Formation of bile ducts in three dimensional culture

三维培养中胆管的形成

• 批准号: 7982912
• 负责人: Keith E Mostov
• 金额: $ 33.6万
• 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/7982912
• 关键词: Biliary; Caliber; Cell Death; 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）：肝内胆管的分支树由上皮细胞的单层（称为胆管细胞）衬里。在发育过程中，肝细胞和胆管细胞来自双性肝细胞前体。某些注定会变成胆管细胞的肝细胞形成一个单一的环形环围环。胆道前体的单层成为双层结构。然后在两层之间发育流明。最终，这些结构重塑为肝内胆管的分支树。我们已经开发了一个三维文化系统，该系统概括了早期发展中的重要事件。这使我们能够以比体内可能更有可能的机械细节来研究这些事件。我们使用稳定的小鼠细胞系，称为肝祖细胞（HPPL）上增殖的肝祖细胞，该细胞能够区分肝细胞和胆管细胞。我们在细胞外基质层的顶部将它们作为单层生长。然后，我们用第二层细胞外基质覆盖单层。叠加后，一些HPPL细胞从原始的单层中移出，然后在原始单层上爬行。这些迁移的细胞最终在第一个基础上形成了第二个单层。然后在两个单层之间形成流明。这个过程与体内胆管形成的早期事件非常相似。然而，细胞从原始单层中迁移需要磷脂酰肌醇3-激酶（PI3K），这是产生磷脂酰肌醇3,4,5-三磷酸磷酸磷酸（PIP3）的酶，以及激酶Akt以及pip3的流形酶Akt。我们将研究PI3K和PIP3如何参与单层中的细胞迁移。我们将检查Akt，其效应GSK-3的作用以及在微管发生过程中的下游过程。我们将研究细胞在迁移后如何重新分化形成第二层极化上皮细胞。在此过程中，我们将测试脂质PIP3和磷脂酰肌醇4,5-双磷酸的作用。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害和/或再生。拟议的工作将大大增加我们对早期胆管发展的理解，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害和/或再生。拟议的工作将大大增加我们对早期胆管发展的理解，并改善这些疾病患者的健康状况。