中文摘要：

目的探讨小凹蛋白1（Cav-1）上调人脐静脉内皮细胞钙敏感受体介导内皮型一氧化氮合酶（eNOS）激活的作用机制。方法培养人脐静脉内皮细胞，取同代细胞随机分为：（1）对照组；（2）钙敏感受体激动剂精胺（2．0mmol／L）＋Ca2＋组；（3）Caveolae结构破坏剂（Filipin，1．5ms／L）4-精胺＋Ca“组；（4）Car-1ShRNA＋精胺＋Ca2＋组；（5）空质粒＋精胺＋Ca2＋组；（6）Filipin不同浓度（1．5、2．0、2．5ms／L）组。免疫印迹检测人脐静脉内皮细胞中eNOS和磷酸化eNOS（p-eNOS）、Cav—1以及eNOS膜蛋白表达；免疫荧光和免疫共沉淀技术检测Cav-1和eNOS表达、共定位以及相互作用。结果不同浓度Filipin不影响eNOS和p-eNOS蛋白表达（P〉0．05）；精胺作用下细胞内p-eNOS表达增加（P〈0．05），此作用可被Filipin（1．5ms／L）或Car-1基因沉默完全阻断（P〈0．05）；Fili—pin（1．5mg/L）或Car-1干扰处理后，eNOS的膜蛋白表达减少（P〈0．05），eNOS的蛋白表达无变化（P〉0．05）；免疫荧光双标显示Filipin（1．5ms／L）或Car-1基因沉默后eNOS在小凹定位减少，核周边局部区域聚集增多。与对照组和精胺＋Ca2＋组比较，Cav-1ShRNA处理组Cav—1和eNOS相互作用减弱（P〈0．05），其他处理组间的相互作用无统计学意义（P〉0．05）。结论人脐静脉内皮细胞中Cav-1上调钙敏感受体介导eNOS激活作用机制可能与Cav-1影响eNOS质膜定位及抑制eNOS向细胞器转位有关。

英文摘要：

Aim To study the mechanisms of caveolin-1 （Cav-1） up-regulating the extracellular Ca2＋-sensing receptor （CaR）-induced endothelial nitric oxide synthetase （eNOS） activation in human umbilical vein endothelial ceils （HUVECs）. Methods Cultured HUVECs, the same generation of ceils were randomly divided into ： （ 1 ） control group ; （2） CaR agonist （ spermine, 2.0 mmol/L） ＋ Ca2 ＋ group ; （ 3 ） caveolae structural damage （ filipin, 1.5 mg/L） ＋ spermine ＋ Ca2＋ group ; （4） Car-1 short hairpin RNA （ Car-1 ShRNA） ＋ spermine ＋ Ca2 ＋ group ; （ 5 ） vehicle ＋ spermine ＋ Ca2 ＋ group; （6） different concentrations （ 1.5, 2.0, 2. 5 mg/L） fiiipin groups. Western blotting experiments were performed to detect protein expression of Cav-1, eNOS, phosphorated eNOS （p-eNOS） and expressions of Cav-I and eNOS membrane proteins. The interaction and co-localization between eNOS and Cav-1 were determined using eo-immunoprecipitates and immunofiuorescence analysis, respectively. Results Different concentrations filipin did not influence the expression of eNOS and p-eNOS protein in HUVECs. In the presence of Ca2＋ , the CaR agonist spermine at concentration of 2. 0 mmol/L resulted in an increase in the p-eNOS in HUVECs （P 〈 0. 05）, the effect of spermine on the increase of peNOS was also completely blocked after acute caveolae disruption with filipin （ 1.5 mg/L） or transfected with Car-1 ShR- NA （ P 〈 0. 05 ）. The expression of the eNOS membrane protein was decreased in HUVECs after cells were treated by filipin （ 1.5 mg/L） or transfected with Cav-1 ShRNA. Simultaneously, total protein level of eNOS was unaffected. Immunocytochemical results demonstrated that filipin （ 1.5 mg/L） or transfected with Car-1 ShRNA decreased eNOS localization in caveolae, increased in the local area surrounding the nucleus. Compared with control group and spermine ＋ Ca2＋ group, the interaction of eNOS and Cav-1 in Car-1 ShRNA group was attenuated （ P ?