氧化高密度脂蛋白通过MAPKs信号转导途径诱导ECV304细胞表达组织因子  

Effects of oxidized high density lipoprotein on tissue factor expression in ECV304 cell line

作　　者：卜梓斌[1,2,3] 姜志胜[1,2] 马珍妮[3] 董宁征[3] 赵战芝[1,2] 季顺东[3] 沈飞[3] 江淼[3] 王静[1] 谢丽倩[3] 冯雪娟[1,2] 陈晶晶[1,2] 阮长耿[3] 

机构地区：[1]南华大学心血管病研究所 [2]南华大学动脉硬化学湖南省重点实验室,湖南衡阳421001 [3]苏州大学附属第一医院江苏省血液研究所,卫生部血栓与止血重点实验室,江苏苏州215007

出　　处：《中国病理生理杂志》2009年第4期636-641,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.30570766No.30470693);湖南省教育厅重点科研资助项目(No.05A043);动脉硬化学湖南省高校科技创新团队资助项目(No.湘教通[2008]244);南华大学归国留学人员科研启动基金资助项目

摘　　要：目的:探讨氧化高密度脂蛋白(oxHDL)对人脐静脉内皮细胞株ECV304细胞表达组织因子(TF)的影响及其机制。方法:实验分为阴性对照组、阳性对照组(加组胺)、HDL、oxHDL 4组,ECV304细胞经不同浓度oxHDL、HDL(10-120 mg/L)和组胺(1×10-9-1×10-4mol/L)处理不同时间(2-8 h)后,采用实时定量PCR(RQ-PCR)和Western bloting方法检测TF表达。同时,分别应用SP600125[c-Jun氨基端激酶(JNK)特异性抑制剂]、SB203580[p38丝裂素活化蛋白激酶(p38MAPK)特异性抑制剂]、PD98059[细胞外信号调节激酶(ERK1/2)特异性抑制剂],观察其对oxHDL和组胺诱导的ECV304细胞表达TF的影响。结果:正常ECV304细胞未检测到TF,经组胺处理1 h后TF mRNA表达明显增加,在1×10-5mol/L时最高,约为1×10-9mol/L时的4.8倍。oxHDL以时间和浓度依赖性方式诱导ECV304细胞TF mRNA表达,在oxHDL20 mg/L时最高,为10 mg/L时的1.8倍,在第6 h时TF mRNA表达水平为第2 h的5.3倍(P<0.05),并在20 mg/L时显著刺激ECV304细胞TF蛋白表达,为40 mg/L时的1.5倍,而HDL没有此效应。MAPKs(JNK、p38 MAPK、ERK1/2)抑制剂可部分解除oxHDL诱导ECV304细胞TF表达的效应,TF分别下调表达至原来的19.0%、5.0%和13.0%(P<0.05)。结论:oxHDL能以浓度和时间依赖性方式诱导ECV304细胞产生TF,其机制可能通过JNK、p38 MAPK和ERK1/2所介导。AIM： To investigate the expression of tissue factor （TF） induced by oxidized high density lipoprotein （oxHDL） in human umbilical vein cell line, ECV304, and the related mechanisms. METHODS： Four main groups were designed： the negative, the positive （ECV304 with histamine）, the HDL group and the oxHDL group. Quantitative real - time polymerase chain reaction （ RQ - PCR） and Western blotting were used to detect the expression level of TF. The specific inhibitors of MAPKs, SP600125 （c -jun terminal NH2 kinase, JNK）, SB203580 （p38 MAP kinase, p38 MAPK）, PD98059 （extracellular signal -regulated kinase, ERK1/2） were used to investigate the underlying mechanisms. RESULTS： The TF expression in normal ECV304 cell line was not detected. Histamine administration resulted in a significant expression of TF in ECV304 cell line, with strongest effect after 1 h co - incubation at concentration of 1 ×10^-5 mol/L histamine （ about 4.8 - fold higher expression of TF compared with that of 1×10^-9 mol/L histamine）. Expression level of TF was detected after stimulated with oxHDL in dose - and time - dependent manners. The highest expression of TF mRNA was found at 20 mg/L oxHDL and 6 h co - incubation, with 1.8 - fold and 5. 3 - fold increase in TF expres- sion, respectively, compared with that at 10 mg/L oxHDL and 2 h co - incubation. 20 mg/L oxHDL also caused an apparent augmentation of TF protein expression, about 1.5 - fold higher compared with that stimulated by 40 mg/L oxHDL. HDL co - incubation did not cause a detectable expression of TF protein. The mRNA levels of TF in ECV304 cell line induced by oxHDL were decreased by 95.0%, 81.0%, 87.0%, respectively （all P 〈0. 05） after application of inhibitors against p38MAPK, JNK and ERK1/2. CONCLUSION： The results suggest that oxHDL stimulates TF expression in ECV304 cell line in both dose - and time - dependent manners, in which MAPKs signal transduction may play an important role.

关 键 词：组织因子 脂蛋白类 HDL p38 MAPK通路 ERK 1/2通路 JNK通路 

分 类 号：R363[医药卫生—病理学]