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首页> 外文期刊>Physiological Reports >Delivery of shRNA via lentivirus in human pseudoislets provides a model to test dynamic regulation of insulin secretion and gene function in human islets
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Delivery of shRNA via lentivirus in human pseudoislets provides a model to test dynamic regulation of insulin secretion and gene function in human islets

机译：通过慢病毒在人假胰岛中递送shRNA提供了测试人胰岛中胰岛素分泌和基因功能的动态调节的模型

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s Rodent islets are widely used to study the pathophysiology of beta cells and islet function, however, structural and functional differences exist between human and rodent islets, highlighting the need for human islet studies. Human islets are highly variable, deteriorate during culture, and are difficult to genetically modify, making mechanistic studies difficult to conduct and reproduce. To overcome these limitations, we tested whether pseudoislets, created by dissociation and reaggregation of islet cell suspensions, allow for assessment of dynamic islet function after genetic modulation. Characterization of pseudoislets cultured for 1?week revealed better preservation of first‐phase glucose‐stimulated insulin secretion (GSIS) compared with cultured‐intact islets and insulin secretion profiles similar to fresh islets when challenged by glibenclamide and KCl. qPCR indicated that pseudoislets are similar to the original islets for the expression of markers for cell types, beta cell function, and cellular stress with the exception of reduced proinflammatory cytokine genes ( IL1B, CCL2, CXCL8 ). The expression of extracellular matrix markers ( ASPN, COL1A1, COL4A1 ) was also altered in pseudoislets compared with intact islets. Compared with intact islets transduced by adenovirus, pseudoislets transduced by lentivirus showed uniform transduction and better first‐phase GSIS. Lastly, the lentiviral‐mediated delivery of short hairpin RNA targeting glucokinase ( GCK ) achieved significant reduction of GCK expression in pseudoislets as well as marked reduction of both first and second phase GSIS without affecting the insulin secretion in response to KCl. Thus, pseudoislets are a tool that enables efficient genetic modulation of human islet cells while preserving insulin secretion.
机译：啮齿动物胰岛被广泛用于研究β细胞和胰岛功能的病理生理学，但是，人类和啮齿动物的胰岛之间存在结构和功能上的差异，这凸显了人类胰岛研究的必要性。人胰岛高度可变，在培养过程中会变质，并且难以进行基因改造，从而使机理研究难以进行和繁殖。为了克服这些限制，我们测试了由胰岛细胞悬液的解离和重新聚集产生的假胰岛是否允许在遗传调节后评估动态胰岛功能。与培养完整的胰岛相比，培养1周的假胰岛的特性显示，第一阶段葡萄糖刺激的胰岛素分泌（GSIS）的保存性更好，当受到glibenclamide和KCl攻击时，胰岛素分泌特征类似于新鲜的胰岛。 qPCR表明假胰岛与原始胰岛相似，除了促炎细胞因子基因（IL1B，CCL2，CXCL8）减少外，细胞类型，β细胞功能和细胞应激标志物的表达均相似。与完整胰岛相比，假胰岛中细胞外基质标志物（ASPN，COL1A1，COL4A1）的表达也发生了变化。与由腺病毒转导的完整胰岛相比，由慢病毒转导的假胰岛显示出均匀的转导和更好的第一阶段GSIS。最后，慢病毒介导的短发夹RNA靶向葡萄糖激酶（GCK）的传递实现了假胰岛中GCK表达的显着降低，以及第一阶段和第二阶段GSIS的显着降低，而不会影响对KCl的胰岛素分泌。因此，假胰岛是一种能够在维持胰岛素分泌的同时有效地调节人类胰岛细胞的基因的工具。

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