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

 描述(申请人提供)：肌成纤维细胞是胃肠道(GI)中一种有影响力的间质细胞，它调节从细胞增殖到许多重要的过程。 和沿隐窝-绒毛轴的分化、粘膜修复、纤维化和癌症的发展[7-9]。调控肌成纤维细胞功能的信号通路已经在体外进行了研究[10，11]。然而，由于现有共培养和动物模型的限制，这些信号事件对覆盖上皮细胞的实际生理影响在很大程度上仍然是推测的。体外共培养模型缺乏正常的肠壁结构和周围细胞群，而这些都是胃肠道微环境的基本要素[9]。利用条件性基因打靶的动物模型既不是器官特有的，也不是特定于细胞类型的，因为肌成纤维细胞 缺乏唯一的细胞标记[12，13]。目前的研究努力受到体内模型的限制，无法有效地研究肌成纤维细胞在正常和病理条件下如何调节上皮细胞的功能。这项建议涉及开发一种利用小鼠结肠镜检查的新技术，该技术将允许在体内实时研究肌成纤维细胞-上皮细胞 互动。这项技术首先涉及从小鼠结肠组织中分离出原代肌肉成纤维细胞，然后在细胞培养中生长。原代肌成纤维细胞将在内窥镜下通过注射到免疫能力强的同基因小鼠的结肠壁中重新植入。这项技术允许有针对性地操纵小鼠结肠某一段的肌成纤维细胞亚群，可以很容易地重新鉴定、连续评估，并在相同的实验条件下直接与邻近组织进行比较。这项技术可以应用于任何现有的小鼠模型，以研究远端结肠的间质-上皮相互作用。该建议旨在1)证明肌成纤维细胞可以成功和可重复地植入结肠壁，并可以在活的、免疫活性强的同基因小鼠中保持存活，并作为原则的证明，2)该模型可以用于研究基质-上皮细胞的相互作用，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.