寡聚态β-淀粉样肽_(1～42)可通过胶质-炎症反应损伤神经元  

EFFECT OF INFLAMMATORY RESPONSES IN MICROGLIA INDUCED BY OLIGOMERIC β-AMYLOID_(1-42) ON NEURONAL CELLS

作　　者：潘晓东[1] 陈晓春[1] 朱元贵[1] 陈丽敏[1] 张静[1] 

机构地区：[1]福建医科大学附属协和医院神经内科福建省老年医学研究所,福州350001

出　　处：《解剖学报》2008年第6期804-809,共6页Acta Anatomica Sinica

基　　金：福建省科技厅重大课题(2003F009);福建省卫生厅中医药重点课题(WZZ10604)资助项目

摘　　要：目的观察寡聚态β-淀粉样肽1～42(Oligo-Aβ1～42)诱导的小胶质细胞条件培养液对Neuro-2A神经元细胞的影响,探讨炎症介质在胶质介导的神经元损伤中的作用。方法以Oligo-Aβ1～42诱导BV-2细胞,制备小胶质细胞条件培养液,四甲基偶氮唑蓝法(MTT)测定Neuro-2A神经细胞的活力,AO-EB染色荧光显微镜观察计数细胞凋亡和坏死率;酶联免疫吸附试验(ELISA法)测定小胶质细胞培养上清中的肿瘤坏死因子(TNF-α)、白介素1β(IL-1β)及前列腺素E2(PGE2)水平;Griess法检测上清NO水平;免疫印迹法测定小胶质细胞胞质环氧合酶2(COX-2)、诱导型一氧化氮合酶(iNOS)蛋白表达水平。结果Oligo-Aβ1～42诱导的小胶质细胞条件培养液(Aβ-CM)明显引起Neuro-2A神经细胞损伤,MTT法显示神经元活力随Aβ诱导剂量的增加而逐渐下降(P<0.01),并且浓度为1.0μmol/L和5.0μmol/L的Aβ-CM组比同浓度Aβ单纯组神经细胞的存活率更低,分别为(53.75±3.95)和(34.61±2.72)(Aβ-CM组与Aβ单纯组相比P<0.05,P<0.01);联合作用组(Aβ-CM+Aβ)神经细胞存活率下降更明显,两因素方差分析显示,Aβ诱导的小胶质细胞条件培养液与单纯Aβ(1.0μmol/L)有协同作用[F(3,39)=53.16,P<0.001]。AO-EB荧光观察计数显示,低浓度(0.2μmol/L)Oligo-Aβ1～42诱导的小胶质细胞条件培养液可引起神经细胞发生凋亡性损伤,较高浓度(1.0～5.0μmol/L)Oligo-Aβ1～42诱导不仅引起神经细胞凋亡,还引起细胞发生坏死性改变。进一步研究显示,低浓度(0.2～5.0μmol/L)的Oligo-Aβ1～42明显增加小胶质细胞培养上清中TNF-α、PGE2、NO的产量及胞质COX-2和iNOS蛋白表达水平(P<0.05,P<0.01),呈现一定的量效关系。结论低浓度Oligo-Aβ1～42可通过胶质-炎症反应加剧神经元损伤,其作用可能与Oligo-Aβ1～42诱导小胶质细胞产生炎症介质及胞质内COX-2、iNOS蛋白表达水平增高有关。Objective To investigate the effect and possible mechanisms of Oligomeric β-amyloid1-42-induced inflammatory responses in microglia on neuronal cells. Methods BV-2 microglial and Neuro-2A neuronal-like cells were cultured in vitro. Conditioned media from Oligo-Aβ1-42 stimulated BV-2 cells （Aβ-CM） was performed. Neuro-2A cells viability were measured by MTY assay. And the percentage of apoptotic or necrotic neuronal cells were evaluated by epifluorescence microscopy after stained with AO-EB. Tumor necrosis factor-α （TNF-α）, interleukin-1β （IL-1β） and prostaglandin E2 （PGE2） levels were determined by ELISA assay. Nitric oxide （NO） levels were detected by Greiss assay. The levels of inducible nitric oxide synthase （iNOS） and cyclooxygenase-2 （COX-2） proteins were detected by Western blotting. Results Treatment of neuronal cells with conditioned media from Oligo-Aβ2-stimulated in BV-2 cells （Aβ-CM） resulted in a more dramatic decrease in the viability of Neuro-2A cells when compared to the Oligo-Aβ1-42 alone group when the concentration of induced Oligo-Aβ1-42 was at 1.0 and 5.0tmlol/L （ P 〈 O.O5.P 〈 0.01）. The combined groups（Aβ-CM ＋ Aβ） showed signiflcant interaction between Aβ-CM and Aβ（1.0umol/L） by two-way ANOVA. Further, the percentage of apoptotic and necrotic neuronal cells was increased gradually and significantly with increasing concentrations of Aβ in CM by AO-EB stain assay. Meanwhile, the levels of extracellular TNF-α, IL-1β, PGE2 and NO in conditioned media from Oligo-Aβ1-42 stimulated microglial cells were significantly elevated in a dose-dependent manner（0.2- 5.0μmol/L）. Furthermore, Oligo-Aβ1-42 significantly increased the productions of iNOS and COX-2 protein levels in the microglial cells （ P 〈 0.05 or P 〈 0.01 ）. Conclusion Oligomeric β-amyloid1-42 at a low dosage can aggravate the damage of neuronal cells via elevated levels of inflammatory mediators in microglia.

关 键 词：寡聚态β-淀粉样肽1-42 小胶质细胞 神经元 神经炎症 阿尔茨海默病 条件培养液 小鼠 

分 类 号：R742.5[医药卫生—神经病学与精神病学]