Formation of bile ducts in three dimensional culture

三维培养中胆管的形成

• 批准号: 8080226
• 负责人: Keith E Mostov
• 金额: $ 33.27万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8080226
• 关键词: Biliary; Caliber; Cell Death; Cell Polarity; Cell Shape; Cells; Collagen; Development; Disease; Duct (organ) structure; Enzymes; Epithelial Cells; Event; Extracellular Matrix; Gel; Glycogen Synthase Kinase 3; Health; Hepatic; Hepatocyte; Immigration; Integrins; Intrahepatic bile duct; Laminin; Lead; Learning; Life; Lipids; Liver; Mesenchyme; Molecular; Mouse Cell Line; Movement; Natural regeneration; Pathway interactions; Patients; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phosphotransferases; Play; Process; Proliferating; Protein Isoforms; Recycling; Role; Signal Transduction; Stem cells; Structure; System; Testing; Trees; Work; bile duct; biliary tract; cell motility; cell type; cholangiocyte; improved; in vivo; inositol 4,5-bisphosphate; liver function; malformation; matrigel; monolayer; public health relevance

DESCRIPTION (provided by applicant): The branching tree of intrahepatic bile ducts is lined by a monolayer of epithelial cells, known as cholangiocytes. During development hepatocytes and cholangiocytes arise from bipotential hepatoblast precursors. Certain hepatoblasts, which are destined to become cholangiocytes, form a monolayered ring surrounding the portal mesenchyme. This monolayer of biliary precursors becomes a double layered structure. Lumens then develop between the two layers. Eventually these structures remodel into a branching tree of intrahepatic bile ducts. We have developed a three dimensional culture system that recapitulates important events in early development. This enables us to study the these events with much greater mechanistic detail than would be possible in vivo. We use a stable mouse cell line termed Hepatic Progenitor cells Proliferating on Laminin, (HPPL), which is capable of differentiating into hepatocytes and cholangiocytes. We grow these as a monolayer on top of a layer of extracellular matrix. We then overlay the monolayer with a second layer of extracellular matrix. After overlay some of the HPPL cells move up out of the original monolayer and crawl on top of the original monolayer. These migrating cells eventually form a second monolayer on top of the first. Lumens then form between the two monolayers. This process closely resembles the early events in bile duct formation in vivo. However, migration of the cells out of the original monolayer requires phosphatidyl inositol 3- kinase (PI3K), the enzyme that generates phosphatidyl inositol 3,4,5-trisphosphate (PIP3), as well as the kinase Akt, which is downstream of PIP3. We will study how PI3K and PIP3 are involved in migration of cells out of the monolayer. We will examine the role of Akt, its effector GSK-3 and downstream processes during tubulogenesis. We will study how after migration the cells redifferentiate to form a second layer of polarized epithelial cells. We will test the role of the lipids PIP3 and , phosphatidyl inositol 4,5-bisphosphate in this process. Formation of bile ducts is essential to life. Many diseases involve developmental malformation of bile ducts, while other diseases involve damage and/or regeneration of bile ducts. The proposed work will greatly increase our understanding of early bile duct development and lead to improved health for patients with these diseases. PUBLIC HEALTH RELEVANCE: Formation of bile ducts is essential to life. Many diseases involve developmental malformation of bile ducts, while other diseases involve damage and/or regeneration of bile ducts. The proposed work will greatly increase our understanding of early bile duct development and lead to improved health for patients with these diseases.

描述（由申请人提供）：肝内胆管分支树由单层上皮细胞（称为胆管细胞）排列。在发育过程中，肝细胞和胆管细胞由双能成肝细胞前体细胞产生。某些注定要变成胆管细胞的成肝细胞在门脉间充质周围形成单层环。胆汁前体的单层变成双层结构。然后在两层之间形成管腔。最终这些结构重塑成肝内胆管的分支树。我们已经开发了一个三维的文化体系，概括了早期发展中的重要事件。这使我们能够研究这些事件与更大的机制细节比可能在体内。我们使用一种稳定的小鼠细胞系，称为层粘连蛋白上的肝祖细胞（HPPL），它能够分化为肝细胞和胆管细胞。我们将其作为单层生长在细胞外基质层上。然后，我们用第二层细胞外基质覆盖单层细胞。覆盖后，一些HPPL细胞从原始单层中向上移动并爬到原始单层的顶部。这些迁移的细胞最终在第一个单层上形成第二个单层。然后在两个单层之间形成管腔。这个过程非常类似于体内胆管形成的早期事件。然而，细胞从原始单层中迁移出来需要磷脂酰肌醇3-激酶（PI 3 K），产生磷脂酰肌醇3，4，5-三磷酸（PIP 3）的酶，以及激酶Akt，其位于PIP 3的下游。我们将研究PI 3 K和PIP 3如何参与细胞从单层迁移出来。我们将研究Akt的作用，其效应GSK-3和下游过程中tubulogenesis。我们将研究迁移后细胞如何再分化形成第二层极化上皮细胞。我们将测试脂质PIP 3和磷脂酰肌醇4，5-二磷酸在这一过程中的作用。胆管的形成对生命至关重要。许多疾病涉及胆管的发育畸形，而其他疾病涉及胆管的损伤和/或再生。拟议的工作将大大增加我们对早期胆管发育的理解，并改善这些疾病患者的健康状况。 公共卫生相关性：胆管的形成对生命至关重要。许多疾病涉及胆管的发育畸形，而其他疾病涉及胆管的损伤和/或再生。拟议的工作将大大增加我们对早期胆管发育的理解，并改善这些疾病患者的健康状况。