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维持正常的胃肠道生理功能依赖于存在于胃肠道中的免疫系统、微生物群生态系统和内源性神经系统。功能不成熟的胃肠道不利于新生儿存活。肠神经系统(enteric nervous system,ENS)在调节胃肠道的功能方面发挥重要作用[1]。ENS回路的破坏可导致全身性胃肠道症状。ENS与免疫系统、肠道菌群之间存在大量相互作用[2-3]。最近的研究揭示了生命早期三者之间的相互作用及其对早期发育的影响,以及潜在作用机制。本文就近年来这一领域的进展进行综述。
胃肠道在解剖学上由肠系膜、浆膜、肌层、粘膜下层、固有层和上皮组成,由多个外周神经元群支配。ENS是外周自主神经系统的分支[4],位于肠壁内,由肠道神经元和神经胶质细胞组成,可调节胃肠道活动[5]。ENS分为两个神经丛,肌间神经丛的细胞体位于肠道的环形和纵形肌肉层之间,协调胃肠蠕动所需的收缩与舒张。而粘膜下神经丛的细胞体更接近管腔,深入黏膜,协调分泌、吸收和局部血流,其分化晚于肌间神经丛[6]。这些神经丛可感知管腔刺激并以适当的效应信号作出反应,以促进消化、运动和宿主防御[7]。ENS主要源于迷走神经嵴,在胚胎形成期间,一系列复杂的分子细胞机制协调神经嵴细胞迁移,增殖和分化,定植于肠腔内,定向神经元生长,建立相互连接的神经元和胶质细胞网络[8-9]。最近发现,来自人和小鼠胚胎的肠神经元表达特定的化学物质/递质、受体和离子通道,并具有电活性[10],此外,胚胎肠神经元已经开始通过神经递质(包括乙酰胆碱和5-羟色胺(5-HT))相互形成功能连接,有助于生后肠神经元的存活和分化[11]。在妊娠第14周,空肠和近端结肠黏膜下神经丛中开始出现一个均匀的肌间神经丛和少量的神经元。到妊娠第23周时,肠神经元在黏膜下神经丛和肌间神经丛中浓缩为离散的神经节,同时环形肌和粘膜出现了更高密度的神经纤维。可见,从妊娠后14周到23周,ENS逐渐成熟,变得更加精细。故ENS的蓝图在出生时就存在,并在新生儿期出现活跃的神经元形态和环路形成过程 [12],而ENS网络的成熟发展可持续到成年[13]。在小鼠中,神经源性胃肠道运动发生在出生后早期,直至断奶后才完全成熟[14]。生后第一周,肠神经元具有层状树突,并逐渐成熟为层状形态。考虑到树突的经典信号接收作用,树突形状的变化可能提示生后神经元输入来源的变化。肠神经元轴突投射也在出生后第一周发生变化[15]:出生时,大多数肠神经元向尾侧投射其轴突,吻侧轴突投射的发育只有在出生后才明显,这意味着产前无法提供协调蠕动所必需的双向信号交流。尽管小鼠模型可在一定程度上模拟人体未成熟肠道,但与人相比,小鼠出生时包括ENS在内的肠道功能不成熟[4],生命早期人ENS的发育成熟及功能作用尚需进一步研究。
既往认为新生儿在孕晚期可通过胎盘转运免疫球蛋白G(IgG)获得被动免疫[16],故早产儿感染风险高于足月儿。而最新的研究结果表明,胎龄24周的超早产儿即具有与足月儿相似的IgG储备[17]。同时,粘膜免疫在胎儿早期便已建立[18-20]。 结果提示,早产儿对炎症的易感性增加可能不仅仅归因于发育不成熟,还需考虑与免疫成熟和/或免疫调节有关[21]。免疫系统在关键发育期可能与微生物群和ENS同时发育,相互作用。
先天性免疫系统的细胞包括中性粒细胞、巨噬细胞、自然杀伤细胞(natural killer cell,NK) 、树突状细胞和固有淋巴细胞(innate lymphoid cells,ILCs)。ILCs对宿主防御至关重要,尤其对于获得性免疫不成熟的新生儿。第3组ILCs(ILC3s)主要存在于肠道和皮肤中,母体和新生儿微生物群可调节ILC3s的发育和功能,参与新生儿疾病。人体和动物试验表明ILC3s是坏死性小肠结肠炎(neonatal necrotizing enterocolitis,NEC)[22-23]、晚发性败血症[24-25]、肺炎[26]和支气管肺发育不良(bronchopulmonary dysplasia,BPD)[27]发病机制中的潜在介质。ILC3s主要产生细胞因子 白细胞介素-17A(Interleukin 17A,IL-17A)和白细胞介素-22(Interleukin 22,IL-22)[28]。IL-22在健康新生儿肠道中的表达相对较低,但在炎症时表达增加[29]。小鼠模型研究表明,在NEC期间用重组IL-22治疗可显著减少炎症并增强上皮再生[29]。在小鼠中观察到,肠ILC3s表达血管活性肠肽受体(vasoactive intestinal polypeptide receptor,VIPR),并对肠神经元释放的血管活性肠肽(vasoactive intestinal polypeptide receptor,VIP)有反应。肠道中的ILC3s定位于肠神经元附近,VIP通过VIPR激活ILC3s分泌IL-22,这一过程可被肠道微生物如分段丝状细菌5-7上调,而VIPR2可通过竞争性抑制阻断该过程。VIP可被喂食强烈诱导,禁食则减少VIP生成。神经元VIP-VIPR- ILC3信号通路是动态控制肠道IL-22产生的重要途径。VIP对ILC3s分泌IL-22的调节可能是一种对喂养启动后可能发生的感染或炎性损伤的防御途径[30]。但也有研究表明VIP可降低 IL-22分泌,促进肠道对脂质的吸收增加[31]。 以上相互矛盾的结果提示VIP增强IL-22分泌可能受到新生儿肠道环境变化的影响,而VIP降低IL-22的分泌,削弱宿主防御,促进营养吸收,可能有助于肠内喂养和新生儿的生长。肠道如何协调对进食的生理和免疫反应,以优化营养摄取,同时维持屏障功能仍不清楚。目前推测,ILC3s受到包括ENS在内的众多因素的调控,新生儿不同肠道环境下,ILC3s的功能有所不同,而ILC3s仅代表了肠道黏膜免疫中的一种细胞类型,整个黏膜免疫细胞需要一个更复杂、精确的调节来维持肠道的稳态,其机制的阐明对新生儿肠道疾病的防治具有重要意义。
巨噬细胞与ENS作用紧密,已证明生命早期就已建立了巨噬细胞-神经元的相互作用[32-33]。巨噬细胞在表型上被描述为M1(经典活化或促炎性巨噬细胞)或M2(替代活化或抗炎巨噬细胞)。肠道巨噬细胞位于肠道的不同层内,最大的巨噬细胞群主要在固有层的绒毛内,可随机移位。在粘膜下层的巨噬细胞与粘膜下层神经元和血管密切相关,与肌间神经丛一起形成一个有效调节肠道的整合回路。在外肌层肌间神经丛及浆膜中也存在少许巨噬细胞[34]。肠神经元和巨噬细胞具有多种调节肠道稳态的通讯机制,生长因子的共生产生是其中一种,肌层巨噬细胞(muscularis macrophages,MMs)产生肠神经元生长因子骨形态发生蛋白-2(bone morphogenetic protein,BMP-2),而肠神经元和神经胶质分泌集落刺激因子-1(colony stimulating factor-1,CSF-1),一种重要的巨噬细胞生长因子。除介导肠道平滑肌收缩外,乙酰胆碱还是一种抑制肠道炎症的信号分子。研究表明,胆碱能肠神经元向固有层中的神经元相关巨噬细胞发出信号,乙酰胆碱与巨噬细胞上的烟碱型乙酰胆碱受体(nAChR) 受体结合,并显著降低促炎性因子的释放[35]。促炎性肠巨噬细胞参与了新生儿肠道炎症的发病,最典型的是先天性巨结肠相关小肠结肠炎(hirschsprung-associated enterocolitis,HAEC)[36],一种最严重的先天性巨结肠(hirschsprung disease,HSCR)并发症[37]。HSCR是一种先天性神经发育障碍导致的疾病,由于介导神经元迁移的基因缺陷,神经嵴细胞在胎儿发育过程中无法到达结肠,导致无神经节细胞症。患者肠粘膜中缺乏胆碱能神经支配与炎性免疫细胞破坏黏膜免疫和HAEC发生率升高相关[37]。HAEC的发病机制目前尚未完全阐明,可能涉及先天适应性免疫、微生物群和上皮屏障功能[38-42]。
肠上皮细胞(intestinal epithelial cells,IEC)长期暴露于肠腔内致病性和非致病性抗原。向管腔侧的IEC通过分泌粘液和抗菌肽保护宿主。为了维持粘膜稳态,IEC通过模式识别受体感知肠道信号,并协调免疫耐受或免疫激活。上皮屏障的丧失导致病原体的侵入,触发炎性免疫反应,分泌白细胞介素-8(interleukin-8,IL-8),IL-8介导中性粒细胞的趋化,是肠道炎症反应的重要生物标记。有研究发现,从HSCR患者结肠分离的IE中IL-8水平升高,神经纤维密度低[37]。队列研究证实,结肠炎在黏膜神经纤维支配密度稀疏的HSCR患者中更常见[38]。可见,IEC中IL-8表达失调可能导致黏膜神经纤维的异常,这可能是HAEC的病理生理学机制之一[38-39]。综上所述,ENS与肠道免疫细胞密切相关。ENS的改变既是肠道炎症的结果,也可能是调节肠道炎症的参与者。
肠神经元表达多种免疫介质及受体[43-44],使ENS能够与微生物群、局部免疫细胞相互作用。胃肠道中的微生物群是一个复杂的生态系统,包括细菌、病毒和真菌[45]。胎龄足月、经阴道分娩和纯母乳喂养有利于构建健康的微生物群。新生儿在生后早期即可建立主要的微生物群,早期定植菌对于在微生物演替的过程中建立后期菌群至关重要[46-47]。接受配方奶和/或早产母乳,其人乳寡糖(human milk oligosaccharides,HMO)混合物与足月母乳不同,会影响微生物的演替,进而影响菌群与ENS及黏膜免疫的相互作用,以及生命后期的发育和疾病易感性[48]。在新生儿,早期抗生素暴露很常见,会对肠道微生物群落产生重大影响,增加肠道疾病的风险。与成熟期相比,在发育的关键窗口期使用抗生素可对宿主产生更大的影响,例如,给新生小鼠口服万古霉素会对肠道微生物群和 ENS 功能产生深远影响[49]。也有研究报道生命早期接触抗生素,后期易患功能性胃肠道疾病、肥胖、代谢功能障碍和过敏等疾病[50-51]。即便暴露于相同的抗生素,断奶后与新生儿期小鼠的ENS功能变化也不同,提示抗生素在生命不同阶段的不同作用[52],可能与不同时期胃肠道微生物群组成不同有关。微生物群的重大转变是由4月龄左右断奶期间从母乳转变为固体食物引发的。健康足月婴儿在3岁时建立了成熟的微生物组,并在儿童期至青春期和成年期保持稳定[47]。同时,研究发现小鼠断奶后粪便菌群的丰度和群落显著增加[53],固体的引入增加了肠道细菌的底物多样性,使得粪便菌群开始类似于其母亲[48]。最近研究发现,断奶后粘膜下神经发生显著成熟[54],在此期间建立的肠道菌群多样性的增加将产生新的发酵途径,从而影响它们与ENS的相互作用,这是未来值得研究的一个领域。
肠壁中包含控制重要肠道功能的复杂神经元回路,胃肠道内的微生物群可与嵌入肠壁的ENS建立相互作用。已证实ENS是影响宿主生理和行为的微生物群信号的主要靶点[55-61],微生物群会影响 ENS 的活动。如乳酸杆菌菌株可通过激活ENS固有感觉神经元中的钙依赖性钾通道直接调节结肠运动[57-58],ILC3的活化受单核吞噬细胞产生的细胞因子调节,该过程由肠道微生物的刺激诱导[62-63]。活化的ILC3产生的细胞因子支持肠道黏膜上皮细胞产生抗菌肽和粘蛋白,使微生物与肠组织空间分离[64-65],实现屏障作用。由微生物群、ILC3和IEC组成的回路,通过控制肠道共生微生物群和介导对肠病原体的快速保护反应促进肠道屏障功能[66]。值得注意的是,微生物来源的5-羟色胺对调节新生儿ENS很重要 [56]。动物实验表明,无菌小鼠在出生后早期表现出ENS结构和功能的显著异常,定植微生物后上述异常消失 [67]。在ENS发育过程中,5-羟色胺能神经元可影响初始神经发生以及后期神经元的发育和存活,包括表达多巴胺、γ-氨基丁酸和降钙素基因相关肽的神经元 [68]。5-羟色胺是调节大脑功能的重要神经递质,而肠道微生物组可通过“从头合成”或通过影响神经递质相关代谢途径影响神经递质的生成。如产气荚膜梭菌通过表达色氨酸羟化酶-1关键酶调节 5-羟色胺的合成[69]。而儿童神经发育性障碍疾病(如孤独症谱系障碍(autism spectrum disorders,ASD)、注意缺陷多动障碍(attention deficit hyperactivity disorder,ADHD)的发生与多巴胺能系统和5-羟色胺能系统的破坏相关[70]。因此,以上神经递质与肠道微生物组之间的联系可能是 微生物在ASD和ADHD病理生理进程中发挥作用的机制之一[71-72]。此外,ENS可通过传入神经元与中枢神经系统进行通信,传入神经元的感觉信息经过脊髓和迷走神经途径到达中枢,从而感受肠道微生态和功能的变化[73],而肠道微生物组则可通过上述迷走神经途径来调节宿主的情绪和行为反应。在动物模型中,致病菌(如空肠弯曲菌和无丙酸柠檬酸杆菌)感染可诱导焦虑样行为[74],而补充益生菌可减轻 焦虑/抑郁样行为[75]。切断自闭症模型小鼠的迷走神经后,观察到罗伊氏乳杆菌对自闭症行为的诱导效应被阻断[76],因此,我们推测ENS可能参与生命早期发育行为学疾病的发生发展。但目前该领域的研究尚处于起步阶段,需要更多深入的研究以阐明其中的机制,这也将是一个未来的研究热点。
新生儿ENS、黏膜免疫系统和肠道微生物群之间的相互发展、影响,共同调节肠道稳态。ILCs、巨噬细胞均是肠粘膜免疫的重要细胞,与ENS、微生物群构成回路,发挥肠道生理功能,目前相关研究多在成人中进行。近年来,逐渐有关于各系统在生命早期发生相互作用的关键时间及其机制研究。由于其机制的复杂性,且相关研究刚起步,目前对于三者之间的相互作用及作用机制尚不完全清楚。外部因素的影响,如早期暴露抗生素,其是否破坏了ENS、黏膜免疫,导致相关疾病风险增加,可能是未来研究的重点方向,将为新生儿的临床治疗提供思路,改善相关疾病的预后。
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