HL-60细胞IRP_2 mRNA、TfR mRNA、Fn mRNA的表达关系研究

Expression of IRP_2 mRNA, TfR mRNA and Fn mRNA in HL-60 Cells

为了探讨白血病细胞铁代谢及其调节机制,以及铁调节蛋白2(IRP2)在HL-60细胞铁代谢中的作用,将FeCl3或去铁胺(desferrioxamine,DFO)加入含有10%胎牛血清的RPMI1640培养液中,使HL-60细胞处于缺铁或富铁的不同状态下进行培养。分别于细胞培养后第12、24和48小时收集细胞,提取总RNA,采用RT-PCR半定量法测定IRP2mRNA、铁蛋白(Fn)mRNA和转铁蛋白受体(TfR)mRNA等指标的相对表达量。结果表明:①各实验组之间IRP2mRNA的表达量变化不大,组间差异无统计学意义(F组间=1.199,P>0.05);而细胞培养时间对IRP2mRNA的表达有影响,组内差异有统计学意义(F组内=43.418,P<0.01),表现为随时间的延长,IRP2mRNA的表达降低。②各实验组之间TfRmRNA的表达差异有统计学意义(F组内=7.184,F组间=113.926,P<0.01)。在加入DFO的两个实验组中,TfRmRNA的表达升高。在加铁的两个实验组中,与对照组相比较,于12小时时TfRmRNA的表达量上升,24小时时达到高峰,之后迅速下降。③在加铁的两个实验组中,FnmRNA的表达量较高,大约是对照组的2倍,它们与对照组比较,差异具有统计学意义(P<0.05);而在加入DFO的两个实验组中FnmRNA的表达与对照组相比较,无明显的差异(P>0.05)。④IRP2mRNA与TfRmRNA及FnmRNA的表达均不相关(r=-0.005,r=0.074,P>0.05)。结论:①IRP2在转录水平上可能是通过自身不同片段的量的变化,而在转录后水平上是以氧化修饰的方式通过自身降解,对HL-60细胞的铁代谢进行调节。②FnmRNA和TfRmRNA不同程度地参与了对HL-60细胞内铁的调控过程。

To explore the mechanism of iron metabolism and its regulation as well as the roles of IRP2 in ion metabolism of HL-60 cells, HL-60 cells were cultured in RPM1 1640 medium supplemented with 10% heat-inactivated fetal bovine serum,which was treated with ferric chloride （FeCl3 ） or deferoxamine （DFO）. The cells were harvested at 12, 24 and 48 hours of proliferation, and total RNA was isolated; cDNA was synthesized by reverse transcription （ RT）,and relative expression levels of IRP2 mRNA, Fn mRNA and TfR mRNA were determined by RT-PCR. The results showed at follows： （ 1 ） the level of IRP2 mRNA remained constant in all cells, whether or not treated with DFO or FeCl3. However, the expression of IRP2 mRNA decreased when the time of cell culture was prolonged. There was no significant difference between groups （ FB-S = 1. 199, P 〉 0. 05 ）, but there was significant difference among the different time culture （FW-S=43.418, P 〈 0.01 ）. （2） Cells which treated neither with DFO nor ferri chloride showed significant difference from the control （ FW-S = 7.184, FB-S = 113. 926 ; P 〈 0.01 ）. The level of TfR mRNA increased in the cells treated with DFO. Surprisingly, when cells treated with FeCl3 , there was not decline of TfR mRNA expression,but it increased lightly at 12 hours and peaked at 24 hours and declined drastically at 48 hours. （ 3 ） The level of Fn mRNA in the cells treated with FeCl3 was approximately 2-fold as the control cells. In contrast with the control cells,there was significant difference （ P 〈 0.05 ）. The level of Fn mRNA of the cells treated with DFO had little change. As compared with the control cells, no significant difference was seen （ P 〉 0. 05 ）. （4） There was not any significant correlation between IRP2 mRNA and TfR mRNA or Fn mRNA in HL-60 cells （ r = - 0.005 ; r = 0.074 ; P 〉 0.05 ）. It is concluded that （ 1 ） IRP2 may regulate the iron metabolism in HL-60 cells by altering amounts of the IRP2 3.7- or 6.4-kb mRNA at the transcriptional level, or by IRP2 degradation at the post transcriptional level. （ 2 ） Both of Fn mRNA and TfR mRNA participated, more or less, in the iron metabolism in HL-60 cells.