Chloride channels and transporters in human corneal epithelium.
Chloride channels and transporters in human corneal epithelium.
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DOI：
10.1016/j.exer.2010.03.013
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发表时间：
2010-06
影响因子：
3.4
通讯作者：
Zhao, Min
中科院分区：
文献类型：
--
作者：
Cao, Lin;Zhang, Xiao-Dong;Liu, Xiaobo;Chen, Tsung-Yu;Zhao, Min
水和电解质的运输对角膜的清晰度至关重要。最近的研究表明，离子和电解质运输的另一个重要功能是建立引导细胞迁移的创伤电场。我们发现氯离子通量是角膜创伤电流的主要成分。为了阐明氯离子转运的机制，我们研究了人角膜上皮(HCE)细胞的氯离子通道和转运蛋白。我们测试了转化的人角膜上皮细胞系(THCE)、原代培养的人角膜上皮细胞(PHCE)和人类供体角膜。我们首先用RT-PCR方法检测了HCE细胞中CLC(氯离子通道/转运体)家族成员和CFTR(囊性纤维化跨膜电导调节因子)的mRNA表达水平。然后，我们用免疫印迹和免疫荧光共聚焦显微镜证实了选定的CLC家族成员和CFTR的蛋白质表达和分布。最后用电生理技术记录氯电流。在HCE细胞系中检测到ClC-2、ClC-3、ClC-4、ClC-5、ClC-6和CFTRmRNAs。未检测到ClC-1和ClC-7。免疫印迹和免疫染色证实了ClC-2、ClC-3、ClC-4、ClC-6和CFTR在人角膜上皮细胞中的表达和分布。CLC-2优先标记顶层和基底层，而CLC-3和CLC-4仅标记浅层。CLC-6和CFTR标记显示出独特的梯度，在顶层有较强的染色，并向基底层逐渐减少。角膜内皮细胞表达ClC-2、ClC-3、ClC-4、ClC-6，可能还有CFTR。人角膜上皮细胞存在电压依赖性氯离子电流。HCE细胞表达功能性的氯离子通道和转运蛋白。ClC-2、ClC-3、ClC-4、ClC-6和CFTR在人角膜上皮细胞中有不同的表达模式。这些分子及其分布可能在维持静息氯离子通量和调节角膜伤口的氯离子通量方面发挥重要作用，这可能是伤口电信号的主要贡献者。
Transport of water and electrolytes is critical for corneal clarity. Recent studies indicate another important function of transport of ions and electrolytes - establishing wound electric fields that guide cell migration. We found chloride (Cl-) flux is a major component of the corneal wound electric current. In order to elucidate the mechanisms of Cl- transport, we studied Cl- channels and transporters in human corneal epithelial (HCE) cells. We tested a transformed human corneal epithelial cell line (tHCE), primary cultures of human corneal epithelial cells (pHCE), and human donor corneas. We first used RT-PCR to determine expression levels of mRNA of CLC (Cl- channel/transporter) family members and CFTR (cystic fibrosis transmembrane conductance regulator) in HCE cells. We then confirmed protein expression and distribution of selected CLC family members and CFTR with Western blot and immuno-fluorescence confocal microscopy. Finally, Cl- currents were recorded with electrophysiological techniques. The mRNAs of CLC-2, CLC-3, CLC-4, CLC-5, CLC-6, and CFTR were detected in the HCE cell line. CLC-1 and CLC-7 were not detectable. Western blot and immunostaining confirmed protein expression and distribution of CLC-2, CLC-3, CLC-4, CLC-6 and CFTR in human corneal epithelium. CLC-2 preferentially labeled the apical and basal layers, while CLC-3 and CLC-4 labeled only the superficial layer. CLC-6 and CFTR labeling showed a unique gradient with strong staining in apical layers which gradually decreased towards the basal layers. Corneal endothelium was positive for CLC-2, CLC-3, CLC-4, CLC-6 and possibly CFTR. Human corneal epithelial cells demonstrated voltage dependent Cl- currents. HCE cells express functional Cl- channels and transporters. CLC-2, CLC-3, CLC-4, CLC-6, and CFTR had distinct expression patterns in human corneal epithelium. Those molecules and their distribution may play important roles in maintaining resting Cl- fluxes and in regulating Cl- flux at corneal wounds, which may be a major contributor to wound electrical signaling.
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作者：
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