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口服给药是最简单的给药方式,药物是否适合口服给药取决于其在胃肠道中的有效吸收,因此,开发能准确预测口服药物吸收的方法对药物研发相关研究具有非常重要的意义。近几十年来,为了评估临床前开发过程中的口服药物吸收,研究人员们已经建立了多种药物肠吸收的研究方法,主要有体外试验法(in vitro)、在体试验法(in situ)和体内试验法(in vivo)等。动物模型可以模拟整个有机体的生理,但动物模型在肠道生理学以及肠道转运蛋白的表达模式和底物特异性方面与人类有很大不同[1]。2004年,美国FDA估计,92%通过动物试验的药物未能进入市场,原因是动物试验没有预测到的有效性和安全性问题。此外,动物模型往往具有伦理学争议。与动物模型相比,体外培养细胞模型具有操作简便、成本低、无伦理问题等优点[2],研究人员开发了Boyden Chamber和Transwell等模型来模拟肠道的复杂结构和功能。然而,传统的体外培养细胞模型通常缺乏体内特性,例如,流体流动、周期性蠕动、宿主与微生物之间的串扰以及组织之间的串扰[3]。随着微制造和3D打印技术的发展,肠芯片(gut-on-a-chip, GOC)为体外研究肠道疾病提供了新方法[4]。基于肠道功能,肠芯片引入了具有不同部件的模块,例如,用于流体流动的注射泵和用于机械变形的压力系统[5],用于模拟一些肠道功能,使其更具生理相关性。肠芯片突破了传统细胞培养和动物实验的局限性,具有体积小、高度集成化和高通量等特点。本文综述了目前国内外肠芯片模型以及与肠道相关的多器官耦合芯片模型的研究进展,介绍了基于微流控芯片的肠道模型在疾病建模、药物吸收和转运方面的应用。
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常规的肠道体外模型缺乏对组织结构的真实模拟、缺乏微生物群,以及肠道功能的复杂性,例如蠕动和流动。随着微流控技术的飞速发展,基于微流控的肠芯片成为了模拟人体胃肠道的有效途径。如图1所示,肠道微环境的关键因素包括肠道的3D绒毛结构、蠕动运动和肠屏障功能等[26]。为了真实地模拟人体肠道,肠芯片模型应能实现以下功能:①通过体外拉伸细胞培养膜来模拟蠕动样运动;②通过控制流体来再现肠道复杂的3D绒毛结构;③通过控制微流体的设计结构和材料的渗透性来产生生理氧梯度:④通过多细胞共培养的方式,研究肠道与其他组织或肠道-微生物之间的互作关系。
图 1 肠道微环境关键特征[26]
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在过去的10年中,肠道芯片平台已经从简单的2D结构发展到包括更全面的功能,例如绒毛结构、肠道蠕动、氧梯度,甚至免疫系统。在肠道器官芯片发展的过程中,肠芯片的类型主要分为两种:二维夹膜肠芯片和三维绒毛肠芯片。
二维夹膜肠芯片最常见的设备结构包含两个通道(上部和下部),由半透膜(例如聚碳酸酯或聚酯材料)隔开,在膜上生长的肠上皮细胞形成单层上皮的顶端和基底外侧,这种简单的2D模型通常用于评估药物和营养吸收的体外药动学特性。Kimura等[27]开发了一种集成泵式循环装置和光学检测功能的微流控模型,该模型由被胶原蛋白涂层的半透膜分隔的两层通道组成,以抗癌药物环磷酰胺的渗透评价其吸收功能。Shah等[28]设计了一种基于微流控芯片的人类-微生物共培养模型“人与微生物交互系统”(HuMiX),见图2。培养基灌注微腔室与人肠上皮细胞培养微腔室之间用微米多孔膜(孔径1 μm)隔开,人肠上皮细胞培养微腔室与微生物培养微腔室之间用纳米多孔膜(孔径50 nm)隔开,每个微腔室有独立的进出口,该模型还集成了光电二极管用于监测溶解氧的浓度。2D肠道芯片模型可以模拟肠道细胞上的流体剪切应力,以减少对细胞和培养基的需求,但该模型仍缺乏模拟肠道组织关键特征的功能。
三维绒毛结构的实现对于构建体外肠道模型至关重要,肠道内的绒毛微结构不仅是肠上皮层的生理屏障,更重要的是增加了肠表面的吸收面积。虽然有报道称在平面基质上自发形成绒毛,但缺乏对绒毛尺寸和分布的控制,而且重复性差。微型3D支架的集成已经成为更好地再现人类肠道结构的解决方案。Shim等[29]植入了胶原蛋白支架,以重现肠道组织三维绒毛结构,绒毛高度为300 μm,绒毛间距为150 μm。3D条件下Caco-2细胞增殖良好,均能形成肠屏障。此外,将细胞置于灌注培养的3D培养条件下,可显著提高细胞的代谢活性。Costello等[30]利用激光雕刻翻模的技术制作了一种肠道芯片,在这种芯片中细胞依附于所制作的绒毛生长;但是激光雕刻在模具的制作中有一定局限性。激光雕刻利用激光垂直切割平面基底,制作出的模型多为柱状结构,较难制作出更为复杂的3D模型。张忆恒等[31]通过3D打印技术与表面微结构转移方法,成功地模拟了更为复杂的肠道绒毛。
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在肠道中,剪切力在细胞分化中起着重要作用,包括增强黏液产生、增加线粒体活性和提高药物吸收[32]。2D培养时细胞保持在静态条件下,动态参数不容易模拟。在微流控装置中,流体流动可以通过蠕动泵、注射器泵、重力或静水压力以及压力发生器产生,模拟体内液体流动的范围及其在细胞表面的相关剪切应力。与静态条件相比,在连续流动和循环应变下,Caco-2细胞经历细胞分化、极化、绒毛形成、屏障完整性维持、黏液产生等。GOC器件的剪切应力一般取值在0.01 ~ 0.06 dyn/cm2之间,大多数模型中的剪切应力是使用蠕动泵引入的,但这些装置体积庞大且吞吐量低。Tan等[33]用2个微型蠕动泵克服了低吞吐量的限制,每个泵有8个泵管路,允许流体通过16个微通道输送,通过测量微流控芯片中培养的Caco-2细胞氨肽酶活性评估其生长和分化速度,与第21天的静态培养的Transwell系统相比,微流控装置中的Caco-2细胞在第5天显示出更高的氨肽酶活性。尽管该系统具有简单、高通量的特点,但这些模型在重现定义的流速方面是有限的。Kim等[34]开发了一个具有流体流动和施加循环机械应变的肠芯片模型。该装置由两个微通道组成,两侧有两个空心腔室,在空心腔室中施加真空,使分离通道的多孔膜单向延伸。该研究发现,流体流动和周期性机械应变的结合导致了褶皱的形成,这些褶皱再现了肠绒毛的结构,并且该系统中使用的Caco-2细胞显示出极化柱状形态,大小与体内上皮细胞相似。
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在消化过程中,蠕动是由平滑肌与肠神经系统协同作用,在整个胃肠道内产生的食物的不自主的、周期性的推进。蠕动有助于食物消化、营养吸收和肠排空,但也对上皮产生剪切力和径向压力。研究表明,机械拉伸对于准确模拟人体肠道生理、允许细胞分化和防止细菌过度生长至关重要[35]。Jing等[36]提出并构建了一种基于循环变化流体压差原理的新型蠕动肠模型,通过使用多通道、计算机控制的气动泵同时实现了肠道微系统的流体流动和蠕动,考察了蠕动和流体流动对该装置肠上皮细胞生长和分化的影响。结果表明,微流体装置中的周期性蠕动加上流体流动显著促进了肠上皮细胞的增殖以及糖萼和微绒毛的分泌。此外,肠上皮细胞的屏障、吸收和代谢功能以及细胞分化也受到芯片上周期性蠕动和流体流动的影响。Fang等[37]开发另一款包含200个依次连接的横向微孔阵列芯片,通过调节微孔周围的气道内部气压,从而实现孔内类器官的周期性收缩与舒张,模拟肠道的蠕动。研究结果发现,在蠕动环境中长成的人结肠肿瘤类器官具有更均匀的尺寸分布,对该纳米胶束的摄入量显著降低。Grassart等[38]通过循环拉伸模拟肠道蠕动,研究蠕动状态是否会影响人类志贺菌在3D结肠上皮内的传染性。研究表明,与非机械刺激条件相比,拉伸力(蠕动)的应用显著提高了约50%的感染率,蠕动运动促进了细菌入侵。
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肠上皮富含含氧血液,对于增强绒毛的营养吸收和加速针对病原体的免疫反应至关重要。人体肠道是多种微生物群落的宿主,肠道微生物群在肠道的消化和吸收功能中发挥着重要作用[39]。HuMiX实现了肠道模型中氧气浓度梯度的变化,允许Caco-2细胞和厌氧菌之间的共培养,在体内重现转录、代谢和免疫特征[28]。Shin等[40]开发了一种缺氧-氧气接口(AOI芯片),将缺氧培养基从细菌生长的上通道灌注到肠细胞生长的下通道。实验结果表明,上皮细胞层的存在和管腔微通道中的流量依赖性调节对于在AOI芯片中产生稳态垂直氧梯度是必要且充分的。另一个包含高分辨率溶解氧监测的GOC模型是肠芯片,它有6个传感器盘,在模型的顶部和底部固定有氧淬灭荧光颗粒,允许实时监测氧水平。这种GOC模型有一个中央厌氧室,经常用饱和的5% CO2冲洗,维持上腔内低氧水平。使用这款厌氧肠道芯片,Jalili-Firoozinezhad等[41]也证明厌氧条件比好氧条件在生理上能保持更高的微生物多样性。表1概括了常见的肠道吸收芯片模型及其应用。
表 1 肠芯片模型的主要组成和应用
细胞类型 共培养 膜材料 细胞外基质 剪切力 蠕动
(循环机械应变)氧气梯度 应用 Caco-2 否 聚对苯二甲酸(PET)膜 无 1 μl/h 否 否 肠道吸收的功能[42] Caco-2 鼠李糖乳杆菌GG (LGG) 聚碳酸酯(PC)膜 胶原蛋白 25 μl/h 否 是 宿主-微生物分子相互作用[28] Caco-2 人肠微血管内皮细胞 (HIMEC) 聚二甲基硅氧烷(PDMS)多孔膜 Ⅰ型胶原蛋白和Matrigel混合物 60 μl/h 否 是 概述疾病模型和对策药物筛选[41] Caco-2 LGG PDMS多孔膜 Ⅰ型胶原蛋白和Matrigel混合物 30 μl/h 10%;0.15 Hz 否 肠道的运输,吸收和毒性研究[34] Caco-2 大肠杆菌 PDMS膜 Ⅰ型胶原蛋白和Matrigel混合物 30 μl/h 10%;0.15 Hz 否 肠道-微生物相互作用和疾病模拟[35] Caco-2 人脐静脉内皮细胞 (HUVEC)、大肠杆菌细胞和巨噬细胞 PDMS膜 Ⅰ型胶原蛋白 60 μl/h 15%;0.17 Hz 否 模拟肠道炎症模型和药物筛选[43] 肠道活检类器官 HIMEC PDMS膜 Ⅰ型胶原蛋白和Matrigel混合物 60 μl/h 10%;0.2 Hz 否 模拟正常肠道生理学[44] 人十二指肠类器官(成人供体) HIMEC PDMS膜 Ⅳ型胶原蛋白和Matrigel混合物(上皮侧)、Ⅳ型胶原蛋白和纤连蛋白混合物(血管侧) 30 μl/h 10%;0.2 Hz 否 研究肠道代谢和药物转运[45] Caco-2 否 无 聚乙烯-醋酸乙
烯酯0.1 ml/min 否 否 高通量药物吸收分析和细菌-宿主相互作用的研究[30]
Development and application of in vitro intestinal absorption model based on microfluidic chips
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摘要: 肠道是口服药物吸收的主要部位,肠道的上皮细胞上含有绒毛和微绒毛,它们通过增加表面积等因素促进分泌、细胞黏附和吸收。传统的二维/三维(2D/3D)细胞培养模型、动物模型在研究药物吸收方面发挥了重要作用,但是由于缺乏足够的人体药动学的可预测性或伦理问题等,其应用存在一定局限性。因此,以体外活细胞为基础,模拟人肠道的核心结构和关键功能是构建基于微流控芯片的肠道模型的研究重点。该模型是利用微加工技术制备出的模拟人体肠道的复杂微结构、微环境和生理功能的微流控芯片仿生系统。与2D细胞培养和动物实验相比,肠芯片模型能有效地模拟人体内环境,在药物筛选方面更具特异性。本综述概括了国内外肠芯片模型以及与肠道相关的多器官耦合芯片模型的研究进展,及其在疾病建模、药物吸收和转运方面的应用,并总结了当前肠芯片模拟肠道稳态和疾病面临的挑战,为进一步建立更可靠的体外肠芯片模型提供参考。Abstract: The intestine is the main site of oral drug absorption, and the epithelial cells of the intestine contain villi and microvilli, which promote secretion, cell adhesion, and absorption by increasing surface area and other factors. Traditional two-dimensional/three-dimensional (2D/3D) cell culture models and animal models have played an important role in studying drug absorption, but their application is limited due to the lack of sufficient predictability of human pharmacokinetics or ethical issues, etc. Therefore, mimicking the core structure and key functions of the human intestine based on in vitro live cells has been the focus of research on constructing a microfluidic chip-based intestinal model. The model is a microfluidic chip bionic system that simulates the complex microstructure, microenvironment, and physiological functions of the human intestine using microfabrication technology. Compared with 2D cell culture and animal experiments, the intestinal microarray model can effectively simulate the human in vivo environment and is more specific in drug screening. The research progress and applications in disease modeling, drug absorption and transport of intestinal microarray models and intestine-related multi-organ coupled microarray models at home and abroad were reviewed in this paper. The current challenges of intestinal chip simulating intestinal homeostasis and diseases were summarized, in order to provide reference for the further establishment of a more reliable in vitro intestinal chip model.
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Key words:
- microfluidic chips /
- intestinal absorption model /
- microenvironment /
- applications
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图 1 肠道微环境关键特征[26]
图 3 IOAC装置及对难溶SN38和前药的体外渗透系数与计算的油-水分配系数比较[52]
A.IOAC装置示意图;B.SN38和前药的体外渗透性系数与计算的油-水分配系数比较;1. 7-乙基-10-羟基喜树碱;2. 7-乙基-10-羟基喜树碱-20-十一酸酯;3. 7-乙基-10-羟基喜树碱-10-十一酸酯;4. 7-乙基-10-羟基喜树碱-20-丙酸酯
表 1 肠芯片模型的主要组成和应用
细胞类型 共培养 膜材料 细胞外基质 剪切力 蠕动
(循环机械应变)氧气梯度 应用 Caco-2 否 聚对苯二甲酸(PET)膜 无 1 μl/h 否 否 肠道吸收的功能[42] Caco-2 鼠李糖乳杆菌GG (LGG) 聚碳酸酯(PC)膜 胶原蛋白 25 μl/h 否 是 宿主-微生物分子相互作用[28] Caco-2 人肠微血管内皮细胞 (HIMEC) 聚二甲基硅氧烷(PDMS)多孔膜 Ⅰ型胶原蛋白和Matrigel混合物 60 μl/h 否 是 概述疾病模型和对策药物筛选[41] Caco-2 LGG PDMS多孔膜 Ⅰ型胶原蛋白和Matrigel混合物 30 μl/h 10%;0.15 Hz 否 肠道的运输,吸收和毒性研究[34] Caco-2 大肠杆菌 PDMS膜 Ⅰ型胶原蛋白和Matrigel混合物 30 μl/h 10%;0.15 Hz 否 肠道-微生物相互作用和疾病模拟[35] Caco-2 人脐静脉内皮细胞 (HUVEC)、大肠杆菌细胞和巨噬细胞 PDMS膜 Ⅰ型胶原蛋白 60 μl/h 15%;0.17 Hz 否 模拟肠道炎症模型和药物筛选[43] 肠道活检类器官 HIMEC PDMS膜 Ⅰ型胶原蛋白和Matrigel混合物 60 μl/h 10%;0.2 Hz 否 模拟正常肠道生理学[44] 人十二指肠类器官(成人供体) HIMEC PDMS膜 Ⅳ型胶原蛋白和Matrigel混合物(上皮侧)、Ⅳ型胶原蛋白和纤连蛋白混合物(血管侧) 30 μl/h 10%;0.2 Hz 否 研究肠道代谢和药物转运[45] Caco-2 否 无 聚乙烯-醋酸乙
烯酯0.1 ml/min 否 否 高通量药物吸收分析和细菌-宿主相互作用的研究[30] -
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