Fluorescein-based sensors to purify human α-cells for functional and transcriptomic analyses
Fluorescein-based sensors to purify human α-cells for functional and transcriptomic analyses
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基于荧光素的传感器，用于纯化人类 α 细胞以进行功能和转录组分析
DOI：
10.1101/2022.11.27.518097
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Kulkarni Rohit N.
中科院分区：
文献类型：
--
作者：
Kahraman Sevim;Shibue Kimitaka;De Jesus Dario F.;Hu Jiang;Manna Debasish;Wagner Bridget K.;Choudhary Amit;Kulkarni Rohit N.
胰腺α细胞分泌胰高血糖素，这是一种胰岛素反调节肽激素，对维持葡萄糖稳态至关重要。由于缺乏从胰岛中分离高质量α细胞的经济有效的纯化方法，对人类α细胞功能的研究仍然是一个挑战。在这里，我们使用基于反应的探针二乙酰化Zinpyr 1（DA-ZP 1），介绍了一种新的和简单的方法，从解离的人胰岛细胞中富集活的α-细胞，纯度约为95%。通过胰高血糖素的分选和免疫染色确认的α-细胞培养长达10天以形成α-假胰岛。α-假胰岛可以在培养物中维持而没有显著的活力损失，并且通过分泌适当水平的胰高血糖素来响应葡萄糖挑战。RNA测序分析（RNA-seq）显示，关键α-细胞身份基因的表达水平在培养物中得以维持，而一些基因如DLK 1、GSN、SMIM 24在α-假胰岛中以时间依赖性方式发生改变。总之，我们报告了一种分选高纯度人原代α细胞的方法，可用于下游分析，如功能和转录研究。
Pancreatic α-cells secrete glucagon, an insulin counter-regulatory peptide hormone critical for the maintenance of glucose homeostasis. Investigation of the function of human α-cells remains a challenge due to the lack of cost-effective purification methods to isolate high-quality α-cells from islets. Here, we use the reaction-based probe diacetylated Zinpyr1 (DA-ZP1) to introduce a novel and simple method for enriching live α-cells from dissociated human islet cells with~ 95% purity. The α-cells, confirmed by sorting and immunostaining for glucagon, were cultured up to 10 days to form α-pseudoislets. The α-pseudoislets could be maintained in culture without significant loss of viability, and responded to glucose challenge by secreting appropriate levels of glucagon. RNA-sequencing analyses (RNA-seq) revealed that expression levels of key α-cell identity genes were sustained in culture while some of the genes such as DLK1, GSN, SMIM24 were altered in α-pseudoislets in a time-dependent manner. In conclusion, we report a method to sort human primary α-cells with high purity that can be used for downstream analyses such as functional and transcriptional studies.
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影响因子：
9.3
作者：
Baron M;Veres A;Wolock SL;Faust AL;Gaujoux R;Vetere A;Ryu JH;Wagner BK;Shen-Orr SS;Klein AM;Melton DA;Yanai I
通讯作者：
Yanai I
影响因子：
1.2
作者：
Dorrell, Craig;Abraham, Stephanie L.;Grompe, Markus
通讯作者：
Grompe, Markus
影响因子：
--
作者：
Kalwat, Michael A.;Wiseman, Dean A.;Thurmond, Debbie C.
通讯作者：
Thurmond, Debbie C.
DOI：
10.1152/ajpendo.00344.2014
发表时间：
2015-01-15
影响因子：
5.1
作者：
Elliott, Amicia D.;Ustione, Alessandro;Piston, David W.
通讯作者：
Piston, David W.
影响因子：
4.6
作者：
Zhang Y;Parmigiani G;Johnson WE
通讯作者：
Johnson WE