雌激素及雌激素受体α减轻谷氨酸对神经细胞的损伤  

Effects of estrogen and estrogen receptor α on neuronal injury induced by glutamate

作　　者：朱加应[1] 张广云 王建怡[3] 袁平[3] 胡晓[1] 

机构地区：[1]贵州省人民医院神经内科,贵州贵阳550002 [2]临沂市妇幼保健院儿科,山东临沂276000 [3]贵州省人民医院小儿内科,贵州贵阳550002

出　　处：《中国病理生理杂志》2014年第1期121-127,共7页Chinese Journal of Pathophysiology

基　　金：贵州省科技厅攻关项目(No.黔科合sy[2009]3054);贵州省优秀科技教育人才省长专项资金资助项目(No.黔省专合字[2010]86)

摘　　要：目的:建立谷氨酸诱导的神经细胞损伤模型,观察雌激素及雌激素受体α(estrogen receptorα,ERα)对谷氨酸诱导的神经细胞损伤的作用。方法:原代培养小鼠大脑皮层神经细胞,神经元特异性烯醇化酶(neuron-specific enolase,NSE)免疫组化染色鉴定神经元的纯度。建立谷氨酸诱导的神经细胞损伤模型。用前期实验中已构建成功的ERα重组慢病毒(V-ERα-RFP-flag)感染经谷氨酸诱导的神经细胞。实时荧光定量PCR和Western blotting检测ERαmRNA和蛋白表达水平。实验分为3组:(1)对照组:用空慢病毒(V-RFP-flag)感染经谷氨酸诱导的神经细胞;(2)雌激素组:用雌激素干预经谷氨酸诱导的神经细胞;(3)慢病毒组:用V-ERα-RFP-flag感染经谷氨酸诱导的神经细胞。流式细胞术检测各组神经细胞凋亡率;实时荧光定量PCR及免疫荧光染色检测各组神经细胞中N-甲基-D-天冬氨酸受体1(N-methyl-D-aspartate receptor 1,NMDAR1)及囊泡膜谷氨酸转运体蛋白1(vesicular glutamate transporter protein 1,VGLUT1)的变化。结果:成功原代培养神经细胞,经NSE免疫组化方法鉴定神经元纯度大于90%。成功建立经谷氨酸诱导的神经细胞损伤模型。MOI=7的V-ERα-RFP-flag感染神经细胞72 h后,在荧光显微镜观察下可见到红色荧光表达,与对照病毒相比,能增加神经细胞中ERαmRNA和蛋白表达水平(P<0.01)。与对照组相比,雌激素组和慢病毒组细胞凋亡率降低(P<0.05),且实时荧光定量PCR结果显示,雌激素组和慢病毒组的NMDAR1和VGLUT1 mRNA表达降低(P<0.05),免疫荧光实验结果提示NMDAR1和VGLUT1阳性的细胞数减少(P<0.01)。结论:雌激素和ERα能减轻谷氨酸对神经细胞的损伤效应,该保护作用可能通过抑制NMDAR1和VGLUT1的表达来实现。AIM： To observe the effects of estrogen and estrogen receptor ot （ERot） on the neuronal damage induced by glutamate （Glu）. METHODS ： Primarily cultured mouse neurons were used in the study. The model of neuro- nal injury was established by Glu induction. The neuron-specific enolase （NSE） was identified by the immunohistochemical method to determine the purity of the neurons. The recombinant lentiviral vector containing ERa （V-ERot-RFP-flag） was constructed. Real-time quantitative PCR and Western blotting were used to detect the mRNA and protein expression of ERct in the neurons. The neurons were divided into 3 groups： （ 1 ） control group： empty lentivirus （V-RFP-flag） was used to in- fect the injured cells ; （2） estrogen group ： estrogen intervention was applied to the injured ceils; （ 3 ） lentivirus group ： V- ERct-RFP-flag was used to infect the injured cells. The apoptosis of the neurons was detected by flow cytometry. The chan- ges of N-methyl-D-aspartate receptor 1 （ NMDAR1 ） and vesicular glutamate transporter protein 1 （ VGLUTI ） were deter- mined by the methods of real-time quantitative PCR and immunofluorescence. RESULTS ： The neurons were primary cul- tured with the purity of over 90%. The injury model of the neuron was successfully induced by Glu. V-RFP-flag and V- ERct-RFP-flag （carrying red fluorescence） at MOI =7 were used to infect the neurons. After 72 h, the red fluorescence was observed significantly under a fluorescence microscope. Compared with the control lentivirus, V-ERa-RFP-flag signifi- cantly enhanced the mRNA and protein levels of ERot. Compared with control group, the apoptotic rates of the neurons in estrogen a＇rouD and lentivirus ~rouD were decreased, and the mRNA expression of NMDAR1 and VGLUT1 in estrogen ~roupand lentivirus group was reduced. The numbers of NMDAR1 and VGLUT1 positive cells were also decreased. CONCLU- SION： Estrogen and ERa reduce Glu-induced damage in the neurons by inhibiting the expression of NMDAR1

关 键 词：雌激素 雌激素受体仪 谷氨酸 神经元 

分 类 号：R741.05[医药卫生—神经病学与精神病学]