摘要
目的:研究血管内皮细胞蛋白激酶C对CD44分子表达的调控机制。方法:以人脐静脉血管内皮细胞(humanumbilical vein endothelial cells,HUVECs)为研究模型。利用免疫沉淀法制备Raf-1激酶的提取物,以Western blot及增强化学发光法检测Raf-1激酶活性;放射自显影检测CD44的磷酸化情况;RT-PCR方法检测CD44基因表达。结果:与未处理的细胞相比较,加入10ng/ml的Phorbol-myristate-acetate(PMA)后,CD44磷酸化随时间延长而明显增强,尤以30分钟时最显著。HUVECs的PKC活化后,Raf-1Kinase活性明显增强,加入Calphostin C后其活性则被抑制;10ng/ml PMA处理1分钟,即能看到CD44基因表达明显升高(P<0.05),而先加入0.05μmol/L Calphostin C、30μmol/L RKIP,5μmol/L PD98059、10μmol/LGenistein预处理HUVECs,则能抑制PMA对CD44基因表达的上调作用。结论:PKC活化通过对CD44的磷酸化作用而影响CD44基因表达,其信号传导途径为PKC→Raf-1Kinase→MEK→ERK。此路径相关酶的抑制剂能抑制CD44基因的表达。
Objective:To study the mechanism of protein kinase C regulating CD44 gene expression in vascular endothelial cells. Methods: Human umbilical vein endothelial cells (HUVECs) were taken as study model. The extract of Raf-1 kinase by immuno- precipitation was used for the Western blot analysis, and its activity was determined by enhanced chemiluminescene assay. CD44 gene expression was detected with RT-PCR, and phosphorylation was measurated by autoradiography. Results: CD44 phosphorylation in HU- VECs was enhanced by 10ng/ml PMA treatment as compared with untreated cells, which reached the highest level at 30 minutes. Raf- 1 kinase activity increased significantly after exposure to 10ng/ml PMA, and 0.05 μmol/L Calphostinc could inhibit the role of Protein kinase C(PKC). CD44 gene expression level increased obviously after exposure to 10 ng/ml PMA ( PKC activator) for only 1 minute (P 〈0. 05), but 0. 05 μmol/L Calphostin C(PKC inhibitor), 30 μmol/L RKIP (Raf-1 kinase inhibitor), 5 μM PD98059(mitogenactivated protein kinase/extracellular signal regulated kinase inhibitor), 10 μmol/L genistein( extracellular signal regulated kinase inhibitor) could inhibit the up-regulation of PKC on CD44 gene expression. Conclusion:PKC activity effected CD44 gene expression by CD44 phosphorylation, and one of signal transduction pathways is PKC/Raf-1 kinase / MEK/ERK. The enzyme inhibitors above could inhibit the CD44 gene expression.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2007年第11期975-978,共4页
Chinese Journal of Immunology