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目的探讨蛋白激酶C(PKC)激动剂佛波酯(PMA)和抑制剂钙磷酸结合蛋白(Calphostin C)对荷脂ECV304内皮细胞黏附能力影响的机制。方法采用体外培养和直接计数法观察内皮细胞黏附能力变化;PepTag Non-Radioactive Assay法定性定量细胞膜上PKC活性状态;RT-PCR和Western blot检测黏附相关指标细胞间黏附分子-1(ICAM-1)、I-κBα和ezrin表达的变化。结果100nmol/L PMA在激活细胞膜PKC活性的同时,可以与氧化型低密度脂蛋白(ox-LDL)协同增强ICAM-1和ezrin表达,但下调I-κBα的表达,并使内皮-单核细胞的黏附能力增强;300nmol/L Calphostin C基本上可以逆转50μg/ml ox-LDL诱导的酶活化和对ICAM-1、I-κBα和ezrin表达的调节,即PKC活性减弱,ICAM-1和ezrin表达下调,I-κBα表达上调,内皮细胞黏附能力明显降低。结论PMA、Calphostin C→PKC→NF-κB/I-κB→ICAM-1→Adhesion可能是黏附信息传递整合的一条重要途径,而黏附分子→ezrin→细胞骨架途径则可能起到加强内皮-单核细胞间黏附能力的作用。
Objective To investigate the mechanism of PKC agonist phorbol ester (PMA) and inhibitor of calcium phosphate binding protein (Calphostin C) on the adhesion of ECV304 endothelial cells. Methods The changes of adhesion ability of endothelial cells were observed by in vitro culture and direct counting method. PepTag Non-Radioactive Assay method was used to qualitatively determine the state of PKC activity on the cell membrane. The expression of ICAM-1 was detected by RT-PCR and Western blot. , I-κBα and ezrin expression changes. Results 100 nmol / L PMA could enhance the expression of ICAM-1 and ezrin simultaneously with oxidized low density lipoprotein (ox-LDL) while down-regulating the expression of I-κBα and activating endothelial cell-monocyte ; 300nmol / L Calphostin C can basically reverse the enzymatic activation induced by 50μg / ml ox-LDL and regulate the expression of ICAM-1, I-κBα and ezrin, that is, the decrease of PKC activity and the expression of ICAM-1 and ezrin Down-regulated, the expression of I-κBα was up-regulated, and the adhesion ability of endothelial cells was significantly decreased. Conclusions PMA, Calphostin C → PKC → NF-κB / I-κB → ICAM-1 → Adhesion may be an important pathway for the adhesion information transfer integration. However, the adhesion molecule → ezrin → cytoskeleton pathway may play an important role in enhancing endothelial- The role of intercellular adhesion capacity.