Höner zu Bentrup Kerstin, Ramamurthy Rajee, Ott C Mark, Emami Kamal, Nelman-Gonzalez Mayra, Wilson James W, Richter Emily G, Goodwin Thomas J, Alexander J Stephen, Pierson Duane L, Pellis Neal, Buchanan Kent L, Nickerson Cheryl A
Department of Microbiology and Immunology, Program in Molecular Pathogenesis and Immunity, Tulane University Health Sciences Center, New Orleans, LA 70112, USA.
Microbes Infect. 2006 Jun;8(7):1813-25. doi: 10.1016/j.micinf.2006.02.020. Epub 2006 Apr 27.
In vitro cell culture models used to study how Salmonella initiates disease at the intestinal epithelium would benefit from the recognition that organs and tissues function in a three-dimensional (3-D) environment and that this spatial context is necessary for development of cultures that more realistically resemble in vivo tissues/organs. Our aim was to establish and characterize biologically meaningful 3-D models of human colonic epithelium and apply them to study the early stages of enteric salmonellosis. The human colonic cell line HT-29 was cultured in 3-D and characterized by immunohistochemistry, histology, and scanning electron microscopy. Wild-type Salmonella typhimurium and an isogenic SPI-1 type three secretion system (TTSS) mutant derivative (invA) were used to compare the interactions with 3-D cells and monolayers in adherence/invasion, tissue pathology, and cytokine expression studies. The results showed that 3-D culture enhanced many characteristics normally associated with fully differentiated, functional intestinal epithelia in vivo, including better organization of junctional, extracellular matrix, and brush-border proteins, and highly localized mucin production. Wild-type Salmonella demonstrated increased adherence, but significantly lower invasion for 3-D cells. Interestingly, the SPI-I TTSS mutant showed wild-type ability to invade into the 3-D cells but did not cause significant structural changes to these cells. Moreover, 3-D cells produced less interleukin-8 before and after Salmonella infection. These results suggest that 3-D cultures of human colonic epithelium provide valuable alternative models to study human enteric salmonellosis with potential for novel insight into Salmonella pathogenesis.
用于研究沙门氏菌如何在肠道上皮引发疾病的体外细胞培养模型，若能认识到器官和组织在三维（3-D）环境中发挥功能，且这种空间背景对于构建更逼真模拟体内组织/器官的培养体系至关重要，将会从中受益。我们的目标是建立并表征具有生物学意义的人结肠上皮三维模型，并将其应用于研究肠道沙门氏菌病的早期阶段。人结肠细胞系HT-29在三维环境中培养，并通过免疫组织化学、组织学和扫描电子显微镜进行表征。利用野生型鼠伤寒沙门氏菌和一个同基因SPI-1三型分泌系统（TTSS）突变衍生物（invA），在黏附/侵袭、组织病理学和细胞因子表达研究中比较它们与三维细胞和单层细胞的相互作用。结果表明，三维培养增强了许多通常与体内完全分化的功能性肠上皮相关的特征，包括连接蛋白、细胞外基质和刷状缘蛋白的更好组织，以及高度局部化的黏蛋白产生。野生型沙门氏菌对三维细胞的黏附增加，但侵袭显著降低。有趣的是，SPI-I TTSS突变体表现出侵入三维细胞的野生型能力，但并未对这些细胞造成显著的结构变化。此外，三维细胞在沙门氏菌感染前后产生的白细胞介素-8较少。这些结果表明，人结肠上皮的三维培养为研究人类肠道沙门氏菌病提供了有价值的替代模型，有望为沙门氏菌发病机制提供新的见解。
