| Intestinal Co-culture System to Study TGR5 Agonism and Gut Restriction
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Author:
Date:
2021-03-20
[Abstract] The activation of the Takeda G-protein receptor 5 (TGR5, also known as the G protein-coupled bile acid receptor 1, GPBAR1) in enteroendocrine L-cells results in secretion of the anti-diabetic hormone Glucagon-Like Peptide 1 (GLP-1) into systemic circulation. Consequently, recent research has focused on identification and development of TGR5 agonists as type 2 diabetes therapeutics. However, the clinical application of TGR5 agonists has been hampered by side effects of these compounds that primarily result from their absorption into circulation. Here we describe an in vitro screening protocol to evaluate the TGR5 agonism, GLP-1 secretion, and gut-restricted properties of small molecules. The protocol involves differentiating gut epithelial and endocrine cells together in transwells to ...
[摘要] [摘要]肠内分泌L细胞中的Takeda G蛋白受体5（TGR5，也称为G蛋白偶联胆汁酸受体1，GPBAR1）的激活导致抗糖尿病激素胰高血糖素样肽1的分泌。 （GLP-1）进入全身循环。因此，最近的研究集中于鉴定和开发TGR5激动剂作为2型糖尿病治疗剂。但是，TGR5激动剂的临床应用受到这些化合物的副作用的阻碍，这些副作用主要是由于它们吸收进入循环系统所致。这里我们描述一个体外 筛选协议以评估TGR5激动剂，GLP-1分泌和小分子的肠道限制性特性。该协议涉及在跨孔中将肠道上皮细胞和内分泌细胞一起分化，以评估TGR5激动剂的药效学和化合物对肠道单层的毒性。作为概念的证明，我们证明了该协议在评估有效的TGR5激动剂自然存在的胆汁酸代谢物的性质中的应用。该协议改编自Chaudhari等人。（202 1 ）。
[背景和d ] GI道的肠壁是由几个不同类型的细胞，每一个特定的和，有时独有的功能的（阿莱尔等人。，2018） ...
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| Adhesion and Invasion Assay Procedure Using Caco-2 Cells for Listeria monocytogenes
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Author:
Date:
2017-05-05
[Abstract] Listeria monocytogenes is an important Gram-positive foodborne pathogen that is a particular problem in ready-to-eat food. It has an ability to survive in harsh conditions like refrigeration temperatures and high salt concentrations and is known to cross intestinal, placental and blood-brain barriers. Several cancerous cell lines like cervical, liver, dendritic, intestinal and macrophages have been used to study in vitro propagation and survival of listeria in human cells. Human intestinal epithelial cells have been used to study how listeria crosses the intestinal barrier and cause infection. The protocol in this articles describes the procedures to grow Caco-2 cells, maintain cells and use them for adhesion and invasion assays. During adhesion assay the cells are ...
[摘要] 单核细胞增生利斯特氏菌是一种重要的革兰氏阳性食源性病原体，是即食食品中的一个特殊问题。它具有在诸如制冷温度和高盐浓度的恶劣条件下生存的能力，并且已知可以穿过肠，胎盘和血脑屏障。已经使用了诸如子宫颈，肝脏，树突状细胞，肠和巨噬细胞的几种癌细胞系来研究人细胞中李斯特菌的体外扩增和存活。人肠上皮细胞已被用于研究李斯特菌如何穿过肠屏障并引起感染。本文中的方案描述了生长Caco-2细胞的过程，维持细胞并将其用于粘附和侵袭测定。在粘附测定期间，将细胞与李斯特菌孵育30分钟，但是在侵袭测定中，细胞生长在感染后的几个时间点被停止以监测细胞中李斯特菌的生长和存活率。
背景 ...
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| 3D Stroma Invasion Assay
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Author:
Date:
2017-03-20
[Abstract] We have developed a 3D co-culture system composed of fibroblasts and colorectal cancer cells that enables us to study the desmoplastic reaction. This method also enables us to study the influence of the desmoplastic reaction on the migration of colorectal cancer cells through the surrounding stroma. This protocol has been previously published (Coulson-Thomas et al., 2011) and is described here in more detail.
[摘要] 我们开发了一种由成纤维细胞和结肠直肠癌细胞组成的3D共培养系统，使我们能够研究脱发反应。这种方法还使我们能够研究脱发反应对结肠直肠癌细胞通过周围基质的迁移的影响。以前已经公布了该协议（Coulson-Thomas等人，2011），并且在此更详细地描述。
背景 癌症的进展依赖于癌细胞和周围细胞如成纤维细胞，炎性细胞和内皮细胞之间的复杂的串扰，其形成癌症微环境。成纤维细胞是主要的细胞外基质产生细胞并且负责组织的结构形成。肿瘤周围的成纤维细胞被癌细胞“激活”成肿瘤相关成纤维细胞（TAFs），并在肿瘤发生和转移中发挥关键作用。在一些癌症中，TAF上调细胞外基质表达，产生一种主要由胶原纤维和蛋白多糖组成的无组织基质，其影响癌细胞增殖，迁移和扩散。这被称为脱发反应，并且在癌细胞生长期间，不同的肿瘤可能表现出各种等级的发育不良。
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