中文摘要：

目的：观察T型钙通道阻断剂镍对胚胎神经干细胞的调节，分析T型钙通道在神经干细胞增殖和分化中的作用。 方法：实验于2004—08／2006—01在南方医科大学神经生物学教研室完成。选取清洁级成年雌性Wistar孕鼠1只，分离大脑半球，制成单细胞悬液，利用无血清培养技术，在添加碱性成纤维细胞生长因子、表皮生长因子、B27 supplement的DMEM／F12培养基中培养胚胎神经干细胞，当神经球增大至50～200个细胞／球时，即可传代。取第3代胚胎神经干细胞，在培养基中分别加入0，0．25，0．5，1．0mmol／L的氯化镍，以BrdU免疫荧光、四唑盐、流式细胞仪检测细胞增殖指数以及DCX和S-100β阳性细胞数。 结果：①神经干细胞体外培养的鉴定：取原代和传代培养的细胞进行免疫荧光鉴定．发现均有细胞表达nestin抗原。传至第3代时，其阳性比例可达99％。②诱导分化及鉴定：分化2d后，镜下观察细胞长出突起，已初步具有神经元和胶质细胞形态。免疫荧光染色显示分化细胞呈现DCX和S—100β阳性。③细胞增殖检测结果：加入0.25，0．5，1．0mmol/L的氯化镍处理细胞，MTT检测其吸光度值分别为未加入氯化镍的62．09％，48．20％，34．85％，呈浓度依赖性降低（P均〈0．001）；免疫荧光检测BrdU阳性细胞数分别为未加入氯化镍的85．75％，74．25％，57．01％（P〈0．05）。流式细胞仪检测加入0．5mmol／L氯化镍的单细胞悬液其增殖指数显著高于未加入氯化镍的单细胞悬液（66．11％，1．016％，P〈0．001）。④镍对细胞分化的影响：单细胞悬液加入0．25，0．5，1．0mmol／L氯化镍后，BrdU阳性细胞数量减少（P均〈0．001）；DCX阳性细胞数分别比未加入氯化镍的单细胞悬液增加23％，52％，58％（P〈0．05或0.01）；S—100β阳性细胞数增加85％，105％，110％（P〈0．05或0．01）。 结?

英文摘要：

AIM： To observe the regulation of T-type calcium channel blocker nickel on the embryonic neural stem cells, and further elucidate the function of Ttype calcium channel in the proliferation and differentiation of embryonic neural stem cells. METHODS： The study was carried out in the Department of Neurobiology, Southern Medical University from August 2004 to January 2006. One adult Wistar pregnancy rat of cleaning class were selected to separate the cerebral hemispheres and prepare the monocenular suspension, and the dissociated embryonic neural stem cells were euhured in serum-free DMEM/F12 medium containing epidermal growth factor （EGF）, basic fibroblast growth factor （bFGF） and B27 supplement. The neurosphere would be passaged when it grow to 50-200 cells. After adding 0, 0.25, 0.5 and 1.0 mmol/L nickel chloride in culture medium of the third passage cells, the MTF method, BrdU immunofluorescent double labeling and flow cytometry were used to test the cellular proliferation index and the number of DCX and S-100β positive cells. RESULTS：①Identification of in vitro cultured embryonic neural stem cells： Primary and passaged cells were immunoblotted, and both showed nestin-positive. After three passages, the proportion of nestin-positive cells reached to 99%. ②Identification of differentiated cells： Following two days of culture in differentiation medium, neuron-like and astrocyte-like cells were observed, which expressed DCX and S-100β, respectively. ③Cell proliferation test result： After treatment with 0,25, 0.5 and 1.0 mmol/L nickel chloride, the number of optical density （OD） from MTT test was 62.09%, 48.20% and 34.85% of controls, respectively, presenting concentration-depend decreased manner （P 〈 0.001）; The number of positive-BrdU cells from immunofluorescence test was 85.75%, 74.25% and 57.01% of controls （P 〈 0.05）. Flow cytometry graphs demonstrated that the proliferation index was higher in the monocellular suspension adding 0.5 mmol/L nickel chloride than i