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LRRC8A细胞模型的构建及其生理特性研究

周雁红¹, 郑锴², 夏忠雪¹, 姜晓明³, 狄文慧¹, 徐连秀¹, 应超¹, 郝峰^1,*

¹吉林医药学院检验学院，吉林 132013；²吉林医药学院管理学院，吉林 132013；³北华大学药学院，吉林 132013

摘要

本文旨在构建容积调控性阴离子通道主要成分LRRC8A的细胞模型，并应用该模型研究LRRC8A的生理特性。构建LRRC8A和YFP-H148Q/I152L真核表达载体，应用脂质体转染、抗生素筛选和有限稀释，获取共表达LRRC8A和YFP-H148Q/I152L的Fisher大鼠甲状腺滤泡上皮(Fischer rat thyroid, FRT)细胞。倒置荧光显微镜观察目的基因表达情况，荧光淬灭动力学实验检测LRRC8A和YFP-H148Q/I152L的功能。获得用于研究LRRC8A容积调控性阴离子通道的细胞模型，并应用该细胞模型研究LRRC8A的生理特性，包括阴离子转运特性、渗透压对LRRC8A的开放、阴离子转运速度的影响以及氯离子通道抑制剂对LRRC8A的作用。结果显示：(1)成功获得共表达LRRC8A和YFP-H148Q/I152L的FRT细胞，该细胞模型可用于LRRC8A容积调控性氯离子通道生理特性的研究。(2)在低渗状态下，LRRC8A容积调控性阴离子通道激活，可转运阴离子，如：碘离子和氯离子等；YFP-H148Q/I152L可用于研究阴离子的转运速度；渗透压是LRRC8A容积调控性阴离子通道开放的调控因素，其开放与渗透压呈负相关；氯离子通道抑制剂对LRRC8A通道的转运功能具有抑制作用，并呈剂量依赖关系。上述结果提示，本研究成功构建LRRC8A细胞模型，且应用该模型研究显示LRRC8A具有经典的容积调控性阴离子通道的特性。

关键词： LRRC8A; 容积调控性阴离子通道; 渗透压; FRT细胞

分类号：Q291

Establishment and application of a cell model for LRRC8A physiological characteristic study 

ZHOU Yan-Hong¹, ZHENG Kai², XIA Zhong-Xue¹, JIANG Xiao-Ming³, DI Wen-Hui¹, XU Lian-Xiu¹, YING Chao¹, HAO Feng^1,*

¹Laboratory Medical College, Jilin Medical College, Jilin 132013, China；²Management College, Jilin Medical College, Jilin 132013, China；³Pharmacy College, Beihua University, Jilin 132013, China

Abstract

The aim of the present study was to establish a cell model of  volume-regulated anion channel subunit LRRC8A and investigate the physiological characteristics of LRRC8A. The eukaryotic expression vectors of LRRC8A and YFP-H148Q/I152L were constructed and transfected into Fischer rat thyroid (FRT) cells by Lipofectamine 2000. The FRT cell lines co-expressing LRRC8A and YFP-H148Q/I152L were obtained by antibiotic screening. The expression of LRRC8A and YFP-H148Q/I152L in FRT cells was detected by the inverted fluorescence microscope. The fluorescence quenching kinetic experiment was done to verify the function and effectiveness of the cell model. Then the cell model was utilized to study the physiological characteristics of LRRC8A, such as the characteristics of anion transport, the opening of LRRC8A by osmotic pressure, the effect of anion transport velocity, and the effect of chloride channel inhibitors on LRRC8A anion channel. The results of the inverted fluorescence microscope showed that LRRC8A was expressed on the cell membrane and YFP-H148Q/I152L was expressed in the cytoplasm. The results of fluorescence quenching kinetic test showed that under the condition of low osmotic state, LRRC8A could transport some kinds of anions, such as iodine and chloride ions. Osmotic pressure played a key role in the regulation of LRRC8A volume-regulated anion channel opening. Chloride channel inhibitors inhibited ion transport of LRRC8A channel in a dose-dependent manner. It is suggested that LRRC8A has the characteristics of classic volume-regulated anion channels by using the cell model of FRT cells co-expressing LRRC8A and YFP-H148Q/I152L.

Key words: LRRC8A; volume-regulated anion channels; osmotic pressure; FRT cells

收稿日期：2018-08-29　　录用日期：2019-03-11

通讯作者：郝峰　　E-mail: haof863@126.com

DOI: 10.13294/j.aps.2019.0045

引用本文：

周雁红, 郑锴, 夏忠雪, 姜晓明, 狄文慧, 徐连秀, 应超, 郝峰. LRRC8A细胞模型的构建及其生理特性研究[J]. 生理学报 2019; 71 (4): 555-561.

ZHOU Yan-Hong, ZHENG Kai, XIA Zhong-Xue, JIANG Xiao-Ming, DI Wen-Hui, XU Lian-Xiu, YING Chao, HAO Feng. Establishment and application of a cell model for LRRC8A physiological characteristic study . Acta Physiol Sin 2019; 71 (4): 555-561 (in Chinese with English abstract).