Sealing and Healing of Epithelial Gaps during Cell Shedding and Disease

描述(由申请人提供)：肠道上皮对不希望的腔内容物对身体的侵袭具有屏障作用。使用免疫组织化学和一种新的体内共聚焦和双光子显微镜方法来研究活的绒毛，我们发现肠上皮被细胞脱落和潜在的不完善的细胞包装引起的不连续所打断。令人惊讶的是，这些无细胞的缝隙被一种维持上皮屏障的不渗透物质填满。我们的结果表明，在使用细胞迁移修复细胞层的缝隙之前，上皮可以封闭上皮屏障。虽然细胞重建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，该机制的扰动导致疾病。我们在这份R21拨款申请中的目标是，在建议该项目值得全额RO1级资金之前，解决我们认为将证实或否认我们的发现的重要性的基本点。我们将使用体内共聚焦显微镜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过程中和之后维持屏障功能的机制。具体地说，我们将使用酶消化、免疫组织化学和显微分析工具来确定哪类材料封闭了缝隙并维持了上皮屏障。我们还将测试肌球蛋白轻链激酶的激活是否介导了缝合缝隙的钱包缝合。在第二个目的中，我们将询问细胞在哪里丢失，沿着绒毛产生间隙，使用带有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一群细胞的命运。第三个目标是一项初步的共聚焦内窥镜研究，评估未患病的人类回肠组织上皮间隙的特征、位置和频率。这些研究的结果将确定缝隙封闭和愈合所涉及的分子，并为我们在以后的工作中确定健康和患病组织(特别是肠道通透性改变的疾病)中缝隙的重要性奠定基础。去年，我们发现了一种新的方法，让肠道衬里提供一种屏障，保护身体免受管腔内容物的影响，在这种方式下，身体能够暂时用不透水的物质填补肠道衬里细胞层的缺口，而不是立即使用另一个(新的)细胞来填补缺口。利用先进的显微技术研究小鼠和人类肠道的活体组织，我们将致力于了解小鼠肠道缝隙封闭的潜在机制，并评估人类肠道缝隙的位置和频率。这些研究的结果将使我们能够评估人类疾病差距的重要性，作为下一步。

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.