An efficient two-step subcellular fractionation method for the enrichment of insulin granules from INS-1 cells.
An efficient two-step subcellular fractionation method for the enrichment of insulin granules from INS-1 cells.
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一种有效的两步亚细胞分级分离方法，用于从 INS-1 细胞中富集胰岛素颗粒
DOI：
10.1007/s41048-015-0008-x
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发表时间：
2015
影响因子：
--
通讯作者：
Xu T
中科院分区：
文献类型：
--
作者：
Chen Y;Xia Z;Wang L;Yu Y;Liu P;Song E;Xu T
胰岛素是血糖稳态的关键调节因子之一。99%以上的胰岛素是由胰腺β细胞分泌的。在每个β细胞内，胰岛素在分泌前被包装成胰岛素分泌颗粒（insulin secretary granules, isg）。胰岛素分泌是一个复杂但组织良好的动态过程，包括未成熟ISG （iISG）从反式高尔基网络出芽、iISG成熟和成熟ISG （mISG）与质膜融合。然而，参与这一过程的分子机制在很大程度上是未知的。因此，在确定iisg和misg的不同特征和蛋白质含量之前，分离和富集它们是至关重要的。在这里，我们开发了一种高效的两步亚细胞分离方法，用于从INS-1细胞中富集iISGs和mISGs: OptiPrep梯度纯化，然后Percoll溶液纯化。结果表明，该方法可以成功区分和富集iisg和misg。该方法可以很容易地适用于其他细胞或组织中SGs的研究，从而为阐明颗粒成熟和分泌机制提供了有用的工具。
Insulin is one of the key regulators for blood glucose homeostasis. More than 99% of insulin is secreted from the pancreatic β-cells. Within each β-cell, insulin is packaged and processed in insulin secretary granules (ISGs) before its exocytosis. Insulin secretion is a complicated but well-organized dynamic process that includes the budding of immature ISGs (iISGs) from the trans-Golgi network, iISG maturation, and mature ISG (mISG) fusion with plasma membrane. However, the molecular mechanisms involved in this process are largely unknown. It is therefore crucial to separate and enrich iISGs and mISGs before determining their distinct characteristics and protein contents. Here, we developed an efficient two-step subcellular fractionation method for the enrichment of iISGs and mISGs from INS-1 cells: OptiPrep gradient purification followed by Percoll solution purification. We demonstrated that by using this method, iISGs and mISGs can be successfully distinguished and enriched. This method can be easily adapted to investigate SGs in other cells or tissues, thereby providing a useful tool for elucidating the mechanisms of granule maturation and secretion.