胞二磷胆碱对原代培养的中脑多巴胺能神经元的保护作用及其机制  

NEUROPROTECTIVE EFFECTS AND MECHANISM OF CITICOLINE ON PRIMARY CULTURED DOPAMINERGIC NEURONS OF MESENCEPHALON

作　　者：姜晓燕[1] 龚平生[2] 张卫民[3] 王志成[1] 李晶[4] 赵刚[1] 龚守良[1] 

机构地区：[1]吉林大学公共卫生学院卫生部放射生物学重点实验室 [2]吉林大学分子酶学工程教育部重点实验室,长春130021 [3]吉林省卫生厅卫生监督所,长春130062 [4]吉林大学第一临床医院神经内科,长春130021

出　　处：《神经解剖学杂志》2006年第5期511-516,共6页Chinese Journal of Neuroanatomy

基　　金：科技部"中国-奥地利"科技合作项目2004-2006ⅦA15

摘　　要：为了研究胞二磷胆碱(citicoline,CC)对6-羟多巴胺(6-OHDA)诱导的Parkinson病体外细胞模型中的多巴胺能神经元的保护作用及其机制,本研究采用MTT法检测细胞活力;应用Fluo3/AM荧光染料,并通过流式细胞仪检测细胞内钙离子[Ca2+]i浓度;罗丹明123检测线粒体膜电位(ΔΨm);罗丹明123和PI荧光双染,在荧光显微镜下观察细胞膜通透性和细胞凋亡;乳酸脱氢酶(LDH)试剂盒检测培养上清中LDH的漏出;TH免疫组化染色鉴定多巴胺能神经元。实验分成三组:(1)对照组:即原代培养细胞;(2)6-OHDA损伤组:即原代培养细胞加6-OHDA;(3)CC保护组:原代培养细胞中分别加2、1、0.1、0.01和0.001mmol/LCC后,再加6-OHDA。结果显示:与6-OHDA组相比,1、0.1、0.01和0.001mmol/LCC组细胞活力增加(P<0.01);[Ca2+]i浓度下降和ΔΨm升高(P<0.01);除了0.001mmol/LCC组外,各CC组LDH漏出率明显低于6-OHDA组(P<0.01)。以上结果提示:在体外Parkinson病细胞模型中,CC可能通过保护神经元细胞膜,提高线粒体膜电位,增加细胞活力,降低[Ca2+]i浓度,减少LDH漏出等途径削弱6-OHDA的神经毒性作用,发挥其对神经元的保护作用。To study the ncuroprotective effects and mechanism of citicoline （CC） on the toxicity induced by 6-hydroxydopamine （6-OHDA） towards dopantinergic meseneephalie neurons in primary culture of Parkinson＇s disease （PD） cell model in vitro, the rate of cell viability was evaluated with （3-（4,5-dimcthylthioazol-2-yl）-2,5-diphenyl-tetrazolium bromide （MTT） assay; the concentrations of intracellular free Ca^2＋ （ [ Ca^2 ＋ ] 1 ） was examined by using the fluorescent dye Fluo3/AM and detected by flow cytometer. By measuring thc intracellular Rhodamine123 fluorescence density, mitochondrial membrane potential （ △ψm） was evaluated; and with rhodamine 123 and PI double staining, the cell membrane penetrability and apoptosis were observed by fluorescence microscope. Using a laetate dehydrogenase （LDH） assay kit, the transudatory amount of LDH into the culture medium was measured. The dopaminergic neurons were identified by tyrosine hydroxylase （TH） immunocytochemistry. The experiment was divided by three groups： （ 1 ）control group, making up of primary culture neurons; （2）6-OHDA group, it was composed of primary euhure neurons and 6-OHDA; （3） CC protective group, whieh was composed of primary culture neurons, various concentrations involved 2,1,0.1,0.01 and 0. 001 mmol/L CC and 6-OHDA. The results showed that the late of cell viability of neurons in group 1,0.1,0.01 and 0. 001 mmol/L CC were increased as compared with 6-OHDA （P 〈 0.01 ）, the concentrations of [ Ca^2＋]1 were decreased and the percentage of △ψm were increased （P 〈 0.01 ）. The transudatory amount of LDH in CC group （ except 0. 001 mmol/L CC group） was lower than that of 6-OHDA group. The present results suggest that CC showed a nero,）protective effect by protecting cell membrane,elevating the level of △ψm, increasing the rate of cell viability of neurons, decreasing the concentrations of [ Ca^2 ＋ ]1 and the transudatory amount of LDH to reduce neurotoxic effect of 6-OHDA on do

关 键 词：胞二磷胆碱 6-羟多巴胺 多巴胺能神经元 PARKINSON病 神经保护 原代培养细胞 

分 类 号：R742.5[医药卫生—神经病学与精神病学]