Identification and purification of functional human β-cells by a new specific zinc-fluorescent probe
Identification and purification of functional human β-cells by a new specific zinc-fluorescent probe
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DOI：
10.1177/002215540104900412
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发表时间：
2001-04-01
影响因子：
3.2
通讯作者：
Pattou, F
中科院分区：
文献类型：
--
作者：
Lukowiak, B;Vandewalle, B;Pattou, F
胰腺β细胞含有大量锌。我们利用这一点，尝试从胰岛制剂中定位、定量和分离产生胰岛素的细胞。我们的研究旨在确定一种无毒的锌敏感荧光探针，它能够选择性地标记存活的β细胞中不稳定的锌，并在可见光谱中呈现激发和发射波长，使这一技术能被大多数仪器所应用。我们测试了新港绿（Newport Green），这是一种在485nm处可被激发的探针，其解离常数在微摩尔范围内，对锌的亲和力较低。新港绿的亲脂性酯化形式的负载容易、快速、特异，且对细胞无毒。共聚焦显微镜显示出与分泌颗粒相关的强烈荧光。回归分析表明锌荧光与胰岛数量（r = 0.98）以及锌荧光与胰岛素含量（r = 0.81）之间具有良好的相关性。通过测定每DNA的锌荧光，我们能够从纯度和活力两方面评估用于胰岛同种异体移植的不同胰岛制剂的质量。对解离的新港绿标记细胞进行细胞分选，根据每DNA的胰岛素含量和免疫细胞化学分析判断，可使β细胞得到清晰的分离。这种锌探针是第一种能够在可见光谱中特异性标记活细胞的探针，无论是在临床还是基础研究方面，对于β细胞实验都显得非常有前景。
Pancreatic beta -cells contain large amounts of zinc. We took advantage of this to try to localize, quantify, and isolate insulin-producing cells from islet preparations. Our study was designed to identify a non-toxic zinc-sensitive fluorescent probe able to selectively label labile zinc in viable beta -cells and to exhibit excitation and emission wavelengths in the visible spectrum, making this technique exploitable by most instruments. We tested Newport Green, a probe excitable at 485 nm with a dissociation constant in the micromolar range corresponding to a low affinity for zinc. The loading of the lipophilic esterified form of Newport Green was easy, rapid, specific, and non-toxic to cells. Confocal microscopy highlighted an intense fluorescence associated with secretory granules. Regression analyses showed a good relationship between zinc fluorescence and islet number (r=0.98) and between zinc fluorescence and insulin content (r=0.81). The determination of Zn fluorescence per DNA enabled us to assess the quality of the different islet preparations intended for islet allografting in terms of both purity and viability. Cell sorting of dissociated Newport Green-labeled cells resulted in a clear separation of beta -cells, as judged by insulin content per DNA and immunocytochemical analysis. This zinc probe, the first able to specifically label living cells in the Visible spectrum, appears very promising for beta -cell experimentation, both clinically and for basic research.