Formation of bile ducts in three dimensional culture

描述(申请人提供)：肝内胆管的分支树由单层上皮细胞组成，称为胆管细胞。在发育过程中，肝细胞和胆管细胞起源于双潜能肝母细胞前体细胞。某些注定要成为胆管细胞的肝母细胞在门静脉间充质周围形成单层环状结构。胆管前体的这种单层变成了双层结构。然后管腔在两层之间形成。最终，这些结构重塑为肝内胆管的分叉树。我们开发了一个三维文化系统，概括了早期发育中的重要事件。这使我们能够比在活体中更详细地研究这些事件。我们使用了一种稳定的小鼠细胞系，称为层粘连蛋白上增殖的肝祖细胞(HPPL)，它能够分化为肝细胞和胆管细胞。我们将它们作为单分子层生长在一层细胞外基质上。然后我们在单层上覆盖第二层细胞外基质。在覆盖后，一些HPPL细胞从原始单层向上移动，并在原始单层的顶部爬行。这些迁移细胞最终在第一层上形成第二层单层。然后在两个单层之间形成管腔。这一过程与体内胆管形成的早期过程非常相似。然而，细胞从原来的单层迁移出来需要磷脂酰肌醇3-激酶(PI3K)，产生磷脂酰肌醇3，4，5-三磷酸(PIP3)的酶，以及PIP3下游的激酶Akt。我们将研究PI3K和PIP3如何参与细胞迁移出单层。我们将研究Akt、其效应器GSK-3及其下游过程在肾小管形成中的作用。我们将研究迁移后的细胞如何重新分化，形成第二层极化的上皮细胞。我们将测试脂质PIP3和磷脂酰肌醇4，5-二磷酸在这一过程中的作用。胆管的形成是生命所必需的。许多疾病涉及胆管发育畸形，而其他疾病涉及胆管损伤和/或再生。这项拟议的工作将极大地增加我们对胆管早期发育的了解，并改善这些疾病患者的健康状况。 公共卫生相关性：胆管的形成对生命是必不可少的。许多疾病涉及胆管发育畸形，而其他疾病涉及胆管损伤和/或再生。这项拟议的工作将极大地增加我们对胆管早期发育的了解，并改善这些疾病患者的健康状况。

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.