摘要
目的研究钙释放激活的钙调素2(Orai2)在人脐静脉内皮细胞(HUVEC)细胞外钙敏感受体(CaR)介导Ca2+内流和一氧化氮(NO)生成中的作用。方法 1)用转染技术将构建的Orai2干扰质粒(Orai2shRNA)转染入HUVEC,用实时定量RT-PCR和Western blot检测Orai2 mRNA和蛋白的表达。2)取2~5代HUVEC,分别以精胺激活CaR(钙池操纵性钙通道(SOC)和受体操纵性钙通道(ROC)均激活)、ROC模拟剂12-O-十四烷酰佛波醋酸酯-13(TPA)+CaR负性变构调节剂Calhex231(激活ROC及阻断SOC)、蛋白激酶C(PKC)抑制剂Ro31-8220、经典型PKCs和PKCμ抑制剂Go6967(阻断ROC及激活SOC)为细胞模型。实验分为3组:特异性质粒转染组(Orai2shRNA组),未转染组(空白对照组),空质粒组(vehicle组),用荧光探针Fura-2/AM、DAF-FM负载方法同步检测[Ca2+]i和NO的生成。结果 1)与control组相比,shOrai2-71干扰后,Orai2 mRNA和蛋白的表达均明显降低,抑制率分别为75.75%和70.58%(P<0.05)。2)与对照组和空质粒组相比,在4种不同处理因素作用下,Orai2shRNA转染组[Ca2+]i△ratio值和NO净荧光强度值均明显降低(P<0.05)。结论 Orai2参与了HUVEC中CaR经SOC和ROC激活介导的Ca2+内流和NO生成。
Objective To study the role of calcium release-activated calcium modulator 2 （Orai2） in extracellular Ca2＋-sensing receptor （CaR）-induced extracellular Ca2 ＋ influx and the production of nitric oxide （NO） in human umbilical vein endothelial cells （HUVEC）.Methods 1） We silenced the expression of Orai2 genes in HUVEC by transfection constructed Orai2 RNA interference plasmids.The expression of Orai2 protein and mRNA levels were determined by Western blotting and real time RT-PCR respectively.2） The second to fifth passage of HUVEC were incubated with CaR agonist spermine（activating store-operates cation channels （SOC） and examined for receptoroperated channels （ROC））,CaR negative allosteric modulator Calhex231 and ROC analogue TPA,protein kinase C （PKC） inhibitor Ro31-8220,PKCs and PKCμ inhibitor Go6967.Those cell models were divided into three groups：Orai2-71 short hairpin RNA group （Orai2shRNA） ; control group and vehicle group.Intracellular Ca2＋ concentration was detected and the production of NO was determined by DAF-FM.Results 1） shRNA targeted at the Orai2 genes decreased Orai2 protein and mRNA levels by 70.58％ and 75.75％ respectively （P ＜ 0.05）.（2）[Ca2＋]i ratio and the net NO fluorescence intensity values of Orai2shRNA group in four different treatments were significantly reduced （P ＜ 0.05）.Conclusions Orai2 participates in CaR-mediated Ca2 ＋ influx and NO production through SOC and ROC activation in HUVEC.
出处
《基础医学与临床》
CSCD
北大核心
2014年第5期595-601,共7页
Basic and Clinical Medicine
基金
国家自然科学基金(31160239
81160018)
