体外肠囊评估黏膜通透性在胃肠道疾病模型
Summary
This protocol describes the use of excised intestinal tissue preparations or “intestinal sacs” as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.
Abstract
上皮屏障是胃肠道的第一先天防御和选择性地调节传输从所述内腔到底层组织区室，限制横跨上皮更小的分子和几乎完全禁止上皮大分子转运的运输。此选择性通过粘膜凝胶层，这限制了亲脂性分子和两个心尖受体和上皮的紧交界蛋白质复合物的运输来确定。 体外细胞培养的上皮的模型是方便的，但作为一种模式，它们缺乏的微生物群，粘液凝胶，上皮细胞和免疫系统之间的相互作用的复杂性。另一方面，可使用“肠囊”进行体内肠吸收或渗透性的评价，但是这些测定法测量总胃肠道吸收，与没有指示点特异性的。 体外通透性测定;是测量任一整体肠完整性或特定分子的比较运输，与肠部位特异性的附加优点的快速和灵敏的方法。在这里，我们描述了肠囊的渗透性编写研究报告和表观渗透率（P 程序 ）的计算 横跨肠屏障的分子。这种技术可以用作评估药物吸收的方法，或审查胃肠疾病的动物模型的区域的上皮屏障功能障碍。
Introduction
胃肠道的肠上皮屏障是在人成年估计在400 米 2的粘膜表面积。因此，不断地暴露从微生物，摄入药物，营养物和细菌毒素挑战。主机必须不仅容许的共生菌和潜在病原体区分，但必须防止从穿越上皮屏障这些物种和它们的分泌的分子，而在同一时间，允许营养物质的吸收。因此，肠上皮细胞的作用是充当选择性屏障的管腔内容1。做到这一点，在部分地通过在粘膜先天性上皮防御系统，它通过包括组成型和诱导机制2响应生物系统的作用。
上皮屏障功能的丧失是病理学是一个数肠胃疾病的特性，在体内上皮屏障功能的检查可通过示踪剂分子的经口管饲，并随后血清分析3进行评估。然而，该技术提供了没有说明的屏障功能障碍的部位。在体外和使用Transwell小系统3分别尤斯灌流室4,5跨上皮电阻体外评估，通常作为上皮屏障功能的替代指标，但缺乏动物模型6的贡献疾病生理学。在这个协议中，我们描述一个离体组织制剂模型，它允许肠道的完整性和可用于在多个级别来评估黏膜屏障功能的直接的和局部的评估。重要的是，这种技术可应用于疾病的动物模型，或可以药理学上操纵以允许的粘膜屏障功能障碍深度询问。
Protocol
Representative Results
Discussion
这里，我们已经详细介绍了分离和制备肠囊的评估黏膜屏障功能体外 。肠囊的准备工作，主要被用于中医药研究，考察候选药物在整个肠道的吸收。然而，该测定是同样适合于肠道疾病的研究。肠道通透性可能因地区和渗透率的网站具体的评估相差很大，可以更好地了解粘膜完整性的消化系统疾病的区域重要性。该测定是健壮和正确的生理条件下，分离的组织保持存活时间长达6小时的验?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was funded by National Health and Medical Research Project Grant APP1021582 and a Hunter Medical Research Institute grant sponsored by Sparke Helmore/NBN Triathlon and the Estate of the late Leslie Kenneth McFarlane.
Materials
| Dekantel Non-absorbable Silk suture | Braintree Scientific | SUT-S 116 | |
| Media 199 (TC199) | Life Technologies | 11043-023 | No phenol red as this interferes with fluorescence |
| Dulbecco's Modified Eagle Medium (DMEM) | Life Technologies | 21063-045 | No phenol red as this interferes with fluorescence |
| N-acetylcysteine | Sigma Aldrich | Use at 10mM in media | |
| Small animal vascular cathether: Physiocath | Data Sciences International | 277-1-002 | |
| FITC-Dextran 4400 MW | Sigma Aldrich | FD-4 | |
| FITC-Dextran 20,000 MW | Sigma Aldrich | FD-20 | |
| FITC-Dextran 70,000 MW | Sigma Aldrich | FD-70 |
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