Xu Hua-Min, Jiang Hong, Wang Jun, Luo Bing, Xie Jun-Xia
Department of Physiology, Medical College of Qingdao University, No. 308 Ningxia Road, Qingdao, China.
Neurochem Int. 2008 May;52(6):1044-51. doi: 10.1016/j.neuint.2007.10.019. Epub 2007 Nov 9.
Elevated iron accumulation has been reported in brain regions in some neurodegenerative disorders. However, the mechanism for this is largely unknown. Divalent metal transporter 1 (DMT1) is an important divalent cation transporter. The aim of the present study is to construct recombinant adenovirus encoding human DMT1 with iron responsive element (DMT1+IRE) and infect MES23.5 dopaminergic cells in order to investigate the relationship between increased DMT1+IRE expression and iron accumulation. The human DMT1 gene was obtained by RT-PCR from tissues of human duodenum. AdDMT1+IRE was successfully constructed and identified by PCR, restriction endonuclease analyses and DNA sequencing, respectively. It was able to efficiently infect MES23.5 cells, which was confirmed by RT-PCR and Western blots. When incubated with 100 microM ferrous iron for 6h, the intracellular iron levels dramatically increased in AdDMT1+IRE infected MES23.5 cells compared to the solely adenovirus infected cells. Meanwhile, the levels of hydroxyl free radicals and malondialdehyde (MDA) in these cells increased. This led to the activation of caspase-3. The apoptosis in AdDMT1+IRE infected cells was shown with hypercondensed nuclei using Hoechst staining. Analysis of DNA extracted from these cells showed the typical "ladder pattern", indicating the formation of mono- and oligonucleosomes. These results suggested that increased DMT1+IRE expression in MES23.5 cells caused the increased intracellular iron accumulation. This resulted in the increased oxidative stress leading to ultimate cell apoptosis.
据报道，在一些神经退行性疾病的脑区中铁蓄积增加。然而，其机制在很大程度上尚不清楚。二价金属转运蛋白1（DMT1）是一种重要的二价阳离子转运蛋白。本研究的目的是构建编码带有铁反应元件的人DMT1（DMT1+IRE）的重组腺病毒，并感染MES23.5多巴胺能细胞，以研究DMT1+IRE表达增加与铁蓄积之间的关系。通过RT-PCR从人十二指肠组织中获得人DMT1基因。分别通过PCR、限制性内切酶分析和DNA测序成功构建并鉴定了AdDMT1+IRE。RT-PCR和Western印迹证实其能够有效感染MES23.5细胞。与仅感染腺病毒的细胞相比，当用100μM亚铁离子孵育6小时时，AdDMT1+IRE感染的MES23.5细胞内铁水平显著升高。同时，这些细胞中羟自由基和丙二醛（MDA）水平升高。这导致了caspase-3的激活。使用Hoechst染色显示AdDMT1+IRE感染的细胞中出现核固缩，表明细胞凋亡。对从这些细胞中提取的DNA进行分析显示出典型的“梯状条带”，表明形成了单核小体和寡核小体。这些结果表明，MES23.5细胞中DMT1+IRE表达增加导致细胞内铁蓄积增加。这导致氧化应激增加，最终导致细胞凋亡。
