Regulated insulin secretion from tissue engineered skin*

描述(申请人提供)：1型糖尿病是一种由于胰岛β细胞自身免疫破坏而缺乏胰岛素的疾病。尽管胰岛素注射提供了生存所需的荷尔蒙，但它们往往无法严格控制血糖水平，从而导致神经病变和心血管疾病等长期并发症。在这里，我们建议使用基因治疗作为一种替代治疗方式，以提供稳定的基线水平的胰岛素，并根据需求调节胰岛素的分泌。具体地说，我们建议开发由转基因角质形成细胞制成的组织工程化皮肤，作为体内释放胰岛素的装置，用于治疗糖尿病。我们将构建逆转录病毒载体，通过外源性注射小有机分子来调节胰岛素的分泌。使用最佳的基因转移方案，我们将转导表皮角质形成细胞，并检测培养细胞和复层表皮组织中活性胰岛素的分泌。将转基因皮肤替代物移植到无症状糖尿病小鼠身上，将用于检测角质形成细胞胰岛素分泌对血糖水平的影响，并确定外源性因素对胰岛素调节的效果。组织工程皮肤将经过基因改造，在两餐之间提供基线剂量的胰岛素，并在饭前提供更高剂量的胰岛素，以模拟β细胞对营养刺激的胰岛素分泌反应。我们的最终目标是创造一种基于细胞的设备，提供更好的血糖水平控制，并克服糖尿病的破坏性长期影响。我们的团队由一名组织工程师(STA)、一名分子生物学家(KLK)和一名糖尿病专家(SGL)组成。这种专业知识的结合对这一多方面项目的成功至关重要，该项目涉及分子生物学、组织工程、组织移植和受控药物输送，以设计一种基于细胞的设备，用于调节重组胰岛素的输送。

DESCRIPTION (provided by applicant): Type 1 diabetes is a disease characterized by lack of insulin due to autoimmune destruction of pancreatic beta-cells. Although insulin injections supply the amount of hormone necessary for survival, they often fail to provide tight regulation of blood glucose levels resulting in long-term complications such as neuropathy and cardiovascular disease. Here we propose to use gene therapy as an alternative therapeutic modality to supply constant baseline levels of insulin and regulate insulin secretion according to demand. Specifically, we propose to develop tissue engineered skin made of genetically modified keratinocytes as an in vivo delivery device to secrete insulin for the treatment of diabetes. We will construct retroviral vectors that regulate insulin secretion through exogenous administration of small organic molecules. Using optimal gene transfer protocols, we will transduce epidermal keratinocytes and examine secretion of active insulin from cells in culture and from stratified epidermal tissues. Transplantation of genetically modified skin equivalents onto athymic diabetic mice will be used to examine the effects of keratinocyte insulin secretion on blood glucose levels and determine the efficacy of insulin regulation in response to exogenous agents. Tissue engineered skin will be genetically modified to provide baseline amounts of insulin between meals and higher amounts before meals to mimic the insulin secretory response of beta-cells to nutrient stimulation. Our ultimate goal is to create a cell-based device to provide better control of blood glucose levels and overcome the devastating long-term effects of diabetes. Our team is composed of a tissue engineer (STA), a molecular biologist (KLK) and a diabetes expert (SGL). This combination of expertise is essential for the success of this multifaceted project, which involves molecular biology, tissue engineering, tissue transplantation and controlled drug delivery to engineer a cell-based device for regulated delivery of recombinant insulin.