, 这些双链DNA的碱基序列与其源细胞的DNA高度一致. 肿瘤患者血清外泌体DNA基因突变的检出率与肿瘤组织DNA基因突变的检出率高度一致[15-17]. 外泌体丰富的内含物及广泛的存在, 使外泌体检测成为一种非常有吸引力的巨大潜在价值的无创诊断方法.
肿瘤细胞有以下特点: 持续增殖、细胞凋亡逃避、免疫逃避、促进血管生成以及高侵袭、高代谢. 外泌体对细胞的调节机制均可与肿瘤的各个特点相关联, 肿瘤细胞分泌的外泌体通过对大分子物质的转移和传递调节临近或远处肿瘤生长的微环境, 对促肿瘤生成及肿瘤增生、转移发挥着不同的调节作用[15,18,19]. 肿瘤细胞分泌的外泌体还可以通过诱导耐药和免疫抑制来促进肿瘤的发展[12]. 肿瘤在生长过程中会不断地将外泌体释放到周围环境中去, 存在于患者的血液, 也可经血液循环到达腹水和胸膜积液等恶性病变体液中, 从而更易获得临床标本, 检测的灵敏性和特异性可以更好. 外泌体含有的信号分子能反映分泌细胞的生理状态和功能状态, 甚至还会包含细胞病态相关的分子信息, 从而提供了丰富的潜在的生物标志物分子源. 外泌体能在4 ℃保存96 h, 或是在-70 ℃下保存更长时间, 这些特点使得外泌体可应用于肿瘤的早期诊断和预后判断. 靶向肿瘤的外泌体的标志物可能为肿瘤疾病的诊断和治疗提供新的指标和途径, 对肿瘤高危人群早期诊断和早期治疗以降低或减缓肿瘤病例的发生发挥重要的作用.
胰腺癌恶性程度高、死亡率高, 近年发病率在全球也呈上升趋势. 因其早期体征不明显, 临床应用的肿瘤标志物如CA19-9等缺乏特异性, 确诊时往往为晚期, 导致胰腺癌患者预后较差. 胰腺癌的早期诊断已成为医学界面临的一大难题. 最新的研究发现, 对外泌体的检测也许可以使这一难题得以解决. Glypican-1(GPC1)被发现富含于外泌体外膜, 通过对外泌体GPC1的检测, 可鉴别早期胰腺癌、晚期胰腺癌、良性胰腺疾病与健康人群, 并显示出了100%的特异性与敏感性. 此外, 对血清中GPC1+的外泌体进行定量, 可间接反映胰腺癌患者体内瘤体的大小, 且对GPC1+外泌体的检测, 更可早于核磁共振发现胰腺上皮细胞的改变[20]. 虽然还需要大规模的临床试验来验证GPC1+外泌体作为肿瘤生物标志物的效果, 但这个发现确实为外泌体成为一种微创的癌症检测生物标志物奠定了基础.
在另一项筛查性的实验中, 研究者进行了胰腺癌患者与健康人血清外泌体的蛋白质和miRNA差异比较分析. 发现胰腺癌患者外泌体中CD44v6、Tspan8、EpCAM和CD104 4种表面蛋白明显高于健康、良性胰腺疾病患者群, 可达到区分胰腺肿瘤与良性胰腺疾病的目的. miR-1246、miR-4644、miR-3976和miR-4306等四种miRNA也在胰腺癌患者外泌体中特异性高表达[21]. 另一项研究[22]发现胰腺癌患者中miR-17-5p和miR-21的含量显著升高, 这些血清外泌体中的miRNA可作为胰腺癌潜在的生物标志物, 尤其是高水平的miR-17-5p与胰腺癌的转移和分期较晚有显著关系. 胰腺癌细胞可分泌分子量为110 kD的可溶形式EGFR蛋白, 外泌体则含有分子量为170 kD的完整EGFR蛋白和分子量为64 kD截短EGFR蛋白, 不同形式的EGFR蛋白在体内可能承担着不同的功能, 可以作为诊断用血清生物标志物和治疗靶点[23].
结直肠癌是常见的消化系统恶性肿瘤之一, 其发病率和死亡率在全球男、女性中均位居第3位, 且女性发病率逐年呈逐年增高趋势. 结直肠癌目前的诊断主要依靠内窥镜, 但其效率低, 准备程序复杂, 且是一种高成本的侵入性检查, 不易被人们接受. 尽管治疗手段不断进步, 但肿瘤复发仍是导致治疗失败的主要原因之一, 也是增加患者病死率的重要因素. 因此, 结直肠癌诊疗技术仍需提高, 寻找早期诊断及预测预后的生物标志并研究其机制具有重要的意义. KRAS基因在结肠癌患者的突变率为30%-40%, KRAS基因突变导致EGFR信号通路持续激活, 加速肿瘤细胞的增殖, 促进肿瘤细胞的生长和扩散. 研究[18]发现, 在突变型KRAS基因结肠癌细胞分泌的外泌体中含有突变表达的蛋白, 外泌体将突变表达的蛋白转移至野生型KRAS基因的结肠癌细胞, 促进野生型KRAS基因结肠癌细胞的生长. 随着血液为基础的肿瘤标志物的开发, 发现EpCAM、CLDN7和CD44等分子高表达于结直肠癌患者外泌体表面, 与肿瘤的进展密切相关[24,25]. 日本的研究者[26]近期开发了一种新的高敏感的荧光方法(Exo Screen), 定量外泌体表面膜蛋白, 并使用该方法对结直肠癌外泌体进行了CD147(用于鉴定结直肠癌细胞的表面抗原)的定量, CD147的定量可替代CEA和CA19-9成为新的结直肠癌肿瘤标志物. 外泌体中miRNA在结直肠癌患者中存在特异性的高表达, miRNA-17-19集群的高表达与结直肠癌的复发相关, miRNA-19a在结直肠癌患者外泌体中同样表达量显著增加, 且miRNA-19a高表达的患者预后较差[27]. 此外, 结直肠癌细胞分泌的外泌体含有27种细胞周期相关的mRNA, 可有效促进内皮细胞的生长以及血管形成, 从而促进肿瘤的生长和转移[28].
胃癌作为一种常见的恶性肿瘤, 在亚洲具有较高的发病率. 胃癌的临床诊断方法与结肠癌相同, 主要依靠内窥镜, 早期胃癌的症状并不明显, 且临床常用的肿瘤标志物对胃癌患者特异性与敏感性均较差, 因此, 胃癌往往发现于晚期, 并伴有不同程度的转移. 研究者[29]发现胃癌细胞高表达CD97并可经外泌体分泌的方式调节肿瘤细胞的MAPK信号通路, 促进肿瘤细胞的增殖与侵袭. 外泌体中CD97的检测成或许能成为一种有潜在价值的胃癌诊疗工具. 胃癌细胞能选择性地将let-7包装到外泌体中, 并释放至周围环境中, 促进胃癌的恶性进展[30]. 胃癌细胞外泌体的Cbl-b和Cbl-c, 可作用于抗肿瘤的T细胞, 抑制AKT的活性以及Caspase3、Caspase8和Caspase9的激活. 外泌体与免疫细胞之间的相互作用揭示了肿瘤抑制免疫功能的潜在机制[31]. 在另一项被认为有潜在胃癌诊断价值的lnRNA的研究中, 通过对胃癌患者、胃上皮异型增生与健康人的比较发现, LINC00152在胃癌患者的血液中可被检测到高表达, 且手术前后有明显变化. 血清LINC00152主要存在于外泌体中, 在外泌体的保护下, 可以稳定保存, 血清外泌体LINC00152具有胃癌标志物的潜能[32].
肝癌在中国的发病人数占世界发病人数的一半以上, 具有恶性程度高、病情进展快和发病隐匿等特点. 虽然甲胎蛋白已普遍用于筛查工作, 但其仍具有敏感度偏低(40%-65%)、特异性不足(76%-96%)的缺陷, 不能成为一种理想型的肿瘤生物标志物. 寻找一种新型的生物标志物应用于肝癌的早期诊断, 是临床中急需解决的问题. 在研究肝癌细胞侵袭的过程中, 发现高侵袭性的肝癌细胞所分泌的外泌体, 对周围非侵袭性肝癌细胞的侵袭能力有着明显的诱导作用, 非侵袭性的肝癌细胞摄取其外泌体后, PI3K/AKT和MAPK信号通路被激活, MMP-2和MMP-9分泌增加, 侵袭能力增强[33]. 多种蛋白被检出在侵袭性肝癌细胞外泌体中高表达, 如: MET蛋白, S100A4, CAV1和CAV2. 特别是MET蛋白的高表达, 在肝癌的侵袭中可能发挥了重要作用. 这使得预测肝癌是否有转移成为一种可能. 研究者对肝炎、肝硬化、肝癌三种患者血清外泌体的miRNA进行检测, 发现miR-18a、miR-221、miR-222和miR-224在肝癌患者中表达显著增高, 而miR-101、miR-106b、miR-122和miR-195在肝癌患者中表达降低. miRNA在外泌体中明显的差异, 或许会成为诊断肝癌新的生物标志物[34]. 肝癌外泌体中lncRNA(HOTAIR和MALAT1)被认为可用来预测肝癌患者肝移植后的复发情况[34,35].
对外泌体的检测, 在其他消化道系统肿瘤的诊疗中, 也存在着巨大的潜在价值. 如: 食管鳞状细胞癌的研究中发现, miRNA-21在外泌体中的高表达, 表达水平可100%反应肿瘤水平, 外泌体miRNA-21的高表达往往预示着广泛浸润与复发[36]. 十二指肠癌的研究中, 发现转移性十二指肠癌细胞分泌的外泌体富含PABP1蛋白, 细胞不能耐受PABP在胞内的大量囤积, 通过外泌体PABP1蛋白排出胞外是转移性十二指肠癌细胞所具有的特性, 外泌体PABP1是转移性十二指肠癌的分子标志物, 此发现不仅在转移性十二指肠癌的诊断上提供了价值, 也为转移性十二指肠癌的治疗提供了新的思路[37].
尽管消化系统肿瘤临床的诊疗依然以组织活检为金标准, 外泌体作为生物标志物在消化肿瘤的各项研究[20,21,24-64]中均显示出了积极的意义(表1), 与肿瘤的发生、进展、转移密切相关. 目前对肿瘤外泌体的研究正处于早期阶段, 外泌体中丰富的内容物展示出了巨大的研究空间, 已发现的标志物需要临床的验证, 未知的标志物需要在研究中继续探索.
| 肿瘤类型 | 标志物类型 | 外泌体标志物 |
| 胰腺癌 | 蛋白质 | GPC1, CD44v6, Tspan8, EpCAM, CD104, MIF[20,21,38] |
| 核酸 | miR-18a, miR-20a, miR-21, miR-24, miR-25, miR-26, miR-99a, miR-155, miR-185, miR-191, miR-196a, miR-210, miR-1290, miR-1246, miR-3976, miR-4306 miR-4644[21,39-43] | |
| 结直肠癌 | 蛋白质 | CD9, CD44, D62L, CD97, EpCAM, CDH 17, CLDN7, HSP70, CD147[24-26,44,45] |
| 核酸 | Let-7, miR-10b, miR-15b, miR-17, mir-19a, mir-19b, miR-21, mir-23a, miR-29a, mir-92a, miR-142-3p, miR-195, mir-320a, miR-331, miR-332, miR-335, miR-339-3p, miR-375, miR-532, miR-532-3p, miR-652, miR-4437, ΔNp73[27,39,46-52] | |
| 胃癌 | 蛋白质 | CD97, Cbl-b, Cbl-c[29-31] |
| 核酸 | Let-7a, miR-17-5p, miR-21, miR-106a, miR-106b, miR-214, miR-218, miR-221, miR-222, miR-223, LINC00152[28,32,46,53-55] | |
| 肝癌 | 蛋白质 | MET, integrin αvβ5, S100A4, CAV1, CAV2[33,56] |
| 核酸 | miR-9, miR-10b, miR-18a, miR-19a, miR-21, miR-22, miR-25, miR-101, miR-106b, miR-107, miR-122, miR-124, miR125a-5p, miR-125b, miR129-5p, miR-195, miRNA-200a, miR-203, miR-210, miR-216a, miR217, miR-221, miR-222, miR-320a, miR-335, miR-370, miR-486-5p, miR-718, H19, HOTAIR, MALAT1[34,35,46,57-60] | |
| 其他消化系统肿瘤 | 蛋白质 | PABP1[37] |
| 核酸 | Let-7c, miR-18a, miR-21, miR-145, miR-155, miR-183, miR-184, miR-223-5p, miR-375, miR-200c, miR-483-5p[36,61-64] |
外泌体最初被发现时, 曾被人们一直认为只是一种细胞的废弃物. 然而, 随着近几年的研究深入, 人们发现这种膜泡含有细胞特异性的核酸、蛋白质等多种生物活性成分, 并能够作为细胞间通信分子, 在人体中发挥调节作用. 这些发现, 引起了人们对细胞分泌外泌体, 尤其是肿瘤来源外泌体的兴趣.
外泌体在肿瘤的发生、生长与迁移、肿瘤转移前微环境的改变中的机制, 以及在肿瘤早诊、疗效评估、预后分析中的应用价值是目前研究的热点问题.
本文对外泌体在消化系统各肿瘤诊断的近期研究进行了简要总结, 外泌体作为一个新型的研究热点, 已成为了消化系统肿瘤诊断治疗的潜在有效方式.
随着人们对外泌体等细胞囊泡系统及其功能的日渐重视, 基础研究领域, 特别是关于细胞外分泌、跨膜分泌等途径机制相关研究的突破, 将会加深人类对外泌体的全面认识, 其间各种高灵敏度、高通量和高内涵的分析手段也会发挥至关重要的作用, 外泌体的临床应用可能会有良好的前景, 将会在肿瘤的诊断与评估中开创新纪元. 此外, 外泌体的潜在治疗价值也不容忽视. 外泌体以其双层膜结构为基础, 可以在体内稳定的循环, 以外泌体为载体进行靶向治疗也许会成为可能.
外泌体: 是由细胞内的多泡小体与细胞膜融合后以外分泌的形式释放到细胞外的纳米级小囊泡, 直径在30-150 nm之间, 在电镜下呈现球形或杯状的形态, 拥有稳定的双层膜结构, 通过胞吐作用排出细胞外, 并随之进入血液或其他体液系统中, 携带有细胞来源相关的多种蛋白质、脂类、DNA、mRNA、miRNA等, 参与细胞间通讯、细胞增殖、细胞迁移、细胞分化、血管新生和免疫调节等过程.
贾国葆, 副教授, 副主任医师, 温州医科大学附属第一医院消化内科
消化系统肿瘤临床的诊疗依然以组织活检为金标准, 此综述阐述了外泌体作为生物标志物在消化肿瘤的积极意义, 与肿瘤的发生、进展、转移密切相关. 目前对肿瘤外泌体的研究正处于早期阶段, 外泌体中丰富的内容物展示出了巨大的研究空间, 本文为一质量不错, 紧跟医学前沿的较好综述.
手稿来源: 邀请约稿
学科分类: 胃肠病学和肝病学
手稿来源地: 北京市
同行评议报告分类
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编辑:于明茜 电编:李瑞芳
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