Bioluminescent Imaging of Pancreatic Islet Transplants

描述(由申请者提供)：我们的最终目标是通过胰岛移植以有意义的方式为治疗糖尿病做出贡献。我们推测，移植胰岛在植入部位的早期质量损失显著降低了新鲜纯化的胰岛制剂的功能潜力。某些解剖学和免疫学变量会影响植入细胞的早期命运。该提案的总体目标是定义移植位置、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性如何影响细胞的活性和功能。我们假设：1)活性胰岛质量的早期丢失显著降低了新鲜纯化的胰岛制剂的功能潜力；2)细胞活性的损失率取决于植入位置、即刻宿主非特异性炎症反应的活力以及移植细胞结构的组成。其具体目的是：1.建立一种实用的实时生物发光成像方法，作为监测转基因大鼠胰岛素II启动子(RIP)-荧光素酶供体品系小鼠标记的胰岛移植命运的一种方式。2.构建含荧光素酶和绿色荧光蛋白两种报告基因的重组腺病毒和腺相关病毒载体，用于纯化的小鼠和人胰岛的转染。3.应用生物发光成像技术，利用支架基质监测表达荧光素酶的MIN6细胞系移植后的命运。这些目标将通过将实时、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来实现。这种成像方式将使活体小鼠体内“标记的”移植的胰岛或细胞可视化。通过移植后的连续监测，将有可能确定与移植功能结果直接相关的解剖学和免疫学因素如何影响存活的胰岛肿块的命运。

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.