摘 要： 目的 研究铁过载对原代神经元发生衰老的影响。方法 取大鼠原代中脑腹侧神经元进行体外培养，随机分为对照组和实验组，实验组给予100μmol/L枸橼酸铁铵(FAC)处理24 h,对照组用不含FAC的培养液进行处理，通过检测细胞衰老相关β-半乳糖苷酶活性评估细胞衰老状况。结果 与对照组相比，在100μmol/L FAC作用24 h时，实验组原代神经元衰老相关β-半乳糖苷酶活性增加(t=18,P<0.05)。结论 铁过载可诱导原代神经元发生衰老。
关键词 : 神经元;铁;细胞衰老;
Abstract： Objective To investigate the effect of iron overload on the senescence of primary neurons. Methods Primary ventral midbrain neurons of rats were cultured in vitro and were randomly divided into control group and experimental group. The experimental group was treated with ferric ammonium citrate(FAC)(100 μmol/L) for 24 h, and the control group were treated with the medium without FAC. Cell senescence was evaluated based on the activity of β-galactosidase associated with cell senescence. Results Compared with the control group, the experimental group had a significant increase in the activity of β-galactosidase associated with cell senescence after being treated by 100 μmol/L FAC for 24 h(t=18,P<0.05). Conclusion Iron overload can induce the senescence of primary neurons.
Keyword： neurons; iron; cell senescence;
帕金森病是世界第二大神经退行性疾病，典型病理特征是黑质致密带多巴胺能神经元进行性丢失，α-突触核蛋白的聚集和铁沉积[1,2,3]。越来越多的证据表明铁沉积会导致多巴胺能神经元死亡，铁过载是神经元死亡的诱因而非结果[4,5]。除了可以通过氧化损伤途径促进多巴胺能神经元死亡，大量的铁可以促进α-突触核蛋白的表达[6],也可从蛋白翻译后水平影响其磷酸化[7,8],促进蛋白聚积，进而损伤神经元[9,10]。随着年龄增加，多种器官发生铁聚积，脑内铁沉积随之不断加剧[11],过量铁导致的氧化水平升高又会进一步引发铁蛋白释放[12]。细胞衰老是细胞无法进入细胞周期而处于停滞状态，是防止细胞不受调控持续增殖的机制，主要表现为炎性因子的分泌、β-半乳糖苷酶活性增加以及衰老相关分泌表型[13]。随着年龄的增加，衰老细胞在体内逐渐累积，衰老的细胞部分丧失生理活性，影响机体正常生理功能。在主要的神经退行性疾病如阿尔茨海默病、帕金森病以及亨廷顿症病人脑内均发现了衰老的神经元和胶质细胞[14,15]。在疾病状态下的神经元也会出现衰老现象，据文献报道，在γ射线诱导的小鼠胚胎成纤维细胞衰老模型中，衰老细胞的铁水平是正常细胞的30倍之多[16]。另有研究发现，神经毒素百草枯可诱发与帕金森病相关的细胞衰老和神经病理特征[17]。最近有研究表明，诱导胚胎干细胞分化的多巴胺能神经元也观察到衰老表型[18]。已有实验证实，衰老细胞内铁含量明显升高，衰老导致的铁摄取和储存异常会影响铁介导的细胞死亡过程[11,16],衰老的细胞若不及时清除，会进一步损伤周围细胞[19]。为了探讨铁过载能否引发神经元衰老进而引发死亡丢失，本研究用枸橼酸铁铵(FAC)对原代神经元进行处理，检测其衰老发生情况。
1 、材料和方法
1.1、 实验材料
FAC(Sigma),胎牛血清(依科赛(澳洲源)),SA-β-Gal染色试剂盒(CST,9860S),DMEM高糖培养液(Gibco, 1210038)以及DMEM/F12培养液(Hyclone, SH30023.01),D-多聚赖氨酸、B27(Gibco),β-阿糖胞苷、青链霉素混合液、0.01 mol/L磷酸盐缓冲液(PBS)(索莱宝)和倒置显微镜(OLYMPUS,IX73)。
1.2、 实验方法
1.2.1、 中脑腹侧原代神经元培养
将深度麻醉的孕12～14 d的Wistar大鼠用医用乙醇喷洒消毒后，用手术剪沿大鼠的腹中线剪开至腹腔完全暴露，用镊子取出串珠样胚胎，放在预冷的DMEM培养液中，迅速转移至超净工作台上。用尖镊将胎鼠剥离出来，先在解剖显微镜下将中脑组织块取出转移至新鲜的DMEM培养液中，再在镜下修剪掉多余组织块和血管膜，保留蝴蝶状腹侧，加入适量胰蛋白酶，置37 ℃培养箱中孵育消化，5 min后加入终止液终止消化。用1 mL移液枪轻柔地吹打尽量使组织块分散，静置片刻后吸取上清至50 mL离心管中，重新加入新鲜的DMEM培养液5 mL,重复上述步骤3次。将上清以1 000 r/min离心5 min后弃上清，加入含有B27的DMEM/F12完全培养液，轻轻吹打成均匀的单细胞悬液，接种到12孔板上，每孔1×105个细胞，放入细胞培养箱中培养，1 d后更换一半培养液，此后每隔2 d更换新鲜培养液，7 d后成熟的神经元可用于后续实验。经β-阿糖胞苷处理，神经元纯度达90%以上，符合实验要求。
1.2.2 、实验分组及处理
将细胞随机分为对照组和实验组。实验组细胞加入100 μmol/L的FAC作用24 h, 对照组细胞用不含FAC的低血清培养液进行处理。
1.2.3、 细胞衰老相关β-半乳糖苷酶活性检测
应用SA-β-Gal染色试剂盒检测细胞衰老相关β-半乳糖苷酶活性。FAC处理24 h后，用0.01 mol/L PBS润洗细胞2次，每次30 s, 每孔细胞加入1 mL固定液，室温固定15 min, 用0.01 mol/L PBS冲洗细胞2次，每次30 s, 弃掉洗液，每孔加入1.5 mL染色液，用封口膜密封细胞培养板防止染液蒸发，置37 ℃恒温箱中孵育过夜。用0.01 mol/L PBS润洗细胞2次，每次30 s, 在奥林巴斯倒置显微镜明场下观察并获取图像，阳性细胞显示为亮蓝色。
1.3、 统计学分析
应用GraphPad Prism软件进行统计学分析。计量资料结果以x?±s表示，两独立样本比较采用t检验。以P<0.05表示差异有统计学意义。
2、 结 果
与对照组相比较，实验组细胞衰老相关β-半乳糖苷酶阳性染色明显增强(图1),对照组着色细胞比例为(2.077±0.440)%,实验组着色细胞比例为(25.200±1.164)%,两组比较差异具有统计学意义(n=3,t=18,P<0.05)。提示100 μmol/L FAC作用于原代神经元24 h,细胞衰老相关β-半乳糖苷酶活性增加，表明100 μmol/L FAC可以诱导原代神经元发生衰老。
3 、讨 论
铁是维持生命活动的关键微量金属元素之一，在中枢神经系统中发挥重要作用，参与脑内线粒体呼吸、轴突髓鞘化以及神经递质的形成[12]。铁作为活泼金属，也可以催化多种生化反应，增加氧化应激，过量的活性氧损伤细胞膜、核酸及蛋白结构，引发细胞毒性[20]。枸橼酸是铁的天然螯合剂，枸橼酸铁为枸橼酸与铁离子形成的铁盐，是美国食品和药物管理局(FDA)批准的补铁药剂[21]。本实验所用的FAC是枸橼酸铁更具溶解性的形式，添加FAC可模拟高铁环境，以探讨铁过载对神经元细胞衰老的影响。
对于体外培养细胞的衰老研究，衰老相关的β-半乳糖苷酶的活化是常用的生物学特征[22,23]。β-半乳糖苷酶是溶酶体水解酶，通常在pH值为4时表现出活性；而在衰老的细胞中，pH值为6时有活性，利用β-半乳糖苷酶底物进行染色，就能从正常细胞中区分出衰老细胞[24]。本实验采用100 μmol/L FAC处理原代神经元24 h,观察到β-半乳糖苷酶的阳性染色明显增强，提示细胞衰老的发生。
越来越多的证据表明，铁稳态失衡促进神经退行性疾病的发展[25,26,27,28],铁沉积是帕金森病一个主要的病理特征，也是人们普遍认为的发病诱因。有研究证实，细胞内铁过载会增加氧化还原水平[29,30],促进α-突触核蛋白的聚积，进而发挥细胞毒性作用，如二价铁可以通过与α-突触核蛋白mRNA的C端ASP-121、Asn-122、Glu-123位结合，在转录水平促进α-突触核蛋白表达，也可以在转录后水平参与蛋白磷酸化等修饰过程，促进蛋白聚集发挥细胞毒性作用[31,32],并且α-突触核蛋白寡聚过程产生活性氧是铁依赖性的[33]。铁过载增加细胞内氧化应激水平可能是诱导衰老的重要机制。本实验结果表明，铁过载会诱发神经元衰老，这为后续进行神经元变性死亡研究提供了新的思路。但处于衰老状态的神经元细胞是通过何种通路逐渐死亡丢失，神经元衰老与目前已知凋亡、坏死和铁死亡之间是否存在因果或先后关系，衰老在神经元变性死亡过程中的具体机制还有待进一步的研究探讨。
图1 光学显微镜下衰老相关β-半乳糖苷酶染色检查
A:对照组(20倍);B:实验组(20倍);C:对照组(40倍);D:实验组(40倍)。
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