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目的 研究全反式维甲酸 (ATRA)和三氧化二砷 (As2 O3)下调NB4细胞组织因子 (TF)表达的作用机制。方法 用放线菌酮抑制蛋白合成和用放线菌素D阻断RNA合成后 ,用逆转录 聚合酶链反应 (RT PCR)检测ATRA对NB4细胞TFmRNA转录的影响。将含有PML RARα融合蛋白全部编码序列的重组逆转录病毒质粒转染U937细胞 ,以转染空载体的U937细胞为对照 ,用ELISA方法检测ATRA和As2 O3处理的转染PML RARαU937细胞的TF抗原。结果 放线菌酮完全阻断了ATRA对NB4细胞TFmRNA的下调作用 ;ATRA使NB4细胞的TFmRNA的半寿期从正常对照的 6 0min缩短至 30min。转染PML RARα的U937细胞与转染逆转录病毒载体的U937细胞相比 ,其TF表达水平显著升高(P <0 .0 1) ;使用ATRA或As2 O3分别处理转染PML RARα的U937细胞和对照细胞 2 4h ,ATRA和As2 O3均可显著地降低这两个细胞株的TF抗原水平。结论 ATRA对NB4细胞TFmRNA的下调作用依赖于新的蛋白质合成 ,ATRA可能是以间接方式下调APL细胞TFmRNA的转录 ;ATRA处理的NB4细胞TFmRNA的稳定性也有所降低。APL细胞染色体易位产生的融合蛋白PML RARα可能对TF的异常表达有一定的影响。ATRA和As2 O3对TF的下调作用可能不依赖于融合蛋白PML RARα的降解
Objective To study the mechanism of down-regulation of tissue factor (TF) expression in NB4 cells by all-trans retinoic acid (ATRA) and arsenic trioxide (As2O3). Methods Cycloheximide was used to inhibit protein synthesis and actinomycin D was used to block RNA synthesis. Reverse transcription polymerase chain reaction (RT PCR) was used to examine the effect of ATRA on the transcription of TF mRNA in NB4 cells. The recombinant retroviral plasmid containing the entire coding sequence of the PML RARα fusion protein was transfected into U937 cells. U937 cells transfected with the empty vector were used as controls, and the TF antigens of transfected PML RARαU937 cells treated with ATRA and As2O3 were detected by ELISA. Results Cycloheximide completely blocked the down-regulation of TFmRNA by ATRA in NB4 cells; ATRA shortened the half-life of TFmRNA in NB4 cells from 60 min in normal control to 30 min. U937 cells transfected with PML RARα had significantly higher TF expression levels than U937 cells transfected with retroviral vectors (P < 0.01); U937 cells transfected with PML RARα were treated with ATRA or As2O3, respectively. Both the ATRA and As2O3 significantly reduced the TF antigen levels of these two cell lines at 24 h and control cells. Conclusion The down-regulation of TFmRNA in NB4 cells by ATRA is dependent on new protein synthesis. ATRA may down-regulate the transcription of TFmRNA in APL cells indirectly. The stability of TFmRNA in ATRA-treated NB4 cells is also decreased. The fusion protein PML RARα produced by chromosomal translocation of APL cells may have a certain influence on the abnormal expression of TF. The down-regulation of TF by ATRA and As2O3 may not depend on the degradation of the fusion protein PML RARα