Cell differentiation regulates intestinal tight junctions

小肠的主要功能包括从食物中吸收营养和充当过滤系统，选择性地允许有用的物质进入血液，同时防止体液损失和有害物质进入。数以百万计的肠道细胞通过连接蛋白结合在一起，这些连接蛋白同时作为过滤器和粘合剂工作。大多数肠道细胞是吸收细胞，并假定这种细胞类型建立的细胞-细胞连接决定了肠道的总体孔隙度。然而，还有几种其他类型的细胞，主要是非吸收细胞，其过滤特性完全未知，但可能对肠道多孔性有显著影响，本项目将阐明这些细胞类型的功能。只有一种肠道细胞类型的组织将被合成和测试，以确定仅由该细胞类型制造的过滤器的独特成分和孔隙率。该项目将促进对细胞分化和组成如何影响脊椎动物肠道过滤能力的基本理解。这一结果将与人类健康相关，因为几乎所有的过敏性、炎症性和感染性肠道疾病都允许物质的非选择性运动，从而导致疾病和死亡，但没有信息表明这些疾病是唯一的还是同等地影响不同细胞类型的过滤器。结果将通过科学出版物、杂志文章和公共网站上的博客发布。研究、指导和教学机会将提供给国际合作者、博士后研究员、研究生、本科生和高中生，以及当地教师，利用该机构针对代表不足群体的学生的项目。小肠干细胞不断分化为可吸收的肠细胞以及分泌型肠内分泌细胞、潘氏细胞、杯状细胞和其他类型的细胞，形成紧密的连接，封闭细胞旁间隙。干细胞和分泌细胞类型的连接的组成和屏障特性几乎是未知的，因为目前只能在主要由肠细胞或非生理性永生化细胞组成的上皮片中确定通透性。该项目采用了创新的技术，不仅控制干细胞的体外分化以产生主要由一种细胞类型组成的称为肠样细胞的原代培养物，而且还迫使成熟细胞去分化以重新获得干细胞特征。这一显著的技术进步使计划中的实验得以进行，这些实验验证了这样一种假设，即干细胞分化为吸收细胞和分泌细胞会改变紧密连接蛋白的表达和位置，从而导致大分子细胞旁通透性的显著、依赖于细胞类型的变化。主要的连接蛋白occludin，tricellin，和zonula occludens 1在决定细胞类型依赖的细胞旁成分和通透性方面的作用将被检验。高中、本科生、研究生和博士后实习生将参与这个项目，该项目应用现代方法进行研究，增加对细胞分化和非肠道细胞连接如何有助于脊椎动物上皮屏障功能的知识。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

The major functions of the small intestine include absorbing nutrients from food and acting as a filtration system, selectively allowing the passage of useful substances into the blood stream while preventing the loss of body fluids and the entry of harmful materials. The millions of intestinal cells are held together by junction proteins that work simultaneously as a filter and as a glue. Most intestinal cell are absorptive cells, and it is assumed that cell-cell connections made by this cell type determine the overall porosity of the intestine. However, there are several other types of mostly nonabsorptive cells whose filter properties are completely unknown but may contribute significantly to intestinal porosity, and this project will elucidate the function of those cell types. Tissues made of just one intestinal cell type will be synthesized and tested to determine the unique composition and porosity of filters made by only that cell type. The project will advance the fundamental understanding of how cell differentiation and composition affect the filtering capacity of the vertebrate intestine. The results will be relevant to human health because almost all allergic, inflammatory, and infectious diseases of the intestine allow nonselective movements of materials that can lead to illness and death, but there is no information on whether these diseases uniquely or equally affect the filters made by different cell types. Results will be disseminated via scientific publications, magazine articles, and blogs in public websites. Research, mentoring, and teaching opportunities will be provided to international collaborators, a postdoctoral researcher, graduate, undergraduate and high school students, and local teachers, leveraging programs at the institution that target students from underrepresented groups. Small-intestine stem cells continuously differentiate into absorptive enterocytes as well as secretory enteroendocrine, Paneth, goblet and other cell types that form tight junctions sealing paracellular spaces. The composition and barrier properties of junctions of stem-cell and secretory-cell types are virtually unknown, as permeability can presently only be determined in epithelial sheets composed mainly of enterocytes or of nonphysiological immortalized cells. This project employs innovative techniques that not only control in vitro differentiation of stem cells to produce primary cultures called enteroids comprised mainly of one cell type, but also force dedifferentiation of mature cells to reacquire stem-cell characteristics. This remarkable technical advance enables the planned experiments that test the hypothesis that differentiation of stem cells into absorptive and secretory cells alters the expression and location of tight-junction proteins, resulting in significant, cell-type-dependent variation in paracellular permeability of macromolecules. The role of the major junctional proteins occludin, tricellulin, and zonula occludens 1 in determining cell-type-dependent paracellular composition and permeability will be examined. High school, undergraduate, and graduate students and post-doctoral trainees will participate in this project which applies a modern approach to studies that increase knowledge of how cell differentiation and nonenterocyte junctions contribute to the barrier function of vertebrate epithelia.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.