【作者】 张传新；

【导师】 刘玉峰；

【作者基本信息】 郑州大学 ， 儿科学， 2004， 硕士

【摘要】 铁是机体细胞生命活动所必需的微量元素之一。细胞内铁稳态（cellular iron homeostasis）的维持对细胞的生长增殖及正常的机能活动来说都是非常重要的。研究发现，转铁蛋白受体（Transferrin Receptor，TfR）、铁蛋白（Ferritin，Fn）和铁调节蛋白（Iron Regulatory Protein，IRP）参与了细胞内铁稳态（cellular iron homeostasis）的维持活动，其中TfR和Fn的表达都受IRP在转录或转录后水平上的调节。目前已知的铁调节蛋白形式有两种即IRP₁、IRP₂。 白血病是儿童时期较为常见的一种恶性肿瘤疾病，其发病机制目前尚不十分清楚。已有研究证实铁与白血病的发生、发展具有密切的关系。尽管IRP与IRE结合在转录后水平上调节相关基因的表达是当前研究的热点，但对白血病细胞铁代谢及其稳态调控的研究相对较少，尤其是对IRP₂在不同铁状态下调控基因转录作用的研究则更少。 本研究以人白血病细胞株HL-60细胞为实验对象，通过加铁和去铁干预试验，探讨白血病细胞铁代谢及其调节机制以及IRP₂在HL-60细胞铁代谢中的郑州大学2004届研究生毕业论文HL·60红11胞IRpZ mRNA、TI’RmRNA、FnmRNA的表达及其，，肿瘤相关基眯1 WT，mRNA的关系‘JI究作用。同时，探讨HL一60细胞中IRPZ、Tfl丈和Fn与肿瘤基因WTI的关系;以揭示白血病细胞铁代谢机制以及铁在白血病发生发展中的作用。方法 按照实验要求，将FeCI:或去铁胺（Desferrioxamine，DFO）加入含有10%胎牛血清的RPMI一1 640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。实验分为五组，即:FeC13一20组和FeC13一40组（培养液中FeC13终浓度分别为20、40林mol/L）;oFo一50组和nFo一1 00组（培养液中DFo终浓度分别为50、100林mol/L）;对照组（培养液中不加入FeC13和DFO）。细胞培养后，分别于第12小时、24小时和48小时收集各组培养细胞，细胞总RNA的提取参照组织/细胞总RNA提取试剂盒要求进行，提取后液氮冻存。采用RT·PCR半定量法测定IRPZ mRNA、Fn;nRNA、T琅mRNA和wTI:nRNA等指标的相对表达量结果 1.IRP:mRNA:各实验组之间IRP:mRNA的表达量变化不大，经统计学处理，组间差异无统计学意义（F娜庐1.199，P>0.05）。细胞培养时间对IRPZmRNA的表达有影响，经统计学处理，组内差异有统计学意义（F，。二43 .418，尸<0.05），表现为随时间的延长，IRP:mRNA的表达降低。 2.T仅mRNA:各实验组之间的差异有统计学意义（厂w一s二7.184，F。:。.，“113.926，尸<0.01）。在加入去铁胺的DFO一50组和DFO一100组中TfRmRNA的表达升高，并随培养时间的延长和DFO剂量的增加，其表达升高。在加铁的FeC13一20组和FeC13一40组中，与对照组相比较，在12h时TfRmRNA的表达量上升，24h时达到高峰，之后迅速下降。而在48h时，FeCI3一40组的表达量与对照组相比较，约下降2倍，两组的差异具有统计学意义（尸<0.01） 3.Fn mRNA:细胞培养时间对Fn mRNA的表达无明显影响，经统计学处理，组内差异无统计学意义（F，、内月514，尸>仓05）而各实验组之间的差异有统计学意义（F。、。、=209、056，P<0.01）。FeC13一20组和FeC13一40组Fn mRNA的表达量较高，大约是对照组的2倍，它们与对照组比较，差异具有统计学郑州大学2004届研究生毕业论文HL·60幻一l胞IRPZ mRNA、TfRmRNA、FnmRNA的表达及其一与肿瘤相关基l州WT:mRNA的关系研究意义（P<0.05）;而DFO一50组、DFO一100组FnmRNA的表达与对照组相比较，FnmRNA的表达无明显变化（尸>0.05）。 4.IRP:mRNA与T仅mRNA和Fn mRNA的相关性:相关分析结果显示:IRPZ mRNA与T仅mRNA及Fn mRNA的表达均不相关（:=一0.005;r=0.074;P>0 .05）。5.WT，mRNA:各实验组之间T仅mRNA的表达量无明显变化，异无统计学意义（厂，:。.，一l一07，P>0.05）。细胞培养时l’oJ又寸Fn mRNA明显影响，组内差异无统计学意义（F。内=0.5“，尸>0.05）。 组间差的表达无 6.WTI mRNA与IRP:n飞RNA、Fn mRNA以及TfR mRNA的相关性:相关分析结果显示，WT;mRNA的表达与IRPZ mRNA、Fn mRNA以及TfR mRNA的表达均不相关（p>0.05）。结论 1.铁剂和去铁胺是通过IRP:或IRP:mRNA对HL一60细胞的铁代谢产生作用。2.在本实验所用铁剂和去铁胺剂量范围内，铁剂和去铁胺对IRPZ mRNA表达的总量无影响。3.铁剂可能通过影响IRPZ的表达，调控TfR mRNA、Fn mRNA的合成，进而影响HL一60细胞TfR蛋白和Fn蛋白的表达量。4.DFO可能通过影响IRPZ mRNA不同片段的合成，调控TfR mRNA的表达，但对Fn mRNA的表达影响不大。5.IRPZ mRNA与TfR mRNA、Fn mRNA无相关关系。6.铁剂和去铁胺可能不影响HL一60细胞WT，mRNA的表达。7.在Fll mRNAHL一60细胞中，肿瘤基因WT一mRNA与IRPZ mRNA、TfR mRNA、无相关关系。更多还原

【Abstract】 Background and ObjectionIron is an essential factor in many important cell functions, including growth, immunological response and energy production. It is important to maintain cellular iron homeostasis for cell proliferation and normal function. The iron regulatory proteins (IRP1 and IRP1) and the ferritin (Fn) and the transferrin receptor (TfR) involved in the maintenance of cellular iron homeostasis. Fn and TfR are used by cells to adjust intracellular iron concentration to levels in order to be adequate for their metabolic needs. IRP1 and IRP2 might bind to transcripts of ferritin, transferrin receptor to control the expression of iron metabolism proteins at the post-transcriptional level.Tumor cells have a close relation with iron, as other cells do. And leukemia is one of common malignancies. The studies showed iron play a role in the development of leukemia. Although post-transcriptional gene regulation by the interaction of IRPs and IRE on select mRNAs is a very active research area, there is relatively little data on gene transcription which may be modified by IRP2 underdifferent iron status. In order to reveal the mechanism of iron metabolism and the roles of iron playing in leukemia ,we investigated the expression levels and the relationship of IRP2mRNA, TfRmRNA , Fn mRNA and WT1 mRNA under different iron status of HL-60 cells, and probed into the roles of IRP2 playing in HL-60 cells.MethodsHL-60 cells (in 24-well plates) were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum at 37 C in 5% CO2-95% air with 100 U/ml penicillin G and 100 U/ml streptomycin. To examine effects of iron, HL-60 cells were divided into five groups which treated with ferric chloride(FeCL3) or deferoxamine(DFO). HL-60 cells in the control group cultured in control media, in FeCL3-20 group cultured in control media plus of 20 M/L FeCL3, in FeCL3-40 group cultured in control media plus of 40 M/L FeCL3, in DFO-50 group cultured in control media plus of 50 M DFO and in DFO-100 group cultured in control media plus of 100 M DFO. The cells were harvested at 12, 24 and 48h proliferation, and total RNA was isolated; cDNA was synthesized by reverse transcription (RT), and relative amounts of IRP2 mRNA , Fn mRNA and TfR mRNA were determined by RT-PCR.Results1. The levels of IRP2mRNA remained constant in all cells, whether or not treated with DFO or FeCL3.However, they were decreased when the cells prolonged in the media. There was not significant difference among the between-subjects (F=1.199,P > 0.05),but there was significant difference among the within-subjects (F=43.418, P<0.01).2. The levels of TfRmRNA increased in the cells treated with DFO and they are dose-time dependant. In contrast to the control cells which treated neither with DFO nor ferric chloride ,there was significant difference (Fw-s = 7.184, FB-S =113.926; P<0.01). Surprisingly, when cells grown with FeCLs, there wasn’t a decline in expression of TfR mRNA, but it increased lightly at 12 hours andpeaked at 24 hours. This was followed by a drastically decline in its expression so that by 48 hours the levels of TfR mRNA were approximately two times fewer in the cells grown in the presence of 40 uM/L FeCLs than in the control cells. There was significant difference (P <0.01).3. There was not significant difference among the within-subjects (F= 1.514.P > 0.05). It suggested that the levels of Fn mRNA didn’t dependent on time.However, there was significant difference among the between-subjects (F=209.056, P<0.01).The levels of Fn mRNA in the cells treated with FeCLswere approximately two times in the control cells. In contrast with the control cells, there was significant difference (P <0.05). The levels of Fn mRNA of the cells grown in DFO had little change. In contrast to the control cells, there was not significant difference (P > 0.05).4. There wasn’t significant correlation between IR?2 mRNA and TfR mRNA,Fn mRNA in HL-60 cells (P > 0.05 ).5. The levels of WT| mRNA remained constant in all cells whether or not更多还原