大电导钙激活钾通道对大鼠骨髓间充质干细胞增殖的影响  

Effect of Big Conductance Ca^(2+)-Activated K^+ Channel on Proliferations of Rat Bone Marrow Mesenchymal Stem Cells

作　　者：罗家宇[1] 吴方昊 何跃[1] 曾李[1] 肖明朝[1] 

机构地区：[1]重庆医科大学附属第一医院泌尿外科,重庆400016

出　　处：《中国细胞生物学学报》2014年第3期313-319,共7页Chinese Journal of Cell Biology

基　　金：重庆市自然科学基金(批准号:2009BB5411)资助的课题~~

摘　　要：为了观察开放和拮抗大电导钙激活钾通道(big conductance Ca2+-activated K+channel,BKca)对大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)增殖的影响并探讨其机制,该研究分离培养了大鼠BMSCs,采用BKca通道特异性开放剂(NS1619)和拮抗剂(IBTX)干预,MTT、平板克隆测定细胞增殖活力及细胞克隆形成能力;流式细胞术分析细胞凋亡及细胞周期分布;Western blot、定量PCR检测周期蛋白cyclin D1基因和蛋白表达水平;整细胞膜片钳技术分析细胞膜电生理特性。结果显示,NS1619干预组与对照组相比,BMSCs细胞膜K+通道外向电流振幅增大,细胞增殖能力和克隆形成能力增强,凋亡减少。此外,开放BKca通道明显促进细胞从G1期向S期过渡,cyclin D1蛋白及mRNA表达上调,而拮抗BKca通道则相反。推测,BKca通道通过调节细胞周期进程最终影响细胞增殖,该作用可能与其具有调控细胞膜K+电流的电生理特性有关。To investigate the effect of big conductance Ca2＋-activated K＋ channel （BKca） on proliferation of rat bone marrow mesenchymal stem cells （BMSCs） and explore relevant mechanism, BMSCs were isolated from rats bone marrow and expanded in vitro for three generations. BMSCs were treated with BKca specific opener （NS 1619） and blocker （IBTX） separately. Proliferative activity and colony formation ability were determined by MTT and Tablet cloning. Cell cycle distribution and apoptosis were analyzed by flow cytometry. Western blot and Q-PCR were used to detect the mRNA and protein levels of cyclin D 1. Membrane ionic currents were recorded by whole-cell patch clamp technique. Compared with control group, the group of NS1619 exhibited a higher proliferative activity, colony formation ability and lower apoptosis rate. Additionally, the amplitude of outward K＋ currents were enhanced in membrane. The results of flow cytometry, Q-PCR and Western blot suggested that activation of BKca facilitated cell progress from G1 to S phase and improved the expression levels of mRNA and protein of cyclin D 1. However, the result of blockade of BKca was contrary to activation of BKca. According to our speculation, BKca could promote the proliferation of rat BMSCs. The mechanism might be related to the regulationof cell cycle. Furthermore, the electrophysiology properties of BKca play a crucial role in cell proliferation.

关 键 词：大电导钙激活钾通道 骨髓间充质干细胞 细胞增殖 细胞周期 钾离子电流 

分 类 号：R457.7[医药卫生—治疗学]