肖志新 宋立梅 谢俊霞 徐华敏
[摘要]目的 探究枸橼酸铁铵(FAC)、含铁铁蛋白(Ferritin)和去铁铁蛋白(Apoferritin)对原代培养的腹侧中脑神经元中单胺囊泡转运蛋白-2(VMAT-2)和多巴胺转运蛋白(DAT)表达的影响。方法 以FAC、Ferritin和Apoferritin处理原代培养的腹侧中脑神经元24 h后,应用蛋白质免疫印迹(Western Blot)方法检测神经元中VMAT-2和DAT的表达情况。结果 FAC、Ferritin和Apoferritin处理的原代腹侧中脑神经元VMAT-2表达较对照组明显降低,差异具有统计学意义(F=4.295,P<0.05),而DAT的表达没有明显变化。结论 FAC、Ferritin和Apoferritin能够降低原代培养的腹侧中脑神经元的VMAT-2表达,而对DAT表达没有明显影响。
[关键词]铁;铁蛋白质类;中脑;神经元;囊泡单胺转运蛋白质类;多巴胺质膜转运蛋白质类
[中图分类号]R338.2
[文献标志码]A
[文章编号]2096-5532(2021)02-0194-04
[ABSTRACT]Objective To investigate the effect of ferric ammonium citrate (FAC), Ferritin, and Apoferritin on the expression of vesicular monoamine transporter 2 (VMAT-2) and dopamine transporter (DAT) in primary cultured ventral midbrain neurons.Methods After primary cultured ventral midbrain neurons were treated with FAC, Ferritin, or Apoferritin for 24 h, Western blot was used to measure the expression of VMAT-2 and DAT in neurons. Results Compared with the control group, the group of primary cultured ventral midbrain neurons treated by FAC, Ferritin, or Apoferritin had a significant reduction in the expression of VMAT-2 (F=4.295,P<0.05), while there was no significant change in the expression of DAT. Conclusion FAC, Ferritin, and Apoferritin can reduce the expression of VMAT-2 in primary cultured ventral midbrain neurons, with no significant effect on the expression of DAT.
[KEY WORDS]iron; ferritins; mesencephalon; neurons; vesicular monoamine transport proteins; dopamine plasma membrane transport proteins
帕金森病(PD)是第二大常見的神经系统退行性疾病,主要临床表现有运动迟缓、静止性震颤、姿势反射障碍等[1-3]。其病理性特征是黑质(SN)多巴胺(DA)能神经元进行性缺失[4]。迄今为止PD的病因尚未完全阐明,越来越多的证据表明,SN铁的过度沉积可能是PD发病的关键因素之一[5-13]。SN铁增加会产生活性氧和活性氮物质,刺激细胞内α-突触核蛋白形成,导致DA能神经元变性[14-15]。在脑内铁主要与铁蛋白结合,铁蛋白是一种可储存多达4 500个铁原子的蛋白质[16-17],有含铁铁蛋白(Ferritin)和去铁铁蛋白(Apoferritin)两种形式[18]。有研究表明,铁蛋白可能不仅是细胞内的铁储存者,也可能是参与组织和全身铁调控的重要因素[19]。但铁蛋白在DA稳态中起的作用尚不明确。
单胺囊泡转运蛋白-2(VMAT-2)是位于DA能神经元突触囊泡的一种膜蛋白,可以将胞浆内游离的DA转运到囊泡内,并调节随后的释放。它通过向单胺能神经元传递DA小泡来保护DA能神经元[20]。多巴胺转运蛋白(DAT)在SN神经元胞体和树突中大量表达[21-23],可将DA从细胞外迅速转运到突触前神经元胞质内,以维持细胞内外DA稳态。但是在高铁以及存在外源性铁蛋白的状态下,原代培养腹侧中脑神经元中VMAT-2和DAT的表达是否改变尚不清楚。本实验旨在探究高铁、Ferritin和Apoferritin对原代培养的腹侧中脑神经元VMAT-2和DAT表达的影响。现将实验结果报告如下。
1 材料与方法
1.1 实验材料
原代培养的腹侧中脑神经元细胞(取自孕14 d的Wistar大鼠的胎鼠腹侧中脑),DMEM/F12培养液、2.5 g/L的胰酶(美国Hyclone公司),B27营养因子、胎牛血清(美国Gibco公司),青霉素-链霉素溶液(100×,中国索莱宝科技有限公司),D-多聚赖氨酸、枸橼酸铁铵(FAC)、Ferritin、Apoferritin(美国Sigma公司),VMAT-2抗体和DAT抗体(中国上海Abcam公司),ECL发光液(Millipore公司)。
1.2 原代腹侧中脑神经元的培养
实验前将实验器械进行高压处理,提前3 h用D-多聚赖氨酸铺培养板,以高压灭菌水清洗3次放置超净台内备用。将孕14 d的Wistar大鼠以联合麻醉药深度麻醉后,用体积分数0.75的乙醇进行腹部消毒,沿中线剪开,取出串珠样胚胎,放置在预冷的DMEM/F12培养液中。将胚胎移至超净台中显微镜下,在冰上操作,剖开胚胎外膜取出胎鼠置于另一装有DMEM/F12培养液的玻璃皿中。使用眼科镊、眼科剪将中脑部分取出,去除端脑及血管膜,修剪组织留出蝴蝶状的腹侧中脑,将其转移至含预冷DMEM/F12培养液的玻璃皿中。去除DMEM/F12培养液,加入37 ℃预温的2.5 g/L胰酶,在培养箱中消化5 min。加入含有胎牛血清的终止液终止消化。用移液器将组织吹打成单细胞悬液,用蓝枪头吹打约10次,收集单细胞悬液至50 mL离心管中,后套用黄枪头和白枪头重复上述操作。将装有单细胞悬液的离心管以1 000 r/min离心5 min。弃上清,加入含有B27和双抗的DMEM/F12培养液,用吸管吹打成细胞悬液,以2×108/L的密度种板。此后每隔2 d换1次液,第6天细胞成熟可用于实验。
1.3 实验分组及处理
实验分为对照组、FAC处理组、Ferritin处理组和Apoferritin处理组,将原代培养的腹侧中脑神经元培养液换成DMEM/F12基础培养液,对照组细胞只用DMEM/F12基础培养液孵育,FAC处理组细胞加入100 μmol/L的FAC,Ferritin处理组细胞加入80 μmol/L的Ferritin,Apoferritin处理组细胞加入50 μmol/L的Apoferritin。将各组细胞置于37 ℃、含体积分数0.05 CO2的培养箱中孵育24 h。
1.4 VMAT-2和DAT的蛋白免疫印迹(Western Blot)检测
在6孔板中加入100 μL蛋白裂解液,冰上裂解30 min,用刮板将板底的细胞刮下,用移液器转移至1.5 mL的EP管中,在4 ℃下以12 000 r/min离心20 min。用移液器吸取80 μL上清至另一EP管中,使用BCA试剂盒检测上清中蛋白质浓度,加入5×Loading Buffer,95 ℃煮5 min。按照BCA试剂盒检测的蛋白浓度计算SDS-PAGE凝胶电泳的上样量。加入样品后,调节电压至80 V,待样品进入分离胶后,将电压调至120 V,电泳之后将蛋白转至PVDF膜上,以300 mA湿转90 min,切下所需分子量的条带,用含50 g/L脱脂奶粉的TBST室温封闭2 h后,再加入用含50 g/L脱脂奶粉的TBST稀释的VMAT-2抗体(稀释度为1∶1 000)、DAT抗体(稀释度为1∶1 000)和β-actin抗体(稀释度为1∶10 000)孵育相应的条带,4 ℃摇床过夜。用TBST洗条带3次,每次10 min,加入HRP偶联的山羊抗兔二抗(稀释度为1∶10 000),室温孵育1 h,孵育完成后以TBST洗3次,每次10 min。加ECL发光液避光孵育1 min显影。利用Image J软件对条带进行分析,以目的条带与内参照条带的比值作为目的蛋白的相对含量。
1.5 统计学处理
应用GraphPad Prism 5.0软件进行统计学处理,计量资料结果以x2±s的形式表示,多组比较采用单因素方差分析(One way ANOVA检验),并继以Tukey法进行组间两两比较,以P<0.05为差异有统计学意义。
2 结 果
2.1 FAC、Ferritin和Apoferritin对原代培养的腹侧中脑神经元VMAT-2表达的影响
对照组、FAC处理组、Ferritin处理组和Apoferritin处理组细胞内的VMAT-2蛋白表达水平分别为1.175±0.075、0.938±0.044、0.945±0.046和0.921±0.062(n=17)。与对照组相比,FAC处理组、Ferritin处理组和Apoferritin处理组VMAT-2蛋白表达水平明显降低,差异具有统计学意义(F=4.295,q=3.952~4.370,P<0.05),而Ferritin处理组与Apoferritin處理组相比差异无显著意义(q=0.417,P>0.05)。
2.2 FAC、Ferritin和Apoferritin对原代培养的腹侧中脑神经元DAT表达的影响
对照组、FAC处理组、Ferritin处理组和Apoferritin处理组细胞内DAT蛋白表达水平分别为0.910±0.055、0.931±0.679、0.937±0.056、0.958±0.088(n=12),各组间比较,差异均无显著性(F=0.084,q=0.297~0.706,P>0.05)。
3 讨 论
PD是全球第二大神经退行性疾病,其发病与年龄老化、氧化应激、炎症反应、环境因素、遗传因素等有关。越来越多的证据表明,SN铁过度沉积参与了PD的发病,过多的铁激活的小胶质细胞会释放大量的神经炎性因子,增加了DA能神经元的变性[24]。在脑内,铁主要与铁蛋白结合,铁蛋白两种亚型以互补的方式储存细胞内的铁[25]。
有研究提出DA作用的区域概念,VMAT-2和DAT在调节这些区域之间DA转移中起着核心作用[26-27]。VMAT-2将突触前神经元合成的DA摄取到突触囊泡中,释放到突触间隙,从而作用到相应的受体。而DAT可以将突触间隙的DA重新摄取到突触前神经元。它们共同调节DA活性,从而改变DA的含量[28]。本文研究结果显示,给予高铁会导致原代培养的腹侧中脑神经元的VMAT-2表达降低。有实验研究表明,当VMAT-2水平降低约95%时,小鼠表现为DA稳态失调,而且神经元对多种有毒化合物的敏感性增强[29-34]。这提示高铁导致的VMAT-2表达降低可能影响了DA的释放,从而引起DA稳态失调。本文结果还显示,给予铁蛋白后,细胞VMAT-2的表达也会下降,说明腹侧中脑神经元VMAT-2的表达变化不是铁依赖性的,铁蛋白和铁均可通过影响VMAT-2的表达参与DA稳态调控。Western Blot结果显示,经高铁及铁蛋白处理的腹侧中脑神经元DAT的表达没有发生明显变化,提示高铁及铁蛋白并不影响DA重新摄取到突触前神经元。以上结果提示,高铁及铁蛋白能够减少突触前神经元的DA释放,但并不影响DAT介导的DA重新摄取,这就导致了DA的稳态失调,可能增加了神经元对有毒物质的敏感性。本实验以原代细胞为模型,更好地探究了高铁诱导的DA代谢和转运情况,为开发影响DA稳态的铁螯合剂提供了新的依据,也为PD的治疗提供了新的思路。
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(本文編辑 马伟平)