1、【关键词】 TPA; K562细胞; 线粒体铁蛋白; 运铁蛋白受体; 铁蛋白Expression of Mitochondrial Ferritin in K562 Leukemic Cell du ring TPA Induced Cell Differentiation Abstract Mitochondrial ferritin (MtF), a new player in iron metabolism, first identified in 2001, is highly homologous to ferritin both structurally and functiona
2、lly. Preliminary studies have suggested that MtF might play very important roles in the regulation of mitochondrial iron homeostasis. Leukemic cells, just like other malignant cells, demand more iron for their greater proliferation potential. However, little is known about what roles MtF might play
3、in leukemic cell iron metabolism and cell proliferation. The aim of this study was to investigate the expression of MtF, transferrin receptor 1 (TfR1) and ferritin (Fn) mRNAs in K562 leukemic cells during TPA induced cell differentiation and to explore the interrelationship between the expression le
4、vels of these iron metabolism related molecules. K562 cells cultured with or without TPA (16 nmol/L) were collected at 24, 72 and 120 hours respectively. Cell differentiation toward monocyte lineage was confirmed by microscopic study (Wrights staining) and flow cytometry. Semiquantitative RT PCR was
5、 performed to determine mRNA expression, with house keeping gene actin as control reference. This study revealed that over 95% of K562 cells showed morphological features of monocyte/macrophage,and the expression of CD64 on cell surface increased significantly at day 5 with TPA treatment. K562 cells
6、 could express a certain level of MtF before TPA induced differentiation. With increase of TPA induced cell differentiation, MtF mRNA expressions were downregulated progressively. After 5 days of induced cell differentiation, expression levels of MtF and TfR1 mRNA were just % and % of that before TP
7、A treatment. While Fn mRNA expression increased tofolds of that before TPA treatment. It is concluded that MtF expression is downregulated during TPA induced K562 cell differentiation, with concomitant downregulation of TfR1 and upregulation of Fn. The coordinated expression regulation of these key
8、iron metabolism related molecules during cell differentiation may in turn inhibit TfR1 mediated iron uptake via endocytosis and thus adversely affect cell proliferation potential. 线粒体铁蛋白(mitochondrial ferritin, MtF)是2001年才发现的一种铁代谢相关分子,与胞浆铁蛋白具有很高的同源性,也具有亚铁氧化酶活性和铁结合能力,但其表达局限于线粒体1。国外初步研究表明,MtF为线粒体内的铁存储
9、蛋白,MtF的表达可使胞浆内铁向线粒体内转运,降低胞浆可变铁池水平,细胞呈缺铁状态,并通过铁调节蛋白和铁效应元件的相互作用在转录后水平上协调调节运铁蛋白受体1(transferrin receptor 1, TfR1)和铁蛋白(ferritin, Fn)表达水平2。MtF在细胞铁稳态调节方面可能具有十分重要的作用。 中国实验血液学杂志 J Exp Hematol 2007; 15(2)K562细胞诱导分化过程中线粒体铁蛋白表达情况研究线粒体是细胞代谢最为旺盛的细胞器,是血红素和铁硫蛋白合成的主要场所,也是活性氧自由基产生的重要部位3,4。白血病细胞增殖代谢旺盛,对铁的需求大,但目前对白血病细胞
10、线粒体铁代谢了解很少。为此,我们采用TPA体外诱导K562白血病细胞定向分化动态研究了K562白血病细胞诱导分化过程中MtF、TfR1和胞浆Fn mRNA表达的变化情况,探讨MtF在白血病细胞线粒体铁代谢调节方面的作用,为建立针对铁代谢途径的白血病治疗新策略奠定理论基础。 材料和方法 材料和仪器 K562细胞株由四川大学华西第二医院实验中心提供。RPMI 1640培养液为美国Gibco公司产品,特级小牛血清为海克隆生物化学制品有限公司产品,TPA为晶美生物工程有限公司产品,TRIZOL试剂为北京天为时代科技有限公司产品,RevertAidTM First Stand cDNA 合成试剂盒为晶美
11、生物工程有限公司产品,PCR MasterMix 试剂盒为北京天为时代科技有限公司产品,流式细胞仪为Coulter EPICS ELITE ESP产品,PCR仪、凝胶图象分析系统为UVP公司产品。 RT PCR引物 参照有关文献设计1,由Invitrogen公司合成。MtF上游引物序列: 5 TATTTCCTTCACCAGTCCCGG 3;MtF下游引物序列: 5 GCAGGAGACAGCTGACTTTGG 3,MtF扩增产物长度451 bp。TfR1上游引物序列: 5 AGCACAGACTTCACCAGCACC 3; 下游引物序列: 5 GTCCCCAGATGAGCATGTCC 3, 扩增产
12、物长度506 bp。Fn上游引物序列: 5 CCTCCTACGTTTACCTGTCC 3;下游引物序列: 5 GCTTGTCAAA GAGATATTCC 3,扩增产物长度429 bp。内参照 actin上游引物序列: 5 CCAAGGCCAACCGCG AGAG 3,下游引物序列: 5 AGGGTACATGGTGGTGCCGC 3,扩增产物长度587 bp。 K562细胞培养 K562细胞复苏后加RPMI 1640培养液在37、 5% CO2、饱和湿度孵箱中悬浮培养。 实验分组 用无血清RPMI 1640培养液将K562细胞悬液调整至浓度为5105/ml,加入6孔板中,每孔接种量1106个细胞
13、,置于37、 5% CO2、饱和湿度孵箱中进行培养。实验组为TPA诱导分化培养组和对照组,前者根据预实验结果,确定TPA终浓度为16 nmol/ L,对照组为K562细胞普通培养。每孔共设3个复孔。 细胞形态观察 收集各时点的实验组和对照组K562细胞,用PBS洗涤2次,离心涂片后瑞氏染色,凉干后油镜下观察细胞形态,每片计数200个细胞,计算诱导分化率。 诱导分化率=100% 细胞表面CD64表达的流式细胞术检测 收集各时点的实验组和对照组K562细胞,于四川大学华西医院肿瘤生物治疗中心检测细胞CD64表达水平。 半定量RT PCR 细胞总RNA采用TRIZOL试剂盒提取制备,按照产品说明书进
14、行。cDNA第一链合成采用RevertAidTM First Stand cDNA 合成试剂盒。进行逆转录条件为: mRNA模板 g,Oligo dT 1 l,加DEPC H2O至总体积为12 l,70恒温5分钟后,冰上冷却;再加入buffer 4 l, inhibitor 1 l及dNTP 2 l,37恒温5分钟;最后加MMuLV逆转录酶1 l,42 1小时,70 10分钟,冷却。PCR扩增采用2Taq PCR MasterMix 试剂盒。MtF PCR条件为94变性30秒,55退火30秒,72延伸1分钟,30次循环后72恒温10分钟。TfR1的PCR条件为:94 2分钟,94 45秒,60 30秒,72 45秒,30次循环后,72 10分钟。Fn的PCR条件为:94 2分钟,94 45秒,56 30秒,72
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