Cellular models of microvillus inclusion disease

微绒毛包涵体病的细胞模型

• 批准号: 8517115
• 负责人: H Lee Sweeney
• 金额: $ 23.16万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8517115
• 关键词: Actins; Appearance; Binding; Biochemical; Biological Assay; Caco-2 Cells; Cell Culture Techniques; Cell Line; Cell model; Cell physiology; Cells; Cellular Assay; Cessation of life; Code; Cytomegalovirus Infections; Cytoskeleton; Development; Disease; Electron Microscopy; Epithelial Cells; Future; Genes; Goals; Heterozygote; Homozygote; Human; Inclusion Bodies; Intestinal Absorption; Intestines; Kidney Diseases; Kinetics; Lead; Liver diseases; Maintenance; Missense Mutation; Modeling; Molecular Motors; Motor; Movement; Mutation; Myosin ATPase; Outcome; Parenteral Nutrition; Pathogenesis; Pathologic Processes; Proteins; Recycling; Regulation; Role; Staging; Therapeutic Intervention; Time; Vascular Diseases; apical membrane; base; cellular microvillus; design; disease-causing mutation; early onset; functional loss; insight; knock-down; mutant; outcome forecast; research study

DESCRIPTION (provided by applicant): We have developed a cellular model of microvillus inclusion disease. Microvillus inclusion disease (MVID or MID~ MIM#251850) is characterized by the loss of microvilli and the appearance of microvillus inclusion bodies in intestinal epithelial cells. This results in a lss of intestinal absorption, rapidly leading to death without parenteral nutrition (TPN). Te only documented gene involved in this disease in the gene encoding the molecular motor, myosin Vb. The majority of the mutations result in loss of functional myosin Vb protein, while a subset of the mutations are missense mutations that likely alter motor function. We have generated a line of intestinal epithelial cells (Caco-2 cells) in which myosin Vb levels are <5% of wild type and can no longer maintain microvilli when they are polarized. This will serve as a cell culture model for a subset of microvillus inclusion disease, and can be used as a cellular assay for myosin Vb function. We will characterize the impact of MVID-causing missense mutations on the kinetics of the myosin myosin Vb motor. Based on the outcome of the biochemical characterizations, we will create a number of new lines of Caco-2 cells that express representative mutant myosin Vb proteins in the near null (knock down) background, thus creating new cellular models of MVID. Upon differentiation of these cells lines, we will examine the impact the myosin Vb missense mutations on microvilli development/maintenance. The creation of these lines will set the stage for future studies to understand the role of myosin Vb in apical membrane recycling as well as provide a better understanding of the pathogenesis of MVID.

描述（由申请人提供）： 我们开发了微绒毛包涵体疾病的细胞模型。微绒毛包涵体病（MVID或MID~MIM#251850）的特征是肠上皮细胞中微绒毛的丧失和微绒毛包涵体的出现。 这会导致肠道吸收减少，如果没有肠外营养 (TPN)，会迅速导致死亡。 仅在编码分子马达肌球蛋白 Vb 的基因中记录到与这种疾病有关的基因。大多数突变会导致功能性肌球蛋白 Vb 蛋白的丧失，而一部分突变是错义突变，可能会改变运动功能。我们已经生成了一系列肠上皮细胞（Caco-2 细胞），其中肌球蛋白 Vb 水平 < 5% 野生型，当它们极化时不能再维持微绒毛。这将作为微绒毛包涵体疾病子集的细胞培养模型，并可用作肌球蛋白 Vb 功能的细胞测定。我们将描述引起 MVID 的错义突变对肌球蛋白肌球蛋白 Vb 运动动力学的影响。根据生化表征的结果，我们将创建许多新的 Caco-2 细胞系，这些细胞在近零（敲低）背景下表达代表性突变肌球蛋白 Vb 蛋白，从而创建新的 MVID 细胞模型。在这些细胞系分化后，我们将检查肌球蛋白 Vb 错义突变对微绒毛发育/维持的影响。 这些细胞系的创建将为未来的研究奠定基础，以了解肌球蛋白 Vb 在顶膜回收中的作用，并更好地了解 MVID 的发病机制。