Orai3参与了HUVECs外钙敏感受体介导的钙内流和NO生成

发布时间：2018-08-21 09:16

【摘要】：目的:研究Ca2+通道蛋白Orai3在人脐静脉内皮细胞(HUVECs)外钙敏感受体(CaR)介导的钙内流和一氧化氮(NO)生成中的作用和机制。方法:(1)利用转染技术将构建的Orai3干扰质粒(Orai3 shRNA)转染HUVECs,采用激光共聚焦显微镜观察细胞转染效果,实时定量RT-PCR和Western blotting检测Orai3 shRNA转染后Orai3 mRNA和蛋白的表达以及抑制效率。(2)取第2~5代细胞随机分组:特异性质粒转染组即实验组(Orai3shRNA组)、未转染组即空白对照组(control组)和空质粒组(vehicle组),将上述3组细胞分别与CaR激动剂精胺[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-十四烷酰佛波醇-13-乙酸酯(TPA)+CaR负性变构调节剂Calhex 231(激活ROC、阻断SOC)、蛋白激酶C(PKC)抑制剂Ro31-8220及经典型PKCs和PKCμ抑制剂Go6967(阻断ROC、激活SOC)孵育,用荧光探针Fura-2/AM和DAF-FM负载方法同步检测[Ca2+]i和NO生成的变化。结果:(1)实时定量RT-PCR及Western blotting检测结果显示:与control组相比较,shOrai3-77干扰后,Orai3 mRNA的表达明显降低(抑制率为84.5%,P0.05),Orai3蛋白的表达亦明显降低(抑制率为70.68%,P0.05)。(2)[Ca2+]iΔratio值和NO净荧光强度值的测定结果显示:与control组及vehicle组相比,在4种不同处理因素作用下,Orai3 shRNA转染组中[Ca2+]iΔratio值和NO净荧光强度值均明显降低(P0.05),而vehicle组无显著差异(P0.05)。结论:在HUVECs中Orai3参与了CaR经SOC和ROC激活介导的钙内流和NO生成。
[Abstract]:Aim: to investigate the role and mechanism of Ca2 channel protein Orai3 in calcium influx mediated by (HUVECs) receptor (CaR) and nitric oxide (NO) production in human umbilical vein endothelial cells (HUVECs). Methods: (1) the constructed Orai3 interference plasmid (Orai3 shRNA) was transfected into HUVECs by transfection technique and the transfection effect was observed by confocal laser microscope. Real-time quantitative RT-PCR and Western blotting were used to detect the expression and inhibitory efficiency of Orai3 mRNA and protein after Orai3 shRNA transfection. (2) the cells of passage 2 were randomly divided into specific plasmid transfection group (Orai3shRNA group), non-transfection group (control group) and blank control group (control group). In the empty plasmid group (vehicle group), the above three groups of cells were treated with CaR agonist spermine [Ca cell manipulation calcium channel (SOC) and receptor operable calcium channel (ROC)] and the CaR analogue 12-O- tetradecanoyl phorbol alcohol 13 acetate (TPA) CaR negative structural regulation. Calhex 231 (Activation of Roc, C (PKC) inhibitor of SOC), protein Kinase, Ro31-8220) and PKCs and PKC 渭 inhibitor Go6967 (blocking roc, activating SOC) were incubated. Fluorescence probe Fura-2/AM and DAF-FM loading method were used to detect the changes of [Ca2] I and no production simultaneously. Results: (1) the results of real-time quantitative RT-PCR and Western blotting detection showed that: compared with control group, the expression of Orai3 mRNA decreased significantly (inhibition rate was 84.5%, P0.05). (inhibition rate was 70.68%, P0.05). (2) [Ca2] I 螖 ratio value and no net fluorescence intensity value. The results showed that compared with control group and vehicle group, In Orai3 shRNA transfection group, [Ca2] I 螖 ratio value and no net fluorescence intensity value were significantly decreased (P0.05), but there was no significant difference in vehicle group (P0.05). Conclusion: in HUVECs, Orai3 is involved in calcium influx and no production mediated by SOC and ROC activation by CaR.
【作者单位】：新疆地方病与民族高发病教育部重点实验室石河子大学医学院;石河子大学医学院病理生理教研室;石河子大学医学院病理教研室;石河子大学医学院医学机能实验中心;
【基金】：国家自然科学基金资助项目(No.31160239;No.81160018)
【分类号】：R363

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