Bioluminescent Imaging of Pancreatic Islet Transplants

描述（由申请人提供）：我们的最终目标是通过胰岛移植为糖尿病的治疗做出有意义的贡献。我们假设移植胰岛块在植入部位的早期损失显著降低了新纯化胰岛制剂的功能潜力。某些解剖学和免疫学变量影响移植细胞的早期命运。该建议的总体目标是确定移植位置、移植细胞的组成（游离细胞、细胞基质结构或胰岛）以及宿主的免疫反应如何影响细胞活力和功能。我们假设：1)早期丧失活胰岛质量显著降低新纯化胰岛制剂的功能潜力；2)细胞活力丧失的速率取决于移植部位、直接宿主非特异性炎症反应的活力以及移植细胞结构的组成。具体目标是：1。建立一种实时生物发光成像的方法，作为一种监测转基因大鼠胰岛素II启动子(RIP)-荧光素酶供体小鼠移植的“标记”胰岛命运的方式。2. 构建含有RIP和荧光素酶和绿色荧光蛋白两个报告基因的有效腺病毒和腺相关病毒载体，用于转染纯化的小鼠和人胰岛。3.应用生物发光成像技术监测移植的表达荧光素酶的MIN6¿-细胞系的命运使用支架基质。将实时、非侵入性生物发光成像的一般方法应用于胰岛移植的特定领域，将实现这一目标。这种成像方式将允许在活体小鼠中“标记”移植的胰岛或¿-细胞的可视化。通过移植后的连续监测，将有可能确定存活的胰岛肿块的命运如何受到与移植功能结果直接相关的解剖学和免疫学因素的影响。

DESCRIPTION (provided by applicant): Our ultimate goal is to contribute in a meaningful way to the cure of diabetes through islet transplantation. We posit that early loss of transplant islet mass at the implantation site significantly reduces the functional potential of the freshly purified islet preparation. Certain anatomic and immunologic variables impact on the early fate of the implanted cells. The overall goal of the proposal is to define how the transplant location, the make-up of the transplanted cells (free cells, cellular matrix structures, or islets), and the immune responsiveness of the host affect cellular viability and function. We hypothesize that: 1) early loss of viable islet mass significantly reduces the functional potential of the freshly purified islet preparation; 2) that rates of loss of cell viability depend on engraftment site, the vigor of immediate host non-specific inflammatory responses, and the composition of the cellular structures transplanted. The specific aims are: 1. To develop a useful method of real-time bioluminescent imaging as a modality to monitor the fate of transplanted "tagged" islets from transgenic rat insulin II promoter (RIP)-luciferase donor strain mice. 2. To create effective adeno and adeno-associated viral vectors containing the RIP with two reporter genes: luciferase and green fluorescent protein for transfection of purified mouse and human islets. 3.To apply bioluminescent imaging techniques to monitor the fate of transplanted MIN6 ¿-cell lines expressing luciferase using a scaffold matrix. The aims will be accomplished by applying the general methods of real-time, non-invasive bioluminescent imaging to the specific field of islet transplantation. This imaging modality will permit visualization of "tagged' transplanted islets or ¿-cells in the living mouse. Through serial post-transplant monitoring, it will be possible to define how the fate of the viable islet mass is affected by anatomic and immunologic considerations that directly relate to transplant functional outcome.