A Novel In Vivo Mouse Model to Study Myofibroblast-Epithelial Cell Interactions

 描述（由申请人提供）：肌成纤维细胞是胃肠道（GI）的一种有影响力的基质细胞，可调节许多重要过程，包括细胞增殖 以及沿着隐窝-绒毛轴的分化、粘膜修复、纤维化和癌症的发展[7-9]。已经在体外研究了调节肌成纤维细胞功能的信号通路[10，11]。然而，由于现有的共培养和动物模型的限制，这些信号传导事件对上覆上皮的实际生理影响在很大程度上仍然是推测性的。体外共培养模型缺乏正常的肠壁结构和周围细胞群，而这些是GI微环境的基本要素[9]。利用条件性基因靶向的动物模型既不是器官特异性的，也不是细胞类型特异性的，因为肌成纤维细胞 缺乏独特的细胞标志物[12，13]。目前的研究工作受到缺乏体内模型的限制，无法有效地研究肌成纤维细胞如何在正常和病理条件下调节上皮细胞功能。这项建议涉及一种新技术的发展，利用小鼠结肠镜检查，将允许实时在体内研究肌纤维母细胞上皮细胞 交互.该技术首先涉及从小鼠结肠组织中分离原代肌成纤维细胞，随后在细胞培养中生长。然后将原代肌成纤维细胞通过内窥镜注射到免疫活性的同系小鼠的结肠壁中重新植入。该技术允许靶向操纵小鼠结肠段中的肌成纤维细胞亚群，该亚群可以容易地重新鉴定、连续评价并在相同实验条件下与相邻组织直接比较。该技术可应用于任何现有的小鼠模型，以研究远端结肠中的基质-上皮相互作用。该提案旨在1）证明肌成纤维细胞可以成功地和可重复地植入结肠壁中，并且可以在活的、免疫活性的、同基因小鼠中保持活力，并且作为原理证明，2）该模型可以用于研究基质-上皮细胞相互作用，特别是在损伤修复的动物模型的背景下。

 DESCRIPTION (provided by applicant): The myofibroblast is an influential stromal cell of the gastrointestinal (GI) tract that regulates many important processes ranging from cell proliferation and differentiation along the crypt-villous axis, mucosal repair, fibrosis, and the development of cancer [7-9]. The signaling pathways that regulate myofibroblast function have been studied in vitro [10, 11]. However, the actual physiologic impact of these signaling events on the overlying epithelium remains largely speculative due to limitations with existing co-culture and animal models. In vitro co-culture models lack the normal bowel wall architecture and surrounding cell populations that are essential elements of the GI microenvironment [9]. Animal models that utilize conditional gene targeting are neither organ nor cell type-specific since the myofibroblast lacks a unique cell marker [12, 13]. Current research efforts are limited by a paucity of in vivo models to effectively study how the myofibroblast regulates epithelial cell function under normal and pathologic conditions. This proposal involves the development of a novel technique utilizing murine colonoscopy that will allow for the real-time in vivo study of myofibroblast-epithelial cell interactions. The technique first involves the isolation of primary myofibroblasts from mouse colon tissue and subsequent growth in cell culture. Primary myofibroblasts will then be re-implanted endoscopically by injection into the colon wall of an immune-competent, syngeneic mouse. The technique allows for targeted manipulation of a subpopulation of myofibroblasts in a segment of mouse colon that can be easily re-identified, serially evaluated, and directly compared to adjacent tissue under the same experimental conditions. This technique can be applied to any existing mouse model to study stromal-epithelial interactions in the distal colon. The proposal aims to 1) demonstrate that myofibroblasts can be successfully and reproducibly implanted in the colon wall and can maintain viability in live, immune-competent, syngeneic mice, and as a proof of principle, that 2) this model can be used to study stromal-epithelial cell interactions, specifically in the context of an animal model of injury-repair.