EX-VIVO EXPANSION OF HUMAN PANCREATIC EPITHELIAL CELLS

胰岛素依赖型糖尿病是由于胰腺b岛细胞在血糖升高时不能分泌足够的胰岛素而引起的。目前，治疗I型糖尿病的主要方法是终生使用外源性胰岛素。然而，可能的替代疗法已经出现。许多患者通过胰岛移植成功地摆脱了胰岛素依赖。尽管这种方法具有巨大的前景，但在广泛应用方面仍存在重大障碍。其中之一是合适的移植材料供应有限，无法在培养中生长和操纵胰腺上皮细胞。在这个I期申请中，我们建议设计一种用于正常人类细胞扩增的基因保护方案。通过进行一系列确定的基因改变，我们将以一种可逆的方式使细胞不朽，同时保持它们的基因组完整性和细胞特性。这将使我们能够创建HPE克隆群体，可以在培养中扩增，恢复到正常表型，并在动物中进行功能测试。该计划的长期目标是使供体HPE细胞能够在移植前进行操纵和体外克隆扩增。成功将有助于解决更大的难题，在通过胰岛移植减轻胰岛素依赖的患者范围能够大大扩大之前，必须解决这个难题。近期的好处是克隆HPE细胞系的衍生，这将使对胰岛细胞及其祖细胞生物学的基础研究成为可能。建议的商业应用：本文提出的工作具有许多潜在的商业应用。在短期内，功能性HPE细胞系的发展将能够分析β细胞和β细胞前体，从而最终开发出IDDM和NIDDM的生物治疗干预措施。最终，本文所描述的方法可以使自体胰岛移植和同种异体移植的材料无限扩展。

Insulin dependent diabetes mellitus results from an inability of b-islet cells of the pancreas to secrete sufficient insulin in response to elevation of blood glucose. At present, the principle treatment for type I diabetes is life-long supply of exogenous insulin. However, possible alternative treatments have emerged. A number of patients have been successfully rescued from insulin dependence by transplant of pancreatic islets. Despite the enormous promise of this approach there remain significant hurdles to widespread application. Among these is the limited supply of suitable material for transplant and the inability to grow and manipulate pancreatic epithelial cells in culture. In this Phase I application, we propose to devise a genoprotective protocol for expansion of normal human cells. By making a series of defined, genetic alterations, we will reversibly immortalize cells in a manner that preserves both their genomic integrity and their cellular identity. This will allow us to create clonal populations of HPE that can be expanded in culture, reverted to a normal phenotype, and tested functionally in animals. The long-term goal of the proposed program is to enable donor HPE cells to be manipulated and clonally expanded ex vivo prior to transplantation. Success would contribute a piece to the larger puzzle that must be solved before the range of patients that can be relieved of insulin dependence by islet allografting can be greatly expanded. A near-term benefit would be the derivation of clonal HPE cell lines that will enable basic research into the biology of islet cells and their progenitors. PROPOSED COMMERCIAL APPLICATIONS: The work proposed herein has numerous potential commercial applications. In the short term, the development of functional HPE cell lines would enable analysis of beta-cells and beta-cell precursors that could lead to the eventual development of biotherapeutic interventions for IDDM and NIDDM. Ultimately the approaches described herein could allow infinite expansion of material for islet autograft and allograft.