测定细菌和化学物质对开牛结不二病的肠道渗透性的影响
Summary
该协议描述了如何测量卡内沙布迪炎的肠道渗透性。该方法有助于对肠道健康进行与肠道细菌与其宿主相互作用相关的基础生物学研究，并有助于筛选出益生菌和化学制剂，以治疗漏肠综合征和炎症性肠病。
Abstract
在活生物体中，肠道高渗透性是导致许多炎症性肠病 （IBDs） 的严重症状。Caenorhabditis elegans是一种非哺乳动物动物模型，由于其寿命短、透明度高、成本效益高和缺乏动物伦理问题而被广泛用作测定系统。本研究开发了一种方法，通过高通量图像分析系统，研究不同细菌和3，3′-二丁二醇甲烷（DIM）对大肠杆菌肠道渗透性的影响。蠕虫感染了不同的肠道细菌或与DIM共处理48小时，并喂食氟西辛异体酸（FITC）-dextran过夜。然后，通过比较蠕虫体内的荧光图像和荧光强度，对肠道渗透性进行了研究。这种方法还可能识别影响动物模型中肠道渗透性的益生菌和致病性肠道细菌，并有效检查有害或促进健康的化学品对肠道渗透性和肠道健康的影响。然而，该协议在遗传水平上也有相当大的局限性，特别是用于确定哪些基因被改变来控制疾病，因为这种方法主要用于表型测定。此外，这种方法仅限于准确确定哪些致病基物引起炎症或增加蠕虫肠道在感染期间的渗透性。因此，需要进一步深入研究，包括利用突变细菌和线虫研究分子遗传机制，以及细菌的化学成分分析，以充分评估细菌和化学物质在确定肠道渗透性方面的作用。
Introduction
肠道渗透性被认为是与肠道微生物群和粘膜免疫相关的主要障碍之一，并可能受到多种因素的影响，如肠道微生物群修饰、上皮损伤或粘液层改变1。最近的论文报道了通过分析肠道细胞层2的荧光通量率来测量培养的人类肠道细胞的肠道渗透性的有效方案，但研究论文较少，提供了一个合适的程序来测量线虫的肠道渗透性，特别是在C.elegans中，使用FITC-dextran染色。
有两个代表性的协议，用于测量C.elegans的肠道渗透性使用尼罗河红3和二氧化钠（或蓝精灵测定）4，5。在此协议中，我们使用 FITC-dextran（平均分子量 10，000），其分子量远远高于尼罗河红 （MW = 318.37） 和二氧化钠 （MW = 792.85）。FITC-dextran 比尼罗河红或二氧化钠染料更类似于实际大分子营养素，如碳水化合物，这些营养素通过肠道层被吸收。无需荧光显微镜即可轻松评估用二氧化钠（蓝色蓝精灵染料）喂养的C. elegans的肠道渗透性。然而，在蓝精灵测定中，由于缺乏标准化，对肠道渗透性进行定量分析比较困难，应手动评估4、5。在尼罗河红测定中，尼罗河红也污渍细胞中的脂液滴，这可能干扰C.elegans6的肠道渗透性的精确测定。本方案能够快速、准确地定量分析使用各种肠道细菌和化学品治疗的大肠杆菌的肠道渗透性，同时避免不特定的脂质染色。
由于价格实惠、易于操作、动物伦理问题有限、寿命短，是生物领域的一种典型模型，有利于快速实验。特别是，在整个C.elegans基因组公布后，发现C.elegans基因组中近40%的基因与导致人类疾病的基因有正交。此外，透明体允许在生物体内观察，这有利于研究细胞事件和荧光应用在细胞生物学，例如，干细胞染色与DAPI或免疫组织化学9。C. elegans经常被用作实验动物，研究肠道微生物群与宿主之间的相互作用;此外，C.elegans用于筛选促进健康的益生菌10、11、12以及促进肠道健康的膳食化学品13、14。
假单核菌和肠球菌是已知的肠道细菌，对胃肠道系统有负面影响，特别是肠道的结肠上皮细胞15，16。因此，测量这些细菌触发的肠道渗透性是筛选和开发新药所必需的，这些药物可以恢复和减少细菌炎症和感染造成的损害。在这个协议中，我们测试了这些肠道细菌对大肠杆菌渗透性的影响。
我们还报告一个优化的协议，用于测试化学物质的肠道渗透性。为此，我们使用3，3′-二氧化铝甲烷（DIM）作为模型化学，因为DIM是一种生物活性代谢物化合物，来自Indole-3-卡宾醇，存在于布拉西卡食品植物中，据报道对小鼠17、18的IBD有治疗作用。此外，我们最近发现DIM改善肠道渗透功能障碍，在培养的人类肠道细胞以及模型线虫C.elegans19。
在这项研究中，我们使用了三种不同的实验条件。首先，我们测量了不同细菌，P.aeruginosa和E.粪便对肠道渗透性的影响（图1）。其次，我们测量了活和热灭活P.aeruginosa对肠道渗透性的影响（图2）。第三，我们测量了DIM（一种化学模型）对用P.aeruginosa喂养的C.elegans的肠道渗透性的影响（图3）。
本研究的目的是开发优化的规程，以测量大肠杆菌的肠道渗透性，通过处理各种肠道细菌和化学品而改变。
Protocol
Representative Results
Discussion
通过利用这种新方法确定C.elegans的肠道渗透性，结合自动荧光显微镜和定量图像分析，可以确定肠道微生物或化学物质在体内引起的差异，特别是在C.elegans肠道中。该协议适用于肠道渗透性调查，并适用于许多任务，如在应力条件下的活性氧物种 （ROS） 测定和形态检查，由于其方便和易于操作。此外，该方法还可用于确定治疗和预防方法对C.elegans中多种病原体的影响。特别?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了韩国科学技术研究院的校内研究资助（2E29563）的支持。
Materials
| 3,3’-diindolylmethane | Sigma | D9568 | |
| 90×15 mm Petri dishes | SPL Life Sciences, South Korea | 10090 | |
| 60×15 mm Petri dishes | SPL Life Sciences, South Korea | 10060 | |
| Bactor Agar | Beckton Dickinson | REF. 214010 | |
| Formaldehyde solution | Sigma | F1635 | |
| Brain Heart Infusion (BHI) | Becton Dickinson | REF. 237500 | |
| Caenorhabditis elegans N2 | Caenorhabditis Genetics Center (CGC) | Wild type | |
| Cholesterol | Sigma | C3045 | |
| Costa Assay Plate, 96 Well Black With Clear Flat Bottom Non-treated, No Lid Polystyrene | Corning Incorporated | REF. 3631 | |
| Dimethyl sulfoxide | Sigma | D2650 | |
| Enterococcus faecalis KCTC 3206 | Korean Collection for Type Culture | KCTC NO. 3206 | Falcutative anaerobic |
| Escherichia coli OP50 | Caenorhabditis Genetics Center (CGC) | ||
| Fluorescein isothiocyanate – dextran | Sigma | FD10S | |
| Harmony software | PerkinElmer | verson 3.5 | |
| Luria-Bertani LB medium | Merck | VM743185 626 1.10285.5000 | |
| Magnesium sulfate heptahydrate | Fisher Bioreagents | BP2213-1 | |
| Fluoromount aqueous mounting medium | Sigma | F4680 | |
| Operetta CLS High-Content Analysis System | PerkinElmer | HH16000000 | |
| Peptone | Merck | EMD 1.07213.1000 | |
| Pseudomonas aeruginosa PA01 | Korean Collection for Type Culture | KCTC NO. 1637 | |
| Sodium Chloride | Fisher Bioreagents | BP358-1 | |
| Stereo Microscope | Nikon, Japan | SMZ800N | |
| Yeast extract | Becton Dickinson | REF. 212750 |
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