Sealing and Healing of Epithelial Gaps during Cell Shedding and Disease

性状（由申请方提供）：肠上皮对不良管腔内容物侵入身体提供屏障。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的方法，允许活绒毛的研究，我们发现，肠上皮细胞脱落和潜在的不完善的细胞包装所造成的不连续性。令人惊讶的是，这些无细胞间隙充满了维持上皮屏障的不可渗透物质。我们的研究结果表明，上皮细胞可以密封上皮屏障之前，使用细胞迁移愈合细胞层的差距。虽然细胞恢复将最终恢复上皮的连续性，我们的假设是，我们观察到的机制是屏障功能的主要捍卫者绒毛细胞脱落后立即，这一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确认或否认我们发现的重要性的基本点，然后再建议该项目值得获得全部RO1级资助。我们将使用体内共聚焦和双光子显微镜在正常和突变小鼠中进行功能研究，并使用免疫组织化学和共聚焦内窥镜进行小鼠和人类样本之间的比较研究。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，免疫组织化学和微量分析工具来确定什么样的材料密封的差距，并维持上皮屏障。我们还将测试肌球蛋白轻链激酶的激活是否介导了愈合间隙的荷包缝合。在第二个目标中，我们将询问细胞丢失的位置以及沿着绒毛产生沿着的位置，使用具有EGFP的隐窝表达嵌合体的嵌合小鼠来测量来自单个隐窝的迁移到活绒毛上的细胞群的命运。第三个目标是初步的共聚焦内窥镜研究，评价非病变人类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间隙密封和愈合的分子，并为我们在以后的工作中定义健康和患病组织（特别是肠道通透性改变的疾病）中间隙的重要性奠定基础。去年，我们发现了一种新的方法，可以让肠道内壁提供一个屏障，保护身体免受管腔内容物的侵害，在这种方法中，身体能够用一种不可渗透的物质暂时填充肠道内壁细胞层的间隙，而不是立即使用另一个（新的）细胞来堵塞差距。使用先进的显微镜方法，让我们研究小鼠和人类肠道中的活组织，我们将努力了解小鼠间隙密封的潜在机制，并评估人类肠道中间隙的位置和频率。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.