中文摘要：

目的：研究小凹蛋白-1（Cav-1）对人脐静脉内皮细胞（HUVECs）细胞外钙敏感受体（CaR）介导钙内流的作用机制。方法：取2～3代HUVECs,采用细胞膜穴样凹陷（caveolae）结构破坏剂非律平（filipin）和甲基-β-环糊精（MβCD）及Cav-1基因沉默,配合CaR激动剂精胺（spermine）和负性变构调节剂Calhex 231。Fura-2/AM负载检测细胞内Ca2＋浓度（[Ca2＋]i）。Western blotting检测HUVECs中Cav-1以及CaR蛋白表达,免疫共沉淀技术检测Cav-1和CaR相互作用。用蔗糖密度梯度离心的方法提取并鉴定caveolae,Western blotting检测caveolae的标志蛋白Cav-1和浮舰蛋白1（flotillin-1）及CaR表达。同时检测胞膜、胞浆和高尔基体标志蛋白：转铁蛋白受体（TfR）、β-肌动蛋白（β-actin）和β-外被体蛋白（β-COP）。检测富含Cav-1膜（CEM）区、非caveolae I区（NCF I）和II区（NCF II）的Cav-1和CaR表达。结果：（1）细胞外液为含钙液时,Calhex 231完全阻断精胺刺激引起的[Ca2＋]i升高（P〈0.05）,MβCD可加强精胺升高[Ca2＋]i的作用（P〈0.05）。不同浓度MβCD处理后各组Cav-1和CaR相互作用的差异无统计学意义（P〉0.05）;（2）免疫共沉淀结果显示,各组Cav-1和CaR蛋白表达的差异无统计学意义（P〉0.05）;（3）与control组比较,spermine＋Ca2＋组、filipin＋spermine＋Ca2＋组和MβCD＋spermine＋Ca2＋组CEM区的Cav-1和CaR蛋白表达均降低（P〈0.05）,NCF I区的Cav-1和CaR蛋白表达增加（P〈0.05）,NCF II区Cav-1蛋白表达增加（P〈0.05）,而CaR蛋白表达的差异无统计学意义（P〉0.05）。结论：在HUVECs中,Cav-1和CaR共定位于同一caveolae区,同时Cav-1对CaR介导的Ca2＋内流有下调作用,其机制可能与Cav-1抑制CaR膜定位、促使其向非caveolae区转位及减弱其对激动剂的反应性有关。

英文摘要：

AIM： To study the role of caveolin-1 （Car-1） in down-regulating the extracellular Ca2 ＋ -sensing receptor （CaR）-mediated Ca2＋ influx in human umbilical vein endothelial cells （HUVECs） and its mechanisms. METH- ODS： HUVECs were collected and cultured to the second or third passage. Filipin was used to induce acute caveolae dis- ruption. Methyl-β-cyclodextrin （MβCD） or shRNA targeting Cav-1 combined with CaR agonist spermine and negative al- losteric modulator Calhex 231 was also used in HUVECs. Intracellular concentration of Ca2＋ （ [ Ca2＋ ]1） Was measured by Fura-2/AM loading. The protein expression of Cav-1 and CaR was examined by Western blotting. The interaction and co- localization of Cav-1 and CaR were determined by the method of co-immunoprecipitation （Co-IP）. Caveolae-enriched mem- brane （CEM） fractions were isolated and identified by detergent-free （Na2C03） sucrose density gradient centrifugation. The protein levels of Cav-1, CaR, flotillin-1, β-coat protein （ β-COP）, β-actin and transferrin receptor （TfR） were detec-ted by Western blotting. Noncaveolar fraction I （ NCF I） and noncaveolar fraction II （ NCF II） in the CEM fractions were separated. RESULTS： Using extracellular buffer with Ca2~ , the increase in [ Ca2 ~ ]i induced by spermine in HUVECs was abolished after inhibition of CaR by its negative allosterie modulator calhex231. Conversely, the effect of spermine on the increased [ Ca2 ~ ] i in HUVECs was further augmented after acute caveolae disruption by MI~CD. No significant differ- ence of the protein levels of CaR and Cav-1 in HUVECs among treating with different concentrations of MI3CD was ob- served. The results of Co-IP showed that the protein levels of CaR and Cav-1 in every group of HUVECs were not signifi- cantly different. Compared with control group, the protein expression of CaR and Cav-I in CEM was decreased in spermine ＋ Ca2 ＋ group, filipin ＋ spermine ＋ Ca2 ＋ group and MIBCD ＋ spermine ＋ Ca2 ~ group, and