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目的研究HDL通过1-磷酸鞘鞍醇受体途径上调COX-2表达的细胞内信号机制。方法培养人脐静脉内皮细胞,HDL不同时间和浓度处理,qPCR和WB检测COX-2、CREB和Sphk2表达情况。人脐静脉内皮细胞转染SiRNASphk2后,qPCR和WB检测HDL对COX-2表达情况。人脐静脉内皮细胞用采用JTE(抑制S1P2)、PTX(抑制Gi/o)、SB203580(抑制p38MAPK)、Stau-rosporine(抑制PKC)及U0126(抑制ERK1/2)抑制剂处理后,qPCR和WB检测HDL对COX-2的表达情况。免疫共沉淀检测p-CREB和Sphk2结合情况,用激光共聚焦进行定位观察。结果 HDL呈时间和剂量上调COX-2、SphK2表达及CREB磷酸化,转染SphK2siRNA后COX-2表达水平下调;采用JTE(抑制S1P2),SB203580(抑制P38MAPK),Staurosporine(抑制PKC),U0126(抑制ERK1/2)抑制剂处理后,HDL对COX-2的上调作用明显抑制;免疫共沉淀和激光共聚焦显示p-CREB和SphK2结合。结论 HDL通过S1P2-G i/o-PKC(P38MAPK/ERK)-SphK2?p-CREB途径影响COX-2的表达。HDL激活内皮细胞核内鞘鞍醇激酶和使CREB磷酸化,从而上调COX-2的表达。
Aim To investigate the intracellular signaling mechanism by which HDL up-regulates the expression of COX-2 through the 1-phospho-sphingosine receptor pathway. Methods Cultured human umbilical vein endothelial cells were treated with HDL at different times and concentrations. The expression of COX-2, CREB and Sphk2 were detected by qPCR and WB. Human umbilical vein endothelial cells transfected with SiRNASphk2, qPCR and WB detection of HDL on COX-2 expression. Human umbilical vein endothelial cells were treated with qPCR and WB after treatment with inhibitors of JTE (S1P2 inhibition), PTX (inhibition of Gi / o), SB203580 (inhibition of p38 MAPK), Stau-rosporine (inhibition of PKC) and U0126 (inhibition of ERK1 / 2) Detection of HDL expression of COX-2. Co-immunoprecipitation detection of p-CREB and Sphk2 binding, laser confocal positioning observation. Results The expression of COX-2, SphK2 and CREB phosphorylation were up-regulated by time and dose in HDL. The expression of COX-2 was down-regulated after transfection with SphK2 siRNA. The expression of COX-2 was inhibited by JTE Inhibition of ERK1 / 2 inhibitor inhibited the upregulation of COX-2 by HDL. Co-immunoprecipitation and confocal laser scanning revealed the binding of p-CREB and SphK2. Conclusion HDL can affect the expression of COX-2 through S1P2-G i / o-PKC (P38MAPK / ERK) -SphK2? P-CREB pathway. HDL activates endothelial nuclin kinase in endothelial cells and phosphorylates CREB, thereby up-regulating COX-2 expression.