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人乳腺MCF10A 细胞系K+ 通道的表达和特性及其与增殖的关系

刘佳, 封爽, 张磊, 吴争, 陈茜, 程为, 王世强, 邹伟

辽宁师范大学生命科学学院，大连 116029； 辽宁省生物技术与分子药物研发重点实验室，大连 116029；大连理工大 学物理与光电工程学院，大连 116026；北京大学生命科学学院，北京100871

摘要

近年来发现，K+ 通道与乳腺癌细胞的增殖和转化密切相关，但机制尚不清楚。本研究室前期报道了K+ 通道阻断剂4- 氨基吡啶(4-aminopyridine, 4-AP)能够抑制人乳腺上皮细胞的增殖，本文则进一步检测几种电压门控K+ 通道(voltage-gatedK+ channel, Kv)在人乳腺上皮细胞系MCF10A 中的表达，运用全细胞膜片钳技术，初步研究了该细胞K+ 通道的特性，观察K+ 通道阻断剂对细胞增殖以及信号通路蛋白活性的影响。结果显示，MCF10A 细胞均有Kv1.1、Kv1.2、Kv1.3 和Kv1.5基因mRNA 的表达，其中Kv1.5 表达量明显高于乳腺癌细胞MCF7。全细胞膜片钳钳制细胞于-60 mV，给予持续时间800 ms、范围从-60 mV 到+60 mV 的去极化刺激电压，步幅为10 mV，然后给予持续150 ms 的-60 mV 的刺激，刺激频率为1 Hz，可记录到一种跨膜电流，该电流具有电压依赖、外向整流的特性，并且能被Kv 通道阻断剂4-AP 阻断，证实该细胞膜存在Kv 通道。此外，4-AP 阻断K+ 通道10 min 后，与增殖相关的有丝分裂原活化蛋白激酶(mitogen-activatedprotein kinases, MAPK)信号通路ERK1/2 蛋白活性增强而p38 蛋白活性减弱；5 mmol/L 4-AP 处理细胞48 h 后，MCF10A的生长抑制率为25.29%。以上结果提示，在人乳腺上皮细胞系MCF10A 细胞膜上存在不同亚型的Kv 通道，该通道可被4-AP 阻断，并且4-AP 能够抑制MCF10A 细胞的增殖，其机制可能与细胞增殖信号通路不同成员的活性调节有关。

关键词： 电压门控K+ 通道; 人乳腺上皮细胞系

分类号：Q291

[Expression and properties of potassium channels in human mammary epithelial cell line MCF10A and its possible role in proliferation.] [Ariticle in Chinese]

Liu Jia, FENG Shuang, ZHANG Lei, WU Zheng, CHEN Qian, CHENG Wei, WANG Shi-Qiang, ZOU Wei

College of Life Science, Liaoning Normal University, Dalian 116029, China； Liaoning Key Laboratories of Biotechnology andMolecular Drug Research and Development, Dalian 116029, China； School of Physics and Optoelectronic Engineering, DalianUniversity of Technology, Dalian 116026, China； College of Life Sciences, Peking University, Beijing 100871, China

Abstract

Voltage-dependent potassium channels (Kv) are involved in proliferation and transformation in mammary epithelial cells. Inprevious studies, several groups have detected various potassium channels in breast cancer cells, and they assumed that potassiumchannels are related to the development of breast carcinoma, although the precise mechanisms are still unknown. We have previouslyreported that 4-aminopyridine (4-AP), one kind of potassium channel (K+ channel) blocker, could affect the proliferation of MCF10Acells. The aim of the present study is to explore the expression and properties of K+ channels in human mammary epithelial cells(MCF10A) and whether Kv channels are required for the proliferation of MCF10A cell. Electrophysiological, MTT analysis, PCR andWestern blot methods were used to identify a K+ conductance which is involved in tumorigenesis and not yet be described in MCF10Acells. A voltage-dependent, outward rectification and 4-AP-sensitive K+ current was observed in these cells. The perfusion of 5 mmol/L4-AP significantly decreased the amplitude of Kv current from (912.5±0.6) pA to (275±0.8) pA (n=5, P<0.01), when cells wererecorded using 800 ms voltage steps from a holding potential of -60 mV to voltage ranging from -60 mV to +60 mV. PCR analysis demonstrated that Kv1.1, Kv1.2, Kv1.3, and Kv1.5 were all expressed in MCF10A and MCF7 cells. Furthermore, the expression ofKv1.5 was much higher in MCF10A than that in MCF7. Inhibitory effect of 4-AP on cell proliferation was dosage-dependent.Incubation with 5 mmol/L 4-AP reduced MCF10A cell proliferation to 25.29% in 48 h. Western blot analysis showed the activation ofERK1/2 which related to cell proliferation was enhanced, while p38 activation was decreased by 4-AP treatment for 10 min. These dataprovided the first evidence of the Kv channels expression in MCF10A cell and 4-AP could inhibit the proliferation of MCF10A throughblocking the potassium channels, and the mechanism may be related to regulating the activity of different members of cell proliferationsignaling pathway of MEK/ERK.

Key words: voltage-gated K+ channel; human mammary epithelial cell line

收稿日期：2010-01-04　　录用日期：2010-05-20

通讯作者：邹伟　　E-mail: weizou60@126.com

引用本文：

刘佳, 封爽, 张磊, 吴争, 陈茜, 程为, 王世强, 邹伟. 人乳腺MCF10A 细胞系K+ 通道的表达和特性及其与增殖的关系[J]. 生理学报 2010; 62 (3): 203-209.

Liu Jia, FENG Shuang, ZHANG Lei, WU Zheng, CHEN Qian, CHENG Wei, WANG Shi-Qiang, ZOU Wei. [Expression and properties of potassium channels in human mammary epithelial cell line MCF10A and its possible role in proliferation.] [Ariticle in Chinese] . Acta Physiol Sin 2010; 62 (3): 203-209 (in Chinese with English abstract).