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首页> 外文期刊>Journal of tissue engineering and regenerative medicine >CD99 as surface anchor for human islet endocrine cell purification
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CD99 as surface anchor for human islet endocrine cell purification

机译：CD99作为人胰岛内分泌细胞纯化的表面锚

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摘要

Rat and human beta cell proteomes were quantified by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), searching for cell surface markers. In human beta cells, CD99 (cluster of differentiation 99) was ranked among the plasma membrane proteins that combine a high molar abundance with a relative degree of selectivity for the endocrine cells of the islets of Langerhans. Therefore, the applicability of CD99 as anchor for islet endocrine cell purification was investigated. The CD99 gene and protein expression were studied using microarray, LC-MS/MS, western blotting, flow cytometry and immunofluorescence, and a protocol was developed for magnetic bead-mediated beta cell enrichment from human pancreas digests using available anti-CD99 antibodies. In human, but not in rat, CD99 protein and mRNA were abundantly expressed by islet endocrine cells but undetectable in exocrine pancreas. The extracellular CD99 epitopes appeared to be trypsin-resistant, enabling the binding of anti-CD99 antibodies to an insulin+/TSQ+ cell subset and efficient coupling of magnetic beads for positive selection of CD99+ cells. A MACS-CD99 purification of human pancreas fractions with low endocrine purity consistently yielded a fourfold enrichment of insulin+/TSQ+ cells and formation of viable and functional endocrine aggregates after 24 h of culture. It is concluded that CD99 is a human beta cell surface marker that, by virtue of its high molar abundance and resistance to tryptic digestion, can be used as anchor for upscalable magnetic bead-mediated islet endocrine cell purification. Copyright (C) 2016 John Wiley & Sons, Ltd.
机译：通过液相色谱 - 质谱/质谱（LC-MS / MS）量化大鼠和人β细胞蛋白质，搜索细胞表面标记。在人β细胞中，CD99（分化簇99）在血浆膜蛋白中排名，所述血浆膜蛋白质与朗格汉兰胰岛胰岛内分泌细胞的相对选择性相结合。因此，研究了CD99作为胰岛内分泌细胞纯化的锚的适用性。使用微阵列，LC-MS / MS，蛋白质印迹，流式细胞术和免疫荧光研究CD99基因和蛋白质表达，并且使用可用抗CD99抗体的人胰腺消化的磁珠介导的β细胞富集开发了方案。在人类中，但不在大鼠中，CD99蛋白质和mRNA被胰岛内分泌细胞大量表达，但在外分泌胰腺中不可检测。细胞外CD99表位似乎是抗胰蛋白酶的，使抗CD99抗体与胰岛素+ / TSQ +细胞的结合能够与磁珠的有效偶联，以阳性选择CD99 +细胞。具有低内分泌纯度的人胰腺级分的MACS-CD99纯化始终如一地产生胰岛素+ / TSQ +细胞的四倍富集，并在培养24小时后形成可行的和功能性内分泌聚集体。得出结论，CD99是一种人β细胞表面标志物，其借助于其高摩尔丰度和对胰蛋白酶消化的抗性，可用作升高的磁珠介导的胰岛内分泌细胞纯化的锚。版权所有（c）2016 John Wiley＆Sons，Ltd。

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