RNA干扰HeLa细胞IKCa1基因对中电导钙激活钾通道电流的影响  

Effects of RNA interference against IKCa1 geng on the immediate conductance Ca^(2+)-activated K^+ current in HeLa cells

作　　者：范凌晔 汪春燕 詹平 刘玲 聂丹 毛熙光 

机构地区：[1]西南医科大学附属医院妇产科,四川泸州646000

出　　处：《泸州医学院学报》2016年第4期340-343,共4页Journal of Luzhou Medical College

基　　金：四川省科技厅应用基础项目(2008JY0014-1)

摘　　要：目的:本研究旨在探讨RNA干扰He La细胞IKCa1基因对中电导钙激活钾通道电流(IKCa1)的影响。方法:采用携带IKCa1基因的p Genesil-IKCa1质粒及p Genesil-HK对照质粒分别转染He La细胞。实验分为Control组(无质粒转染组),p Genesil-HK组和p Genesil-IKCa1组。在荧光显微镜下观察报告基因GFP在He La细胞中的表达情况并将GFP阳性率作为转染效率的评价指标,采用RT-PCR技术检测IKCa1基因的表达水平,应用膜片钳电生理技术记录IKCa1。结果:1 p GenesilHK组和p Genesil-IKCa1组转染率分别为75.3%和72.4%,两组之间阳性率差异无统计学意义(P>0.05);2 p Genesil-IKca1组IKCa1基因的m RNA表达水平明显低于Control组及p Genesil-HK组(P<0.01);3在He La细胞能记录到IKCal电流,该电流具有钙离子依赖性且能被Clotrimazole完全阻断,与Control组及p Genesil-HK组相比,p Genesil-IKCa1组的IKCa1电流电压曲线下移,钳制电压为+60 m V时电流密度为(10.05±3.37)p A/p F,明显低于Control组(21.78±5.54)p A/p F和p Genesil-HK组(20.54±5.29)p A/p F(P<0.01)。结论:通过RNA干扰能有效抑制宫颈癌He La细胞IKCa1电流,为今后开展以IKCal为靶点的宫颈癌的基因治疗提供新的靶点和电生理实验依据。Objective： To detect the effects of RNA interference ageinst IKCa1 gene on the intermediate conductance Ca^（2＋）-activated K＋current（IKCa1 current） in He La cells. Methods： He La cells were transfected with p Genesil-sh RNA plasmids directed against human IKCa1 gene（p Genesil-IKCal） or control p Genesil-sh RNA plasmide（p Genesil-HK）, respectively. The experiment was divided into three groups： control group（non-transfection）, p Genesil-HK group and p Genesil-IKCa1 group. The transfection efficiency was determined by the percentage of GFP-positive cells via fluorescent microscope. The m RNA levels of IKCa1 in He La cells were measured by RT-PCR. The current clamp and whole-cell patch clamp recordings were used to evaluate the electrophysiological characteristics of IKCa1 in He La cells. Results： 1 No difference of transfection efficiency was observed between p Genesil-HK group and p Genesil-IKCa1 group（75.3% vs. 72.4%,P〈0.05）. 2 The m RNA levels of IKCa1 in p Genesil-IKCa1 group was significantly reduced when compared with control group and p Genesil-HK group（P〈0.01）. 3IKCal currents were recorded in He La cells, which were calcium-dependent and inhibited by Clotrimazole. Comparison of current-voltage relationship among three groups showed that there was a significant downward shift in the current-voltage curve in p Genesil-IKCal group. At a clamp voltage of ＋60m V, IKCal current desity in p Genesil-IKCa1 group was significantly reduced（10.05 ± 3.37 p A/p F）when corrpared with that in control group（21.78 ± 5.54 p A/p F） or p Genesil-HK group（20.54 ± 5.29 p A/p F）（P〈0.01）.Conclusion： The IKCa1 current in He La cells could be effectively blocked by RNA interference against IKCa1 gene,which provided additional electrophysiological evidence for IKCa1-targeted therapies in the cervical cancer.

关 键 词：宫颈癌 中电导钙激活钾电流 RNA干扰 膜片钳 

分 类 号：R737.33[医药卫生—肿瘤]