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水通道蛋白在人角膜内皮细胞和上皮细胞系中的下调作用。

Effect of down-regulation of aquaporins in human corneal endothelial and epithelial cell lines.

作者信息

Shankardas Jwalitha, Patil Rajkumar V, Vishwanatha Jamboor K

机构信息

Department of Biomedical Sciences, Graduate School of Biomedical Sciences, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.

出版信息

Mol Vis. 2010 Aug 10;16:1538-48.

PMID:20806077
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC2925904/
Abstract

PURPOSE

The purpose of this study was to determine the effects of down-regulation of Aquaporin 1 (AQP1) and Aquaporin 5 (AQP5) on cell proliferation and migration in human corneal endothelial (HCEC) and human corneal epithelial (CEPI17) cell lines, respectively.

METHODS

AQP1 and AQP5 were down regulated using siRNA following lipofectamine-mediated transfection in corneal endothelial and epithelial cells, respectively. Down-regulation was confirmed using RT-PCR, indirect immunofluorescence, and immunoblot analysis. Total internal reflection fluorescence (TIRF) microscopy was used to detect cell surface aquaporin expression. Cell proliferation was determined by SRB (sulfrodamine B) assay. Cell migration was determined by in vitro wound healing and migration assay.

RESULTS

In HCEC cells, AQP1 was localized to the cytosol as well as cell membrane and its down-regulation resulted in decreased cell proliferation and migration with a significant decrease in phosphorylated ERK (pERK). In CEPI17 cells AQP5 protein expression was also localized to cytosol as well as cell membrane. AQP5 down-regulation resulted in an increase in proliferation and cell migration with no significant difference in pERK.

CONCLUSIONS

AQP1 plays a role in HCEC proliferation and migration via the ERK signaling pathway and therefore may have significant implications in corneal endothelial dysfunction whereas; AQP5 may play an indirect role in human corneal epithelial cell proliferation and migration.

摘要

目的

本研究旨在分别确定水通道蛋白1（AQP1）和水通道蛋白5（AQP5）的下调对人角膜内皮（HCEC）细胞系和人角膜上皮（CEPI17）细胞系中细胞增殖和迁移的影响。

方法

分别在角膜内皮细胞和上皮细胞中，通过脂质体介导的转染使用小干扰RNA（siRNA）下调AQP1和AQP5。使用逆转录聚合酶链反应（RT-PCR）、间接免疫荧光和免疫印迹分析来确认下调情况。采用全内反射荧光（TIRF）显微镜检测细胞表面水通道蛋白的表达。通过磺罗丹明B（SRB）法测定细胞增殖。通过体外伤口愈合和迁移试验测定细胞迁移。

结果

在HCEC细胞中，AQP1定位于细胞质和细胞膜，其下调导致细胞增殖和迁移减少，磷酸化细胞外信号调节激酶（pERK）显著降低。在CEPI17细胞中，AQP5蛋白表达也定位于细胞质和细胞膜。AQP5下调导致增殖和细胞迁移增加，pERK无显著差异。

结论

AQP1通过ERK信号通路在HCEC增殖和迁移中起作用，因此可能对角膜内皮功能障碍有重要影响；而AQP5可能在人角膜上皮细胞增殖和迁移中起间接作用。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ac30/2925904/1ea87f5b052a/mv-v16-1538-f6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ac30/2925904/5929d9858ea4/mv-v16-1538-f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ac30/2925904/36220ef23bd6/mv-v16-1538-f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ac30/2925904/17dd652f53d0/mv-v16-1538-f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ac30/2925904/1a80100506f7/mv-v16-1538-f4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ac30/2925904/f62e393d4e5a/mv-v16-1538-f5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ac30/2925904/1ea87f5b052a/mv-v16-1538-f6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ac30/2925904/5929d9858ea4/mv-v16-1538-f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ac30/2925904/36220ef23bd6/mv-v16-1538-f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ac30/2925904/17dd652f53d0/mv-v16-1538-f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ac30/2925904/1a80100506f7/mv-v16-1538-f4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ac30/2925904/f62e393d4e5a/mv-v16-1538-f5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ac30/2925904/1ea87f5b052a/mv-v16-1538-f6.jpg

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