砷剂和维甲酸对NB4细胞组织因子和凝血酶调节蛋白mRNA及促凝活性的影响

Effects of Arsenic Trioxide or Retinoic Acid on mRNA and Protein Expression of Tissue Factor and Thrombomodulin and Procoagulant activity in NB4 Cells

为了研究三氧化二砷(As2O3)和全反式维甲酸(ATRA)对NB4细胞组织因子(TF)和凝血酶调节蛋白(TM)mRNA及其表达的影响,本研究采用体外细胞培养方法,以As2O3、ATRA处理NB4细胞,用ELISA动态测定其TF、TM抗原表达及促凝活性(PCA)变化,用逆转录-聚合酶链反应(RT-PCR)测定TF和TM的mRNA转录。结果表明:1μmol/L的As2O3和1μmol/LATRA均可使NB4细胞TF抗原及mRNA表达呈时间依赖性渐进性下调;在24、48、72、96和120小时时间点,As2O3组TF抗原的表达水平分别为13.3±1.8,8.6±1.9,10.8±1.5,2.0±0.6和2.6±0.9ng/107;ATRA组TF抗原的表达水平分别为12.4±1.1,11.3±1.8,5.7±1.7,2.8±0.8和2.0±0.6ng/107,与对照组比较具有显著性(P<0.05);在不同的时间点As2O3和ATRA,均可使其PCA下降,As2O3组血凝时间分别为123.5±10.5,156.3±11.6,179.3±15.3,248.9±20.1,312.0±29.8(秒);ATRA组血凝时间分别为76.4±5.6,146.8±10.9,198.2±15.6,265.8±20.6和363.8±31.9(秒);ATRA使NB4细胞TM抗原表达及其mRNA转录上调。结论:As2O3、ATRA可降低TFmRNA转录,下调TF蛋白表达,降低NB4细胞PCA;ATRA增高TM转录及表达,缓解APL患者凝血功能异常。

To investigate the effect of arsenic trioxide （As2O3 ） or all-trans retinoic acid （ATRA） on the mRNA and protein expression of tissue factor （TF） and thrombomodulin （TM） and procoagulant activity （PCA） in NB4 cells. The NB4 cells were cultured in vitro and treatecl with As2O3 or ATRA, expression of TF and TM antigen, and PCA change of treated NB4 cells were detected with ELISA, TF and TM mRNA transcription on the NB4 cells was assayed with reversed transcription polymerase chain reaction （RT-PCR）. The results showed that 1 μmol/L AS203 and 1 μmol/L ATRA both gradually downregulated the expression of TF antigen and mRNA on NB4 cells, a human promyelocytic leukemia cell line, in time-dependent manner, as compared with control. The levels of TF antigen expression in AS203 group were 13.3 ± 1.8, 8.6 ± 1.9, 10.8 ± 1.5, 2.0 ± 0.6 and 2.6 ± 0.9 ng/10^7 respectively; while the levels of TF antigen expression in ATRA group were 12.4 ± 1.1,11.3 ± 1.8,5.7 ± 1.7,2.8 ± 0.8 and 2.0 ± 0.6 ng/10^7 at 24, 48, 72, 96 and 120 hours respectively（ P 〈 0.05 ）. The procoagulant activity （PCA） of NB4 cells was decreased, blood coagulation times were 123.5 ± 10. 5,156.3 ± 11.6,179.3 ± 15.3,248.9 ± 20. 1,312.0 ± 29.8 seconds in AS203 groups, respectively; 76.4 ± 5.6,146.8 ± 10.9,198.2 ± 15.6, 265.8 ± 20.6 and 363.8 ± 31.9 seconds in ATRA groups respectively at 24,48, 72, 96 and 120 hours, （P 〈0.05）. ATRA upregulated TM antigen expression on NB4 cells. It is concluded that the AS203 and ATRA decrease mRNA transcription of TF, downregulate expression of TF and reduce procoagulant activity in NB4 cells. The TM transcription and expression upregulated by ATRA may alleviate dysfunction of coagulation in APL.