摘要
背景:华中五味子酮有强的抗氧化活性,可高效清除羟自由基和超氧阴离子,对处于活性氧应激状态下的细胞具有保护作用。神经元内神经原纤维缠结主要是由异常磷酸化的Tau蛋白组成,与阿尔茨海默病的严重程度呈正相关。目的:探讨五味子酮在APP转基因小鼠神经干细胞诱导分化成神经细胞的过程中对Tau蛋白磷酸化的作用。设计、时间及地点:细胞学体外对照观察,于2006-07/2008-10在郑州大学医学实验中心完成。材料:自中国协和医科大学遗传动物中心购得APP基因突变小鼠30只,五味子酮由河南省中医研究院药物所提供。方法:剪取新生APP转基因小鼠尾尖,常规提取DNA,采用PCR法检测动物是否携带有APP基因,APP转基因阳性鼠记为"APP+",APP转基因阴性鼠记为"APP-"。分别取APP+鼠和APP-鼠海马部位脑组织,体外分离培养神经干细胞,并将纯化的神经干细胞向神经细胞方向诱导分化。将从APP+鼠来源的细胞分为五味子酮组、APP+细胞对照组,将APP-鼠来源的细胞设为APP-细胞对照组。五味子酮组向细胞培养液中加入终浓度为50μmol/L的五味子酮溶液,余2组加入相同容积的PBS,培养24h。主要观察指标:免疫荧光化学和免疫印迹分析测定APP+、APP-细胞Tau蛋白在262、396位点的磷酸化水平。结果:与APP+细胞对照组比较,五味子酮组细胞荧光强度减轻,Tau[Ps262]、Tau[Ps396]的磷酸化水平均降低,且Tau[Ps396]位点的磷酸化水平降低尤为明显。APP-细胞对照组Tau[Ps262]、Tau[Ps396]磷酸化水平均较低。结论:在APP转基因小鼠神经干细胞诱导分化过程中,五味子酮可以明显降低Tau蛋白在396、262位点的磷酸化水平,减轻神经元损伤,但其磷酸化程度并未达到阴性细胞水平。
BACKGROUND： Schisandrone has strong activities of anti-oxidation, effectively scavenges hydroxyl radical and superoxide anion, and has protective effects on cells under active oxygen stress state. Neurofibrillary tangles in neurons is composed of abnormal phosphorylation Tau protein and positively correlated with severity of Alzheimer disease.
OBJECTIVE： To explore the effect of schisandrone on tau protein phosphorylation in the process of differentiation of APP transgenic mouse neural stern cells into neural cells. DESIGN, TIME AND SETTING： The cytological in vitro controlled study was performed at the Experimental Center for Medicine, Zhengzhou University from July 2006 to October 2008.
MATERIALS： A total of 30 APP gene mutation mice were purchased from Heredity Animal Center, Peking Union Medical College Schisandrone was supplied by Medicine Institute, Henan Institute of Traditional Chinese Medicine.
METHODS： DNA was extracted from tails of newborn transgenic mouse to check for APP gene by PCR. APP positive mice were considered as APP~, and APP negative mice as APP-. Brain tissues were obtained from the hippocampus of APP＋ mice and APP mice. Neural stem cells were separated from above-mentioned brain tissues, and were cultured and induced to differentiate into neural cells in vitro. The differentiated cells from APP positive mice （APP＋） were divided into 2 groups： APP＋ cell control group and schisandrone group, and cells from APP negative transgenic mice were assigned as APP cell control group. Ceils in the schisandrone group were treaded with 50μmol/L schisandrone for 24 hours. Cells in the APP＋ and APP- cell control groups were incubated in phosphate buffered saline for 24 hours.
MAIN OUTCOME MEASURES： Western blotting and Immunofluorence staining were used to detect the level of Tau protein phosphorylation with 262, 396 site of tau in APP＋ cells and APP cells.
RESULTS： Compared with the APP＋ cell control group, cell fluorescence intensity became weak in the schisan
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第23期4490-4494,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
河南省科技厅自然科学基金项目(0611043000)~~
