Accurate measurement of pancreatic islet β-cell mass using a second-generation fluorescent exendin-4 analog
Accurate measurement of pancreatic islet β-cell mass using a second-generation fluorescent exendin-4 analog
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DOI：
10.1073/pnas.1109859108
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发表时间：
2011-08-02
影响因子：
11.1
通讯作者：
Weissleder, Ralph
中科院分区：
文献类型：
--
作者：
Reiner, Thomas;Thurber, Greg;Weissleder, Ralph
1型糖尿病的标志是胰岛的产生胰岛素的β细胞的自身免疫性破坏。自身免疫性糖尿病一直难以研究或治疗，因为它通常直到已经发生大量β细胞损失才被诊断出来。成像剂，允许非侵入性可视化的变化，β细胞质量仍然是一个高度优先的目标。我们报告近红外荧光β细胞成像剂的开发和测试。基于exendin-4的氨基酸序列，我们通过在K（12）位置引入非天然氨基酸来创建新肽，其随后可以通过生物正交铜催化点击化学与荧光团缀合。细胞测定证实所得荧光探针（E4（x12）-VT 750）具有高结合亲和力（类似于3 nM）。其在体内的性能进行了评价，使用高分辨率活体成像，组织学，全胰腺可视化，和内窥镜成像。根据活体显微镜，探针迅速结合到β细胞，并通过共聚焦显微镜证实，它被内化。整个胰腺的组织学显示荧光和胰岛素染色之间的密切对应关系，并且在用链脲佐菌素（一种β细胞毒素）处理的小鼠中，成像信号和β细胞质量之间存在极好的相关性。单个胰岛也可以通过内窥镜成像可视化。简而言之，E4（x12）-VT 750显示出与葡萄糖样肽-1受体的强选择性结合，并允许准确测量糖尿病和非糖尿病小鼠中的β细胞质量。这种近红外成像探针，以及未来的放射性同位素标记版本，应该被证明是在实验和临床背景下监测糖尿病，进展和治疗的重要工具。
The hallmark of type 1 diabetes is autoimmune destruction of the insulin-producing beta-cells of the pancreatic islets. Autoimmune diabetes has been difficult to study or treat because it is not usually diagnosed until substantial beta-cell loss has already occurred. Imaging agents that permit noninvasive visualization of changes in beta-cell mass remain a high-priority goal. We report on the development and testing of a near-infrared fluorescent beta-cell imaging agent. Based on the amino acid sequence of exendin-4, we created a neopeptide via introduction of an unnatural amino acid at the K(12) position, which could subsequently be conjugated to fluorophores via bioorthogonal copper-catalyzed click-chemistry. Cell assays confirmed that the resulting fluorescent probe (E4(x12)-VT750) had a high binding affinity (similar to 3 nM). Its in vivo properties were evaluated using high-resolution intravital imaging, histology, whole-pancreas visualization, and endoscopic imaging. According to intravital microscopy, the probe rapidly bound to beta-cells and, as demonstrated by confocal microscopy, it was internalized. Histology of the whole pancreas showed a close correspondence between fluorescence and insulin staining, and there was an excellent correlation between imaging signals and beta-cell mass in mice treated with streptozotocin, a beta-cell toxin. Individual islets could also be visualized by endoscopic imaging. In short, E4(x12)-VT750 showed strong and selective binding to glucose-like peptide-1 receptors and permitted accurate measurement of beta-cell mass in both diabetic and nondiabetic mice. This near-infrared imaging probe, as well as future radioisotope-labeled versions of it, should prove to be important tools for monitoring diabetes, progression, and treatment in both experimental and clinical contexts.