蛋白激酶C活性变化影响内皮-单核细胞黏附

Effects of PKC activity change on adhesion of endothelial cells with monocytes

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 effects of protein kinase C（PKC）agonist PMA and PKC inhibitor Calphostin C on adhesion of endothelial cells with monocytes. Methods The adhesion ability of endothelial cells was observed by culturing in vitro and by directly counting method. Cell membrane PKC activity was quantitated by PepTag non-radioactive assay. The mRNA and pro- tein levels of ICAM-I,I-κBα and ezrin were determined by reverse transcription-polymerase chain reaction（RT-PCR） and Western blot, respectively. Results 100 nmol/L PMA activated cell membrane PKC activity,and synergistically enhanced the expression of ICAM-1 and ezrin in the presence of oxLDL. However, it reduced the content of I-κBα. Together with oxLDL, PMA stimulated the adhesion ability of endothelial cells with monocytes. 300 nmol/L Calphostin C essentially reverse the effects induced by 50/,g/ml oxLDL,causing decrease in PKC activity, down- regulation of expression of ICAM-1 and ezrin,decrease in the adhesion ability of endothelial cells and up-regulation of expression of I-κBα. Conclusions The pathway of PMA, Calphostin C→ PKC→NF-κB/I-κB→ICAM-1→adherence may be an important way by which the adhesion information was conveyed and integrated. In addition, the pathway adhesion molecules→ezrin→cytoskeleton may enhance the adhesion ability of endothelial cells with monocytes.