【作者】 王强；

【导师】 陈景元； 宋海峰； 骆文静；

【作者基本信息】 第四军医大学 ， 劳动卫生与环境卫生学， 2008， 博士

【摘要】 研究背景铅是环境中普遍存在的污染物,在体内可以长期蓄积,对人体可以产生全身性多系统的损伤。它具有很强的神经亲和性,可在神经组织中蓄积,引起神经系统功能长期的不可逆的损害。近些年发现,铅的毒性作用没有安全阈值,即体内有铅便有毒。在人群中更为普遍的是慢性低水平铅中毒。虽然许多国家采取了一些降低环境铅污染的措施,但慢性铅中毒依然是现代城市的居民所面临的主要健康问题之一。血脑屏障(BBB)是中枢神经系统的重要结构,它保证了中枢神经系统所需内环境的高度稳定,是中枢神经系统正常进行各项机能活动的基础;同时,BBB结构和/或功能的变化也是许多疾病病理生理变化的核心过程。铅不仅可以诱导脑微循环损伤改变脑血管内皮细胞的膜结构和紧密连接,使未成熟大脑的BBB发生渗漏,还可能通过脑内皮细胞的胞饮作用增强摄入大量的铅。近些年的研究显示,二价铁离子的跨膜转运体(divalent metal transporter 1, DMT1/NRAMP2/DCT1)也是铅的结合底物,并且铅与DMT1的亲和力比铁高。提示,在BBB铅可以竞争性抑制细胞铁的吸收来干扰细胞内铁的平衡,或者取代铁与铁结合蛋白结合来干扰这些蛋白质的功能。本实验为了克服在体研究不能施加干预措施的局限性,通过对体外BBB模型的建立,进一步研究内皮细胞跨膜转运铅的具体机制以及与胞内铁平衡的关系,为阐明铅在血脑屏障转运的分子机制以及有效地采取防护措施提供新的理论依据和可行的途径。研究目的通过人脐静脉内皮细胞(ECV304)和小鼠胶质瘤细胞(C6)共培养建立体外BBB模型,从细胞水平上研究铅跨膜转运与细胞内铁含量变化的关系,以探索铅在BBB跨膜转运的途径及其可能的调控机制。研究方法1、用ECV304和C6细胞建立Transwell非接触式共培养体外BBB模型,测定跨内皮阻抗(Trans-endotheilal electrical resistance, TEER)和FITC-葡聚糖通过量来评价BBB的功能。2、用钨舟原子吸收法测定铅在体外BBB模型转运的动力学特征;用免疫细胞染色法检测DMT1(IRE)蛋白在细胞内的定位;Western blot法检测细胞内DMT1(IRE)蛋白的表达水平;用DFX诱导细胞低铁来研究铅是否影响细胞内铁的平衡。3、反义寡核苷酸转染干扰DMT1 mRNA和蛋白的表达;稳定高表达DMT1细胞的筛选;进一步用这两种细胞构建体外BBB模型以确定DMT1是否是铅进入内皮细胞的主要通路。4、Western blot法检测细胞内IRP1和p-ERK1/2蛋白的表达水平;免疫共沉淀和免疫细胞化学双染法鉴定IRP1和p-ERK1/2蛋白之间是否存在相互作用;用ERK1/2通路的阻断剂(PD98059)研究IRP1和p-ERK1/2蛋白的相互作用与细胞中DMT1(IRE)蛋白表达调控的关系;钨舟原子吸收法测定PD98059对体外BBB模型转运铅的影响。研究结果1、ECV304和C6细胞非接触式共培养建立体外BBB模型ECV-304在C6胶质细胞的作用下,细胞间可以形成广泛的紧密连接复合体进而抑制外加电场下电流的跨内皮运动,产生了达232.5?cm2的TEER值;并且Papp均数在4、5、6d时均明显低于内皮细胞单独培养组,差异有显著性意义(p<0.05),说明我们建立了一种接近在体状态的体外血脑屏障模型。2、铅在BBB模型中的转运具有主动转运的特点铅的转运受pH值、温度、时间和剂量的影响。随着时间的增加,铅的转运量呈较为明显的上升趋势;并且铅的转运量随着铅剂量的增加而增加;在pH5.5的条件下转运铅的量显著高于pH7.4条件下的转运量;37oC时铅的转运量显著高于4oC时的转运量(p<0.05)。提示铅在BBB的转运是以主动转运为主。3、铅诱导的跨膜转运增加是BBB通透性异常改变的主要原因1μM和5μM Pb(NO3)2对体外BBB模型中ECV304细胞形成的TEER和Papp均没用显著的影响(p>0.05),对内皮细胞紧密连接蛋白claudin-1、ZO-1和occludin mRNA也没有明显的改变。铅可增加ECV 304细胞中DMT1(IRE)蛋白的表达,主要是显著增加了DMT1(IRE)在细胞膜上的表达量;不同时间染铅可以增加ECV304细胞中DMT1(IRE)蛋白表达,而DMT1(IRE)在细胞膜上表达的增加更为明显。结果提示,铅转运量的增加主要与跨膜转运异常密切相关。4、铅诱导的跨膜转运异常增加与DMT1蛋白高表达及其膜定位密切相关30μM FeCl3没有显著降低铅的转运(p>0.05);150μM FeCl3在4h和24h可以显著降低铅的转运(p<0.05)。铅和DFX都增加了细胞总蛋白和膜蛋白中DMT1(IRE)的表达;铁却可以降低铅诱导的DMT1(IRE)的表达。铅和铁均没有显著影响DMT1(IRE) mRNA的表达。用反义寡核苷酸转染以降低DMT1(IRE)蛋白的表达,可以降低BBB模型转运铅量,10h有显著性差异(p<0.05)。转染pcDNA3.0-DMT1重组质粒后,用G418筛选获得稳定转染细胞。稳定转染的ECV-304细胞与C6细胞共培养建立模型,可以增加铅的转运量;4h和24h有显著性差异(p<0.05)。以上结果提示,DMT1蛋白是铅跨膜转运的主要载体,细胞内铁稳态的失衡是铅转运增加的主要诱因之一。5、IRP1与p-ERK1/2之间的相互作用可以负反馈调节DMT1蛋白的表达铅在2h增加了细胞浆和细胞膜IRP1蛋白的表达,以后逐渐降低;铅还可以活化ERK1/2蛋白,而胞浆内ERK1/2的磷酸化却显著降低。铅和DFX都增加了细胞中IRP1和p-ERK1/2的表达;铁却可以抑制铅诱导的细胞浆IRP1和p-ERK1/2的高表达。共聚焦显微镜结果显示,细胞内IRP1与p-ERK1/2之间存在共定位,主要分布在细胞浆。铅和DFX都可以降低胞浆内IRP1与p-ERK1/2相互重叠的区域;铁却可以增加胞浆内IRP1与p-ERK1/2的相互重叠。免疫共沉淀结果显示,铅可增加相互作用的IRP1的表达,降低细胞浆内相互作用的p-ERK1/2。ERK阻断剂(PD98059)有增加总细胞DMT1(IRE)和胞浆IRP1蛋白的表达作用。加入PD98059,体外BBB模型的铅转运量增加,10h时有显著性差异(p<0.05)。以上结果提示,IRP1与p-ERK1/2之间的相互作用可以负反馈调节DMT1蛋白的表达。研究结论1、BBB通过DMT1(IRE)介导的跨膜转运增加铅的转运;2、DMT1(IRE)蛋白是介导铅转运进入大脑的载体,并且DMT1(IRE)在细胞膜上的定位与铅转运增加密切相关;3、DMT1蛋白的表达调控主要发生在转录后水平,是通过IRP/IRE调控模式调节DMT1(IRE) mRNA的翻译;4、细胞内IRP1与p-ERK之间存在相互作用,可以调节IRP1的表达及功能;5、在细胞水平上,ERK阻断剂(PD98059)可以上调DMT1蛋白的表达,促进BBB铅的转运。更多还原

【Abstract】 BackgroundLead is a persistent and common environmental contaminant. It accumulates in human body and exists for a long time. With its entry into the body, lead can cause toxic injury in erythrocytes, liver, renal cortex, aorta, brain, lungs, spleen, teeth, and bones. Studies of effects of lead on children have demonstrated the presence of irreversible neurological alterations at low concentrations. These findings demonstrate that, based on cellular and molecular evidence, there is no threshold for lead toxicity. A chronic, low-level intoxication of lead is more common in the general population. Although a variety of actions have been taken to decrease the use and distribution of lead in the environment, it remains a significant health hazard.Due to its unique structure, the blood-brain barrier (BBB) is capable of limiting the penetration of a variety of substances from the blood into the brain. The BBB plays an important role in the homeostasis and is generally seen as a defence mechanism that protects the brain against various poisonous molecules that may traverse the BBB. The BBB has long been known to be a target for Pb toxicity. Under acute exposure to high levels of Pb, Pb-induced microvascular damage is prevalent with leaky microvessels, as characteristic opening of the interendothelial tight junctions and by enhanced pinocytotic activity seen in the damaged BBB. Studies show that lead is a substrate for the divalent metal transporter 1 (DMT1/ NRAMP2/ DCT1), and Pb2+ has a higher transport affinity than Fe2+. Data suggest that lead may interfere with iron homeostasis through pathways specific intended for iron or the substitute for Fe2+ to alter the function of iron-binding protein.In the present study, we overcome the limitations of the in vivo research and try to explore the mechanism of the transcellular transportation of lead and the relationship between the transportation and cellular iron homeostasis. This study would offer the theoretical basis for elucidating the molecular mechanism of lead transport in the BBB and taking some effective measures to control lead toxicity. Aim:The aim of the present study was, by co-culture ECV304 cells and C6 cells to establish BBB model in vitro, investigate the possible relationship between transcellular transport of lead and cellular iron homeostasis, and further explore the possible pathway of lead-induced transcellular transport and its regulatory mechanism. Methods:1. A model of transwell was developed for the co-culture ECV304 cells and C6 cells to established BBB model in vitro, and the BBB restrictive characteristic was assessed by permeability of FITC-labeled dextran and measurement of transendotheilal electrical resistance (TEER).2. The electrothermal atomization atomic absorption spectrometer was employed to measure the characteristic of lead transport kinetics, the intracellular distribution of DMT1 (IRE) protein was analysed by immunocytochemical method, and deferoximine (DFX), inducing iron deficiency to study the effects of lead on cellular iron homeostasis, was used.3. Co-culture ECV304 cells transfected with antisense oligonucleotides to DMT1and pcDNA3.0-DMT1 vector respectively and C6 cells was used to establish BBB model in vitro respectively, and then the role of DMT1 (IRE) protein in lead trafficking was investigated.4. The expression of IRP1 and p-ERK1/2 were analysed by western blot method, the interaction between IRP1 and p-ERK1/2 was identified by co-immunoprecipitation and immunocytochemical methods, the role of the interaction between IRP1 and p-ERK1/2 on the regulation of DMT1 (IRE) protein was investigated, and the effect of PD98059 on lead transportation in an in vitro BBB model was evaluated.Results:1. An in vitro BBB model by co-culture ECV304 cells and C6 cells was established.Compare to the BBB with only ECV304 cells, TEER value (232.5?cm2) increased significantly in the co-culture. After 4 days of co-culture, apparent permeability coefficiency was significantly lower than that with the only ECV304 cells. Therefore, the in vitro BBB model by co-culture was suitable for the study of molecular changes of endothelial paracellular permeability and transendothelial transport.2. Transportation of lead through the in vitro BBB had the characteristics of active transport.Temperature, pH, time and dose all had effect on the transport of lead. With time passed, the transport of lead was significantly increased. The transport of lead was also significantly increased in high dose than that in low dose. Furthermore, the transport of lead was temperature-dependent and transport was higher at 37°C than at 4°C. Those results suggested that transport of lead might be characterized with active transport.3. Abnormal changes of the BBB permeability was mainly caused by the enhanced transcellular transport of lead.After 24 h, treatment of 1μM or 5μM lead could not significantly affect the TEER and apparent permeability coefficiency compared with the control (p>0.05). Treatment of 1μM or 5μM lead had no significant effect on claudin-1、ZO-1and occludin mRNA at four time points (2 h, 4 h, 10 h, 24 h). Treatment of lead enhanced the expression of DMT1 (IRE) protein, especially in the membrane fraction. At different time points, lead caused the increased expression of DMT1 (IRE), especially in the membrane. Results suggested that there was a correlation of enhanced transport of lead and transcellular transport of the BBB. 4. Lead-induced transcellular transport was related with the expression of DMT1 (IRE) protein and its localization in the membrane.Treatment with 30μM FeCl3 had no significant influence on the transport of lead (p>0.05), while treatment with 150μM FeCl3 could decrease the transport of lead at 4 h and 24 h (p<0.05). Both lead and DFX increased the expression of DMT1 (IRE) protein in whole cell and membrane fraction. Iron inhibited the overexpression of DMT1 (IRE) induced with lead. Iron and/or lead did not affect the expression of DMT1 (IRE) mRNA. Antisense oligonucleotides (MA1) produced significant inhibition of the expression of DMT1 (IRE) protein. In an in vitro BBB model, MA1 treatment inhibited lead transport at all time points, and significant inhibition occurred only at 10 h (p<0.05). The recombined plasmids (pcDNA3.0-DMT1) were transfected into ECV304 cells. Cells were subjected to 200μg/ml of G418 antibiotic selection to obtain stable transfected cells. In an in vitro BBB model, stable transfected cells inhibited lead transport at all time points, and significant inhibition occurred only at 4 h and 10 h (p<0.05). There results suggested that DMT1 was the carrier of lead transport and changes of iron homeostasis were mainly caused by the enhanced lead transport.5. The interaction between IRP1 and p-ERK1/2 negatively regulated the expression of DMT1 (IRE) protein.The expression of IRP1 protein was increased by lead in the membrane and cytoplasm fractions at 2 h, and gradually declined with time passing. Compared to the control, lead significantly enhanced phosphorylation of ERK1/2 in the whole cell, but reduced phosphorylation of ERK1/2 in the cytoplasm fraction. Both lead and DFX increased the expressions of IRP1 and p-ERK1/2 proteins. Iron inhibited the overexpression of IRP1 and p-ERK1/2 induced by lead. Confocal microscopy was used to analyze ECV304 cells stained for IRP1 and p-ERK1/2 distribution. Results demonstrated that there was a cytoplasmic distribution of IRP1 as well as the co-localization with p-ERK1/2. Both lead and DFX could reduce the co-localization with p-ERK1/2, but iron increased the area of the co-localization. Results of co-immunoprecipitation showed that lead increased the interacted portion of IRP1, and decreased the interacted portion of p-ERK1/2. PD98059 could increase the expression of DMT1 (IRE) and cytoplasmic IRP1 proteins. In an in vitro BBB model, PD98059 treatment increased lead transport at all time points, and the significant increase occurred only at 10h (p<0.05). These results suggested that the interaction between IRP1 and p-ERK1/2 negatively regulated the expression of DMT1 (IRE) protein.Conclusions:1. DMT1 (IRE) is important in lead transcellular transport in an in vitro BBB model. 2. DMT1 (IRE) is a carrier of lead into brain, and it’s localization in the membrane plays an important role in lead transcellular transport.3. DMT1 (IRE) mRNA is regulated through stabilization by the IRE-IRP system.4. There exists an interaction between IRP1 and p-ERK1/2 in ECV304 cells, regulating the expression and function of IRP1 protein.5. ERK inhibitor, PD98059, increases an expression of DMT1, enhancing the lead transport in an in vitro BBB model.更多还原