Human intestinal epithelial cell line SK-CO15 is a new model system to study Na(+)/H(+) exchanger 3.
Human intestinal epithelial cell line SK-CO15 is a new model system to study Na(+)/H(+) exchanger 3.
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DOI：
10.1152/ajpgi.00069.2012
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发表时间：
2012-07
期刊：
影响因子：
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通讯作者：
H. Exchanger;B. K. Yoo;M. Yanda;Yi Ran No;C. Chris;Yun;C. Yun
中科院分区：
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作者：
H. Exchanger;B. K. Yoo;M. Yanda;Yi Ran No;C. Chris;Yun;C. Yun
Caco-2细胞系代表吸收极化的肠上皮细胞，在其质膜上表达多种形式的Na(+)/H(+)交换器(NHE)。Caco-2细胞表达主要的顶端NHE亚型NHE3，但NHE3的低表达加上低效的转基因往往阻碍了预期的研究。在本研究中，我们研究了SK-CO15细胞是否可以用于研究NHE3的调控。生长在Transwell贴片上的SK-CO15细胞发育出具有微绒毛的极化上皮细胞。SK-CO15细胞的转染率明显高于Caco-2细胞，在基因转移和基因敲除方面具有优势。SK-CO15细胞表达NHE1、NHE2和NHE3。NHE3在这些细胞中的表达明显高于Caco-2，NHE3占NHE总活性的一半以上。共聚焦免疫荧光和表面生物素化实验证实NHE3在SK-CO15细胞中表达。NHE调节因子NHERF1和NHERF2在这些细胞中表达，而NHERF1和NHERF2是调节NHE3活性的重要因素。地塞米松和溶血磷脂酸(LPA)检测NHE3对SK-CO15细胞的刺激效应。地塞米松作用24~48h可增加NHE3的表达和活性。与Caco-2细胞相似，SK-CO15细胞缺乏LPA受体LPA(5)的表达，但外源LPA(5)的表达导致NHE3的急性刺激。Forsklin能显著抑制SK-CO15细胞中NHE3的活性，进一步证明了这些细胞的有效性。我们认为，SK-CO15细胞具有良好的转染性和内源性NHE3的高表达，是一种比广泛使用的Caco-2细胞更适合于NHE3调控研究的新的细胞模型。
The Caco-2 cell line represents absorptive polarized intestinal epithelial cells that express multiple forms of Na(+)/H(+) exchanger (NHE) in their plasma membranes. Caco-2 cells express the major apical NHE isoform NHE3, but low NHE3 expression together with inefficient transfection often hamper intended studies. In this study, we examined whether SK-CO15 cells could be used to study NHE3 regulation. SK-CO15 cells grown on Transwell inserts developed polarized epithelial cells with microvilli. The transfection efficiency of SK-CO15 cells was markedly higher compared with Caco-2 cells, an advantage in gene transfer and knockout. SK-CO15 cells expressed NHE1, NHE2, and NHE3. NHE3 expression was significantly greater in these cells than Caco-2, and NHE3 comprised more than half of total NHE activity. Apical expression of NHE3 in SK-CO15 cells was confirmed by confocal immunofluorescence and surface biotinylation. NHE regulatory factors NHERF1 and NHERF2, which are important for regulation of NHE3 activity, were expressed in these cells. Stimulatory response of NHE3 in SK-CO15 cells was assessed by dexamethasone and lysophosphatidic acid (LPA). Treatment with dexamethasone for 24-48 h increased NHE3 expression and activity. Similarly to Caco-2 cells, SK-CO15 cells lacked the expression of the LPA receptor LPA(5,) but exogenous expression of LPA(5) resulted in acute stimulation of NHE3. Forskolin acutely inhibited NHE3 activity in SK-CO15 cells, further attesting the validity of these cells. We conclude that SK-CO15 cells with the amenity for transfection and high endogenous NHE3 expression are a new and better cell model for NHE3 regulatory investigation than widely used Caco-2 cells.