一种改良的体外工具，用于分离和鉴定大鼠静止胰岛星状细胞。
A modified in vitro tool for isolation and characterization of rat quiescent islet stellate cells.
机构信息
Department of Endocrinology, Zhongda Hospital, Institute of Diabetes, School of Medicine, Southeast University, Nanjing, China.
Graduate Innovation Platform of Southeast University, Nanjing, China.
出版信息
Exp Cell Res. 2019 Nov 1;384(1):111617. doi: 10.1016/j.yexcr.2019.111617. Epub 2019 Sep 7.
BACKGROUND
Islet stellate cells (ISCs) play a critical role in islet fibrosis, contributing to the progression of pancreatic diseases. Previous studies have focused on fibrosis-associated activated ISCs obtained by standard islet explant techniques. However, in vitro models of quiescent ISCs (qISCs) are lacking. This study aims to develop a method to isolate qISCs and analyze their phenotype during activation.
METHODS
Immunofluorescence staining was applied to localize ISCs in normal human, rat, and mouse islets. qISCs were isolated from rat islets using density gradient centrifugation (DGC) method. qRT-PCR, immunoblotting, proliferation, and migration assays were employed for their characterization.
RESULTS
Desmin-positive ISCs were detected in normal human, rat, and mouse islets. Freshly isolated qISCs, obtained by density gradient centrifugation, displayed a polygonal appearance with refringent cytoplasmic lipid droplets and expressed transcriptional markers indicating a low activation/quiescent state. With increasing culture time, the marker expression pattern changed, reflecting ISC activation. qISCs contained more lipid droplets and exhibited lower proliferation and migration abilities compared to spindle-shaped ISCs obtained by traditional explant techniques.
CONCLUSIONS
This study describes a new method for efficient isolation of qISCs from rat islets, representing a useful in vitro tool to study the biology of ISCs in more physiological conditions.
背景
胰岛星状细胞（ISCs）在胰岛纤维化中发挥关键作用，促进胰腺疾病的进展。先前的研究集中在通过标准胰岛外植技术获得的与纤维化相关的活化 ISCs。然而，缺乏静息 ISCs（qISCs）的体外模型。本研究旨在开发一种分离 qISCs 并分析其在活化过程中表型的方法。
方法
免疫荧光染色用于定位正常人类、大鼠和小鼠胰岛中的 ISCs。使用密度梯度离心（DGC）法从大鼠胰岛中分离 qISCs。qRT-PCR、免疫印迹、增殖和迁移实验用于其特征分析。
结果
在正常人类、大鼠和小鼠胰岛中检测到 desmin 阳性 ISCs。通过密度梯度离心获得的新鲜分离的 qISCs 呈多边形外观，具有折光性细胞质脂滴，并表达转录标志物，表明处于低激活/静息状态。随着培养时间的增加，标记物表达模式发生变化，反映出 ISC 的活化。与通过传统外植技术获得的纺锤形 ISCs 相比，qISCs 含有更多的脂滴，增殖和迁移能力较低。
结论
本研究描述了一种从大鼠胰岛中高效分离 qISCs 的新方法，为研究 ISCs 在更生理条件下的生物学特性提供了有用的体外工具。
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