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）： 我们已经开发了一种微绒毛包含疾病的细胞模型。微绒毛包含疾病（MVID或MID〜MIM＃251850）的特征是微绒毛的丧失和肠上皮细胞中微绒毛包含体的出现。 这导致了肠道吸收的LS，迅速导致没有肠胃外营养（TPN）死亡。 TE仅记录了编码分子运动肌球蛋白VB的基因中涉及该疾病的基因。大多数突变导致功能性肌球蛋白VB蛋白的丧失，而突变的子集是可能改变运动功能的错义突变。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，在极化时无法再维持微绒毛。这将作为微伏属疾病的一部分的细胞培养模型，可用作肌球蛋白VB功能的细胞测定法。我们将表征引起MVID的失误突变对肌球蛋白肌球蛋白VB电机动力学的影响。基于生化特征的结果，我们将创建许多新系列的Caco-2细胞，这些细胞在近零（敲低）背景中表达代表性突变的肌球蛋白VB蛋白，从而创建了MVID的新细胞模型。在分化这些细胞系的情况下，我们将研究肌球蛋白VB错义突变对微维利发育/维持的影响。 这些线的创建将为未来的研究奠定阶段，以了解肌球蛋白VB在顶膜回收中的作用，并更好地了解 MVID的发病机理。