Sealing and Healing of Epithelial Gaps during Cell Shedding and Disease

描述（由申请人提供）：肠上皮为不受欢迎的肠道内容物侵入机体提供屏障。利用免疫组织化学和一种新的体内方法，共聚焦和双光子显微镜，允许研究活绒毛，我们发现肠上皮被细胞脱落和可能不完美的细胞堆积引起的不连续性所中断。令人惊讶的是，这些无细胞的间隙被一种不可渗透的物质填充，这种物质维持着上皮屏障。我们的研究结果表明，上皮可以在使用细胞迁移来愈合细胞层间隙之前封闭上皮屏障。虽然细胞恢复最终将恢复上皮的连续性，但我们的假设是，我们观察到的机制是绒毛细胞脱落后屏障功能的主要捍卫者，而这种机制的扰动导致疾病。我们在R21拨款申请中的目标是解决我们认为将是在建议项目值得完全RO1级别资助之前确认或否认我们研究结果重要性的基本要点。我们将使用体内共聚焦和双光子显微镜对正常和突变小鼠进行功能研究，并使用免疫组织化学和共聚焦内窥镜对小鼠和人类样本进行比较研究。在第一个目标中，我们将质疑在细胞脱落期间和之后维持屏障功能的机制。具体来说，我们将使用酶消化、免疫组织化学和微分析工具来确定哪一类物质密封间隙并维持上皮屏障。我们还将测试肌凝蛋白轻链激酶的激活是否介导了愈合缺口的荷包闭合。在第二个目标中，我们将询问细胞在何处丢失，并沿着绒毛产生间隙，使用具有EGFP隐窝表达马赛克的嵌合小鼠来测量从单个隐窝迁移到活绒毛上的细胞群的命运。第三个目的是初步共聚焦内窥镜研究，评估非病变人回肠组织中上皮间隙的特征、位置和频率。这些研究的结果将定义参与密封和愈合间隙的分子，并为我们在以后的工作中定义健康和病变组织（特别是改变肠通透性的疾病）间隙的重要性奠定基础。去年，我们发现了一种新的方法，可以让肠道内壁形成屏障，保护身体免受肠道内容物的侵害。在这种方法中，身体可以用一种不渗透的物质暂时填补肠道内壁细胞层的空隙，而不是立即用另一个（新）细胞来堵塞空隙。利用先进的显微镜技术，我们可以研究小鼠和人类肠道的活体组织，我们将努力了解小鼠肠道间隙密封的潜在机制，并评估人类肠道间隙的位置和频率。这些研究的结果将使我们在下一步评估人类疾病缺口的重要性。

DESCRIPTION (provided by applicant): The intestinal epithelium presents a barrier to invasion of the body by undesirable luminal contents. Using both immunohistochemistry and a new in vivo method for confocal and two-photon microscopy that allows study of living villi, we find that the intestinal epithelium is punctuated by discontinuities caused by cell shedding and potentially imperfect cell packing. Surprisingly, these cell-free gaps are filled with an impermeable substance that maintains the epithelial barrier. Our results suggest that the epithelium can seal the epithelial barrier prior to use of cell migration to heal gaps in the cell layer. While cell restitution will ultimately restore epithelial continuity, our hypothesis is that the mechanism we have observed is the major defender of barrier function immediately following villus cell shedding, and that perturbations of this mechanism lead to disease. Our goal in this R21 grant application is to address what we believe will be the fundamental points that will confirm or deny the importance of our findings prior to suggesting the project is worthy of full RO1 level funding. We will perform functional studies in normal and mutant mice using in vivo confocal and two-photon microscopy, and use immunohistochemistry and confocal endoscopy for comparative studies between mouse and human samples. In the first aim we will question the mechanisms that maintain barrier function during and after cell shedding. Specifically, we will use enzymatic digestion, immunohistochemistry and micro-analytical tools to define what class of material seals the gaps and sustains the epithelial barrier. We will also test if activation of myosin light chain kinase mediates a purse string closure that heals gaps. In the second aim we will ask where cells are lost and gaps are generated along a villus, using chimeric mice with a mosaic of crypt expression of EGFP to measure the fate of the cohort of cells from an individual crypt that migrate onto a living villus. The third aim is an initial confocal endoscopy study evaluating the characteristics, locale and frequency of epithelial gaps in non-diseased human ileal tissue. The outcome of these studies will define molecules involved in sealing and healing of gaps, and set the stage for us to define the importance of gaps in healthy and diseased tissue (in particular diseases having altered intestinal permeability) in later work. Last year we discovered a new way for the intestinal lining to provide a barrier that protects the body from luminal contents, in which the body is able to temporarily fill a gap in the cell layer lining the gut with an impermeable substance, instead of immediately using another (new) cell to plug the gap. Using advanced microscopy methods that let us study living tissues in mouse and human intestine, we will work to understand the underlying mechanism of gap sealing in mouse, and evaluate the sites and frequency of the gaps in human intestine. The outcomes of these studies will poise us to evaluate the importance of gaps in human disease as a next step.