【作者】 徐华敏；

【导师】 谢俊霞；

【作者基本信息】 青岛大学 ， 生理学， 2008， 博士

【摘要】 帕金森病（Parkinson’s disease,PD）是一种以黑质多巴胺（dopamine,DA）能神经元变性坏死为主要病理特征的中枢神经系统退行性疾病,病因尚未阐明。足够的证据证实铁在帕金森病中是一个关键因素。但铁在黑质的选择性聚积的原因至今还不是十分清楚。新的铁转运蛋白的发现是铁代谢领域的重大突破,为解释这一问题提供了理论依据。二价金属离子转运蛋白1（divalent metal transport 1,DMT1）,也被称为具天然抗性的巨噬细胞蛋白2（nature resistance-associated macrophage protein 2,Nramp2）,是哺乳类的第一个跨膜铁转运蛋白。研究发现DMT1参与脑铁代谢,并且发现在PD病人脑黑质DMT1表达异常增高,这与PD病人脑内铁的异常沉积恰巧是同一个部位。本室前期研究亦表明在1-甲基4-苯基-1,2,3,6-四氢吡啶（1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP）制备的PD模型小鼠上,观察到黑质内铁选择性堆积和DMT1的表达增加,提示DMT1的高表达可能参与了铁聚积。本实验为了验证DMT1的表达增加可以导致铁聚积,选用MES23.5多巴胺能神经细胞系作为神经元的模型,应用分子生物学,免疫荧光,激光共聚焦显微镜,流式细胞仪,HPLC等方法研究了DMT1的高表达在细胞铁聚积中的作用。DMT1的高表达为PD的防治提供了新的药物作用靶点。人参皂苷Rg1是近年来研究较多的一种人参单体,药理研究表明它具有抗神经细胞凋亡、抗衰老、抗氧化、提高免疫力、类雌激素等作用。为探讨Rg1对6-羟基多巴胺（6-hydroxydopamine,6-OHDA）处理的MES23.5细胞铁代谢的影响,本实验应用荧光染料calcein结合激光共聚焦显微镜,逆转录聚合酶链式反应（reverse transcriptase polymerase chainreaction,RT-PCR）,Western blots等方法检测了Rg1对细胞内铁水平和DMT1表达的影响,并进一步研究其可能机制。结果发现:一.DMT1的高表达在MES23.5细胞铁聚积中的作用1.免疫荧光检测结果显示DMT1+IRE和DMT1-IRE在体外培养的MES23.5细胞上均有表达。2.应用RT-PCR的方法从人胚近段小肠中获取人的全长DMT1 cDNA,包括+IRE型和-IRE型,测序结果正确。利用细菌内同源重组法成功构建了携带DMT1编码基因的重组腺病毒载体。经PacI酶切鉴定和PCR鉴定获到重组腺病毒载体pAdEasy1-DMT1+IRE和pAdEasy1-DMT1-IRE。重组腺病毒质粒转染293细胞成功制备了高滴度的重组腺病毒。3.重组腺病毒AdDMT1+IRE或AdDMT1-IRE分别感染MES23.5多巴胺能神经细胞系,24 h后可观察到绿色荧光蛋白（green fluorescent protein,GFP）的表达。Western blots检测到AdDMT1+IRE感染的细胞DMT1+IRE的蛋白水平较病毒对照组有明显的增加;AdDMT1-IRE感染的细胞DMT1-IRE的蛋白水平较病毒对照组有明显升高（P＜0.01）。4.MTT测定结果显示AdDMT1+IRE或AdDMT1-IRE感染的MES23.5细胞在铁孵育（100μmol/L）24 h后,细胞存活率均明显降低,较对照组有统计学意义（P＜0.05）。5.细胞铁含量检测结果显示AdDMT1+IRE或AdDMT1-IRE感染的MES23.5细胞铁孵育（100μmol/L FeSO₄）后细胞内铁含量均高于病毒对照组（P＜0.01）。而细胞内羟自由基和MDA含量较病毒对照组有明显的增加,差别有统计学意义（P＜0.01）。6.MES23.5细胞铁孵育后检测细胞凋亡包括caspase-3的活性,DNA ladder和Hoechst染色,结果显示AdDMT1+IRE或AdDMT1-IRE感染的MES23.5细胞铁孵育后caspase-3的活性明显增加（P＜0.01）,出现明显的凋亡梯度带和核固缩等凋亡的形态学变化（P＜0.05）。7.HPLC结果显示AdDMT1+IRE或AdDMT1-IRE感染的MES23.5细胞铁孵育后细胞内多巴胺含量均较病毒对照组有明显的降低（P＜0.05）。8.pcDNA3.1-DMT1+IRE和pcDNA3.1-DMT1-IRE表达载体构建成功,应用荧光染料calcein结合激光共聚焦显微镜技术检测了细胞的摄铁功能,结果表明pcDNA3.1-DMT1+IRE或pcDNA3.1-DMT1-IRE转染的MES23.5细胞摄铁功能较对照组明显增强（双因素方差分析,F=25.995,P＜0.01,pcDNA3.1-DMT1+IRE转染组vs.对照组;P＜0.01,pcDNA3.1-DMT1-IRE转染组vs.对照组）。9.pcDNA3.1-DMT1+IRE或pcDNA3.1-DMT1-IRE转染的MES23.5细胞铁孵育后可以引起铁介导的细胞氧化应激损伤,包括细胞内ROS含量的增加和线粒体膜电位的降低,差别有统计学意义（P＜0.05）。二.人参皂苷Rg1对6-OHDA处理的MES23.5细胞铁聚积的影响。1.10μmol/L 6-OHDA处理MES23.5细胞24 h后,细胞DMT1+IRE的蛋白表达水平明显高于对照组（P＜0.05）,而Rg1或vitamin E预处理均可以显著抑制6-OHDA诱导的DMT1+IRE的蛋白表达的上调（P＜0.05）。2.采用实时定量PCR的方法检测了不同处理组MES23.5细胞DMT1+IRE的mRNA表达变化,结果同蛋白表达变化,6-OHDA可以上调MES23.5细胞DMT1+IRE的mRNA表达水平（P＜0.05）,而Rg1或vitamin E预处理均可以抑制6-OHDA诱导DMT1+IRE的mRNA表达的上调（P＜0.05）。3.10μmol/L 6-OHDA处理MES23.5细胞24 h后细胞的摄铁功能明显增强,而Rg1或vitamin E预处理均可以抑制6-OHDA诱导细胞内铁水平的增加（双因素方差分析,F=18.102,P＜0.01,6-OHDA处理组vs.对照组;P＜0.01,Rg1或vitamin E预处理组vs.6-OHDA处理组）。4.本实验应用Western blots的方法分别检测了铁调节蛋白1（iron regulatory protein 1,IRP1）和铁调节蛋白2（iron regulatory protein 2,IRP2）的蛋白表达水平。结果显示6-OHDA可以同时增加细胞的IRP1和IRP2的蛋白表达水平（P＜0.05）。而Rg1或vitamin E预处理均可以抑制6-OHDA诱导的IRP1和IRP2的蛋白表达的上调（P＜0.05）。5.本实验应用RT-PCR的方法检测了细胞IRP1和IRP2的mRNA表达水平。结果显示10μmol/L 6-OHDA处理MES23.5细胞24 h后细胞的IRP1和IRP2的mRNA表达均明显上调（P＜0.05）。而Rg1或vitamin E预处理均可以抑制6-OHDA诱导的IRP1和IRP2的mRNA表达的上调（P＜0.05）。差别有统计学意义。6.10μmol/L 6-OHDA处理MES23.5细胞24 h后给予100μmol/L的铁孵育,细胞线粒体跨膜电位明显降低,而Rg1预处理均可以抑制6-OHDA介导的线粒体跨膜电位的降低。综上所述,铁转入蛋白DMT1+IRE和DMT1-IRE在体外培养的MES23.5多巴胺能神经细胞系中均有表达;DMT1的表达增加可能是导致细胞内铁聚积的主要原因之一,这种细胞内铁含量的升高可能是由于细胞的摄铁功能的增加所致。细胞内增加的铁可以通过Fenton反应产生羟自由基,损伤细胞脂质,线粒体,从而导致细胞内羟自由基,脂质过氧化产物丙二醛含量的增加,细胞线粒体膜电位的降低,最终激活caspase-3途径引起细胞的凋亡。而Rg1可以通过IRP/IRE途径来调节6-OHDA诱导的DMT1+IRE的表达上调,降低细胞内铁聚积从而减轻铁聚积对细胞的损伤。更多还原

【Abstract】 Parkinson’s disease(PD) is a progressive neurodegenerative disorder characterized by selectively dopaminergic neurons loss in the substantia nigra pars compacta(SNpc) and the depletion of dopamine(DA) in the striatum.Recently many literatures have confirmed that iron plays a key role in Parkinson’s disease.However,the underlying mechanisms of iron accumulation remain unclear.The identification of new iron transporters provides an approach to investigate cellular mechanism of iron accumulation.DMT1,also known as Nramp2(nature resistance associated macrophage protein 2), is newly identified iron transporter.DMT1 is involved in the brain iron metabolism and highly expressed in the neurons of the substantial nigra in PD,which correlates with the iron abnormally deposited in the same area.Our previous studies showed increased DMT1 expression and elevated iron levels in the SNpc of PD mouse model.This suggested the up-regulation of divalent metal transporter 1(DMT1) might be a contributing factor to iron accumulation.In the present study,we aim to verify the hypothesis that increased DMT1 expression caused the iron accumulation.We chose MES23.5 cells as the experimental neuronal model.Using molecular biology,immunofluorescence,laser confocal scanning microscopy,flow cytometry,HPLC and other methods,we investigated the role of over expressed DMT1 in iron accumulation in MES23.5 cells.The upregulation of DMT1 provided new pharmacologic target to treat the disease. Ginsenoside Rg1 is one of the main components of ginseng,which has a variety of biological functions including but not limited to anti-oxidative,estrogen-like activity. The present study was carried out to elucidate the effect of the antioxidant drug ginsenoside Rg1 on levels of expression of the non-heme iron transporter,divalent metal transporter-1(DMT1),and iron levels in MES23.5 dopaminergic cells after 6-hydroxydopamine(6-OHDA) treatment.The main findings are as follows:Ⅰ.The role of over expressed DMT1 in iron accumulation in MES23.5 cells. 1.DMT1+IRE and DMT1-IRE were expressed on MES23.5 cells.2.Human DMT1+IRE and DMT1-IRE genes were amplified by RT-PCR.Recombinant adenovirus AdDMT1+IRE and AdDMT1-IRE were obtained after packaging and amplification in HEK293 cells.3.Increased DMT1 expression was confirmed by western blots.Data showed that DMT1+IRE protein levels increased significantly in AdDMT1+IRE infected cells compared to AdGFP infected cells(P＜0.01).DMT1-IRE protein levels increased significantly in AdDMT1-IRE infected cells compared to AdGFP infected cells (P＜0.01).4.Cell viability reduced when treated with 100μmol/L ferrous iron in AdDMT 1 +IRE or AdDMT1-IRE infected MES23.5 cells(P＜0.05).However,no significant change was observed in AdGFP infected cells.5.Intracellular iron levels,hydroxyl free radicals and lipid peroxidation increased in AdDMT1+IRE or AdDMT1-IRE infected MES23.5 cells after iron incubation compared to cells infected with AdGFP(P＜0.01).6.When cells were treated with ferrous iron,AdDMT1+IRE or AdDMT1-IRE infected cells showed more activated-caspase-3(P＜0.01),apparent ladder patterns, fragmentation of chromatin and hypercondensed(brightly stained) nuclei(P＜0.05).7.Dopamine contens in AdDMT1+IRE or AdDMT1-IRE infected cells were both decreased after iron treatment compared to cells infected with AdGFP(P＜0.05).8.pcDNA3.1-DMT1+IRE and pcDNA3.1-DMT1-IRE expression vectors were successfully constructed.There was a significant decrease in the fluorescence intensity in pcDNA3.1-DMT1+IRE or pcDNA3.1-DMT1-IRE transfected cells compared to the contról when perfused with 1 mmol/L ferrous iron(Two-way ANOVA,F=25.995,P＜0.01,pcDNA3.1-DMT1+IRE vs.control;P＜0.01, pcDNA3.1 -DMT1-IRE vs.control). 9.The production of ROS and changes of mitochondrial transmembrane potential (△Ψm) were detected by Flow Cytometer.Overexpression of DMT1(DMTI+IRE or DMTI-IRE) increased ROS production and decreased mitochondrial transmembrane potential(△Ψm) compared to the control after iron treatment (P＜0.05).Ⅱ.The role of ginsenoside Rg1 in 6-OHDA-induced iron accumulation in MES23.5 cells1.We examined DMT1+IRE expression levels by western blots in MES23.5 cells. DMT1+IRE protein in 6-OHDA treated MES23.5 cells was up-regulated compared to the control,while pretreated with Rg1 or vitamin E could attenuated this upregulation of DMT1 +IRE(P＜0.05).2.Quantitative PCR was conducted to measure DMTI+IRE mRNA levels.There was an increase of DMT1+IRE mRNA in 6-OHDA treated cells and a decrease of DMT1+IRE mRNA in Rg1 or vitamin E pretreated cells(P＜0.05).3.There was a significant decrease in the fluorescence intensity in 6-OHDA treated cells compared to the control when perfused with 1 mmol/L ferrous iron.The fluorescence intensity restored to the control levels when pretreated with Rg1 or vitamin E(Two-way ANOVA,F=18.102,P＜0.01,cells treated with 6-OHDA vs. control,P＜0.01,cells pretreated with Rg1 or vitamin E vs.cells treated with 6-OHDA).4.IRP1 and IRP2 were upregulated in protein levels after 6-OHDA treatment,when pretreated with Rg1 or vitamin E,the two protein levels restored to the levels of the control(P＜0.05).5.Both IRP1 and IRP2 were upregulated in mRNA levels after 6-OHDA treatment, while when pretreated with Rg1 or vitamin E,the mRNA levels of both IRP1 and IRP2 restored to the levels of control(P＜0.05).6.Increased iron influx by 6-OHDA(10μmol/L) treatment resulted in a reduction of the mitochondrial transmembrane potential after iron treatment.While Rg1 could attenuate the reduction of mitochondrial transmembrane potential.In conclusion,these results suggested that DMT1+IRE and DMT1-IRE were expressed on MES23.5 cells.Increased two forms of DMT1 expression in MES23.5 cells caused the increased intracellular iron accumulation.Elevated cellular iron induced oxidative stress,increased caspase-3 activity and ultimately apoptosis.This supports the function of increased DMT1 expression in iron accumulation and the notion that increased DMT1 results in oxidative stress and apoptosis,processes known to occur in PD.We also reported that Rg1 could attenuate the upregulation of DMT1+IRE and decrease the cellular iron accumulation.And this effect may contribute to the capacity of Rg1 to attenuate the expression of IRP1 and IRP2.更多还原