肠上皮细胞通透性的体外及体内测定方法
Summary
本文提出了两种方法来确定肠道屏障功能。在组织培养井中, 使用上皮表 (伏特/欧姆) 直接测量培养的上皮细胞的测定电阻。在小鼠中, 采用 FITC-葡聚糖灌胃法测定体内肠道通透性。
Abstract
肠道屏障抵御致病微生物和微生物毒素。其功能由紧密的结通透性和上皮细胞完整性调节, 而肠道屏障功能的破坏有助于胃肠道和系统性疾病的发展。本文介绍了两种简单的方法来测量小肠上皮的通透性。在体外, Caco-2BBe 细胞被镀在组织培养井作为单层和测定电阻 (三) 可以用上皮 (伏/欧姆) 计测量。这种方法是令人信服的, 因为它的用户友好的操作和重复性。在体内,小鼠孕期 4 kDa 荧光素异硫氰酸酯 (FITC)-葡聚糖, 在采集的小鼠血清样本中测定 FITC-葡聚糖浓度, 以确定上皮通透性。口服灌胃提供准确剂量, 因此是测定体内肠道通透性的首选方法。这两种方法可以在体外和体内测量小肠上皮的通透性, 从而用于研究疾病与屏障功能的关系。
Introduction
肠道上皮细胞不仅负责养分的吸收, 而且是抵御致病微生物和微生物毒素的重要屏障。此肠屏障功能由紧密的结通透性和上皮细胞完整性1、2、3和上皮屏障功能功能障碍与炎症性肠相关调节疾病 (IBD)。perijunctional 肌动球蛋白环 (PAMR) 位于与紧密交汇点紧密相连的细胞内。PAMR 的收缩是由肌球蛋白光链 (MLC) 调节的, 对于紧密接合渗透率4、5、6、7、8的调节至关重要. 9,10。肿瘤坏死因子 (TNF) 是上调肠上皮 MLC 激酶 (拿) 表达和诱导 occludin 内化11、12、13的肠道屏障丢失的中枢。
离子, 如 Na+和 Cl–可以通过孔隙或泄漏通路14跨越旁细胞空间。在 “漏” 上皮中, 三的变化主要反映改变的紧密结通透性。测量是一种常用的电生理学方法, 用于根据细胞膜的阻抗来量化紧密结通透性, 主要是 Na+和 Cl。不同的细胞类型, 包括肠道上皮细胞, 肺上皮细胞和血管内皮细胞, 已经报告了三的测量。这种方法的优点是, 测量是非侵入性的, 可用于实时监测活细胞。此外, 该方法对药物毒性研究15有参考价值。
Caco-2BBe 细胞是人上皮性大肠腺癌细胞, 其结构和功能类似于分化的小肠上皮细胞: 例如, 这些细胞有微绒毛和与小肠刷相关的酶边境。因此, 培养的 Caco-2BBe 单层膜被用作检测屏障功能的体外模型。
在小鼠中, 研究肠道旁细胞通透性的一种方法是通过测量 FITC-葡聚糖从腔内向血液中交叉的能力。因此, 肠道通透性可以通过 gavaging FITC-葡聚糖直接对小鼠进行评估, 并测量血液中的荧光。下面的协议描述了两种简单的方法来评估小肠上皮通透性的体外和体内。
Protocol
Representative Results
Discussion
协议中有几个关键步骤。Caco-2BBe (画笔边框表示) 细胞始终用于测量, 从 Caco-2 细胞系中选择, 用于表达画笔边界蛋白。Caco-2BBe 细胞有一个绒毛吸收表型时完全分化 (经过大约3周的文化后汇合)17。在测量过程中, 必须避免污染, 并对电极进行灭菌。由于该过程是非无菌的, 在大多数情况下, 测量只能在8小时内执行。在测量过程中, 我们确定了刀片内外两点的电阻值。不同部位的电阻测…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢哈佛医学院的杨百翰和女子医院的杰罗尔德博士, 他对完成这项研究的慷慨帮助。这项工作由中国国家自然科学基金 (赠款号81470804、31401229和 81200620)、江苏省自然科学基金 (赠款编号 BK20180838 和 BK20140319) 支持, 研究创新计划江苏省大学毕业生 (赠款编号 KYLX16-0116)、东吴大学高级研究项目 (赠款编号 SDY2015_06)、克罗恩 & 结肠炎基金会研究奖学金 (赠款号 310801)。
Materials
| 22G gavage needle | VWR | 20068-608 | |
| 4 kDa FITC-dextran | Sigma | 46944 | |
| Avertin | Sigma | T48402 | |
| Black 96-well plates for fluorescence | Fisher | 14-245-197A | |
| C57/B6 mice | Nanjing Biomedical Research Institute of Nanjing University | ||
| Caco-2BBe cells | ATCC | CRL-2102 | |
| Dextran sulphate sodium | MP Biomedicals | 2160110 | |
| DMED with high glucose and sodium pyruvate | Hyclone | SH30243.01B | |
| Epithelial (Volt/Ohm) Meter | Millicell-ERS | MERS00002 | |
| Ethanol | Sinopharm ChemicalReagent | 10009218 | |
| Falcon tube (15 mL) | Corning | 430791 | |
| FBS | Gibco | 10437-028 | |
| Fluorescence microscope | Olympus | FV1000 | |
| Fluorometer | Biotek | Synergy 2 | |
| HBSS | 138 mM NaCl, 0.3 mM Na2HPO4, 0.4 mM MgSO4, 0.5 mM MgCl2, 5.0 mM KCl, 0.3 mM KH2PO4, 15.0 mM HEPES, 1.3 mM CaCl2, 25 mM glucose | ||
| IFNg | PeproTech | 315-05-20 | |
| Modular Tissue Embedding Center | Leica | EG1150H | |
| Serum collection tubes | Sarstedt | 41.1378.005 | |
| T75 flask | corning | 430641 | |
| TNF | PeproTech | 315-01A | |
| Parraffin | Sigma | A6330-1CS | |
| Polycarbonate membranes (Transwell) | Costar | 3413 | |
| Pressure pump | AUTOSCIENCE | AP-9925 | |
| Rotary Microtomy | Leica | RM2235 | |
| Trypsin-EDTA | Gibco | 25200-056 | |
| Xylene | Sinopharm ChemicalReagent | 10023418 |
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