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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 effect and mechanism of ox-LDL on the expression of tissue factor (TF) in human umbilical vein endothelial cell line ECV304. Methods: The experiment was divided into negative control group, positive control group (plus histamine), HDL, oxHDL 4 group and ECV304 cells with different concentrations of oxHDL, HDL (10-120 mg / L) and histamine 1 × 10-4mol / L) for different time (2-8 h), the expression of TF was detected by real-time quantitative PCR (RQ-PCR) and Western blotting. In addition, SP600125 [c-Jun N-terminal kinase (JNK) -specific inhibitor], SB203580 [p38MAPK specific inhibitor], PD98059 [ERK1 / 2 ) Specific inhibitor] to observe the effect of oxHDL and histamine on the expression of TF in ECV304 cells. Results: TF was not detected in normal ECV304 cells. The expression of TF mRNA increased significantly at 1 h after histamine treatment and reached the highest at 1 × 10-5 mol / L, which was 4.8 times of that of 1 × 10-9 mol / L. oxHDL induced TF mRNA expression in ECV304 cells in a time-and concentration-dependent manner, which was the highest at ox-LDL 20 mg / L and 1.8-fold at 10 mg / L, and at the 6th h, TF mRNA expression was 5.3- P <0.05), and at 20 mg / L significantly stimulated TF protein expression of ECV304 cells at a rate of 1.5-fold at 40 mg / L, whereas HDL did not. Inhibition of MAPKs (JNK, p38 MAPK, ERK1 / 2) partially relieved the effect of oxHDL on TF expression in ECV304 cells. TF was down-regulated to 19.0%, 5.0% and 13.0%, respectively. CONCLUSION: OxHDL can induce TF production in ECV304 cells in a concentration-dependent and time-dependent manner. The mechanism may be mediated by JNK, p38 MAPK and ERK1 / 2.