西罗莫司经利阿诺定受体2途径诱导血管内皮细胞钙超载  

Sirolimus Induced Calcium Overload in Vascular Endothelial Cells via Ryanodine Receptor Pathway

作　　者：袁玲[1] 聂卫[1] 高萍[1] 李昕[1] 刘伟伟[1] 崔晓雪[1] 

机构地区：[1]天津市医药科学研究所病理室,天津市300020

出　　处：《中国动脉硬化杂志》2016年第2期129-134,共6页Chinese Journal of Arteriosclerosis

基　　金：天津市卫生局科技基金项目(2013KY37)

摘　　要：目的观察西罗莫司造成血管内皮钙超载引起细胞损伤并探讨其相关机制。方法在西罗莫司处理和利阿诺定稳定利阿诺定受体通道的情况下,采用MTT法检测人脐静脉内皮细胞(HUVEC)细胞存活率,HE染色观察细胞形态,一氧化氮(NO)检测试剂盒测定培养液NO含量,Ca2+敏感的荧光染料Fluo-3 AM标记胞质Ca2+,分别采用流式细胞仪分析和激光共聚焦显微镜观察细胞内Ca2+水平,JC-1荧光探针检测线粒体膜电位,Annexin VFITC/PI染色法检测细胞凋亡率。结果西罗莫司能降低细胞存活率(P<0.01)。西罗莫司500 nmol/L刺激24 h后,HE染色发现细胞肿胀,核固缩、核碎裂及胞浆空泡;NO含量降低,细胞内Ca2+含量增高,线粒体膜电位降低,细胞凋亡率增加(P均<0.01)。与西罗莫司500 nmol/L组相比,利阿诺定50μmol/L干预组细胞存活率明显增加(P<0.01),细胞内Ca2+含量降低(P<0.05),细胞培养液NO含量由28.33±4.18μmol/L增至47.03±3.87μmol/L(P<0.01),线粒体膜电位水平由0.24±0.03增至0.45±0.04(P<0.01),细胞凋亡率由43.3%±2.0%降至30.7%±0.9%(P<0.01)。结论西罗莫司可能通过内质网利阿诺定受体途径增加细胞内Ca2+水平,造成钙超载和线粒体损害,继而引起细胞凋亡。Aim To investigate the effect and discuss its potential mechanism of sirolimus induced calcium overload and apoptosis in vascular endothelial cells. Methods Human umbilical vein endothelial cells（ HUVEC） were exposed to sirolimus with or without intervention treatment with ryanodine. Effects on cell viability were assessed by the MTT assay. Effects on the production of nitric oxide（ NO） were detected by nitric oxide assay kit. The intracellular calcium ion（ Ca2 ＋） concentration was assayed with Fluo-3 AM staining,changes of mitochondrial membrane potential were detected by JC-1 fluorescence labeling,HUVEC apoptosis rate was analyzed by Annexin V FITC / PI staining. Results Compared with the normal control group,the cell survival rate was decreased significantly in the treatments of sirolimus injury group（ P〈0. 01）. In sirolimus 500 nmol / L injury group,cell swelling,cytoplasm vacuoles,part of the nucleus pycnosis,nuclear fragmentation were observed by HE staining,levels of intracellular free Ca2 ＋and cell apoptosis rate were increased（ P〈0. 01）,levels of NO and mitochondrial membrane potential were reduced（ P〈0. 01）. The intervention treatments,ryanodine,significantly reduced the sirolimus 500 nmol / L treatment-induced Ca2 ＋（ P〈0. 05） and cell apoptosis（ 43. 3% ± 2. 0% reduced to 30. 7% ± 0. 9%,P〈0. 01）. Ryanodine also increased the sirolimus-treated cells＇ viability（ P〈0. 01）,production of NO（ 28. 33% ± 4. 18 μmol / L increased to 47. 03 ± 3. 87 μmol / L,P〈0. 01） and the level of mitochondrial membrane potential（ 0. 24 ± 0. 03 increased to 0. 45 ± 0. 04,P〈0. 01）. Conclusion The possible mechanism of sirolimus induced calcium overload and apoptosis in HUVEC might be related to the increase of intracellular Ca2 ＋via ryanodine receptor pathway.

关 键 词：西罗莫司 利阿诺定受体 血管内皮细胞 钙超载 

分 类 号：R96[医药卫生—药理学]