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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-60mV,给予持续时间800ms、范围从-60mV到+60mV的去极化刺激电压,步幅为10mV,然后给予持续150ms的-60mV的刺激,刺激频率为1Hz,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10min后,与增殖相关的有丝分裂原活化蛋白激酶(mitogen-activated protein kinases,MAPK)信号通路ERK1/2蛋白活性增强而p38蛋白活性减弱;5mmol/L4-AP处理细胞48h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。
In recent years, it has been found that the K + channel is closely related to the proliferation and transformation of breast cancer cells, but the mechanism is still unclear. Our laboratory previously reported that K + channel blocker 4-aminopyridine (4-AP) can inhibit the proliferation of human mammary epithelial cells. In this paper, we further examined several voltage-gated K + channels , Kv) in human mammary epithelial cell line MCF10A. The whole cell patch clamp technique was used to study the characteristics of K + channel and the effect of K + channel blocker on cell proliferation and signal pathway protein activity. The results showed that MCF10A cells were Kv1.1, Kv1.2, Kv1.3 and Kv1.5 mRNA expression, Kv1.5 expression was significantly higher than breast cancer cells MCF7. Whole-cell patch clamps were used to clamp cells at -60 mV for a duration of 800 ms with depolarization stimulation voltage in the range of -60 mV to +60 mV in 10 mV increments followed by -60 mV stimulation for 150 ms at a stimulation frequency of 1 Hz A transmembrane current was recorded that was voltage-dependent and outward rectified and blocked by the Kv channel blocker 4-AP, confirming the presence of Kv channels in the cell membrane. In addition, after 4-AP blocked K + channel for 10min, the activity of ERK1 / 2 protein associated with proliferation-associated mitogen-activated protein kinase (MAPK) signaling pathway increased and the activity of p38 protein decreased. After 48h, the growth inhibition rate of MCF10A was 25.29%. The above results suggest that there are different subtypes of Kv channels in human mammary epithelial cell line MCF10A, which can be blocked by 4-AP, and 4-AP can inhibit the proliferation of MCF10A cells, which may be related to cell proliferation signaling pathway Different members of the regulation of activity.