不同浓度dbcAMP对SH-SY5Y细胞向γ-氨基丁酸能神经元分化潜能的影响  

Influence of different concentrations of dbcAMP in differentiation potentiality of SH-SY5Y cells to GABAergic-like cells

作　　者：王登莉[1] 周莉[1] 于升[1] 吴江[2] 赵慧[1] 

机构地区：[1]吉林大学基础医学院组织学与胚胎学系,吉林长春130021 [2]吉林大学第一医院神经内科,吉林长春130021

出　　处：《吉林大学学报（医学版）》2014年第5期933-937,I0001,共6页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金资助课题(81171219);吉林大学211工程项目资助课题(2010)

摘　　要：目的:建立具有分化成γ-氨基丁酸能神经元潜能的神经干细胞模型,为γ-氨基丁酸能神经元退行性疾病的研究提供适宜的研究载体。方法:应用双丁酰环腺苷酸(dbcAMP)诱导人神经母细胞瘤细胞(SH-SY5Y细胞),分为0mmol·L-1 dbcAMP组(对照组)和0.3、0.6、1.0及2.0dbcAMP组,观察诱导后各组SH-SY5Y细胞的形态变化;采用Imge-Pro Plus 5.0软件测量神经元样细胞神经突起长度,计算神经突起>30μm细胞所占细胞总数的百分率;采用免疫荧光细胞化学技术检测γ-氨基丁酸能神经元标志性蛋白——谷氨酸脱羧酶65(GAD65)的表达,并计算其免疫反应阳性率。结果:形态学观察,对照组SH-SY5Y细胞呈多边形、圆形或梭型,胞膜光滑,边界清晰;各浓度dbcAMP组随着dbcAMP浓度的增加和诱导时间的延长,SH-SY5Y细胞胞体变小、突起变长;1.0mmol·L-1 dbcAMP组细胞互相交织,表现出成熟神经元的表型。SH-SY5Y细胞诱导培养72h后,与对照组(31.4%±4.2%)比较,0.3、0.6、1.0和2.0dbcAMP组神经突起>30μm细胞所占细胞总数的百分率(40.1%±5.7%、47.5%±6.2%、73.1%±3.2%和74.3%±6.1%)明显升高(P<0.05或P<0.01)。SH-SY5Y细胞诱导培养72h后,与对照组(10.2%±2.1%)比较,0.3、0.6、1.0和2.0dbcAMP组GAD65阳性细胞表达率(22.1%±2.4%、46.9%±3.2%、70.7%±3.4%和72.3%±3.7%)明显升高(P<0.05或P<0.01)。结论:SH-SY5Y细胞具有分化为γ-氨基丁酸能神经元样细胞的潜能,1.0mmol·L-1是dbcAMP的最佳诱导浓度。Objective To establish a nerve stem cell model possessing the potentiality of differentiating into 7-aminobutyric acid neuron-like cells（GABAergic-like cells）, and provide eligible investigative vector for study on GABAergic neurons degenerative diseases. Methods Dibutyryl cyclic adenosine monophosphate （dbcAMP） was applied to induce human neuroblastoma SH-SY5Y cells, and the SH-SYSY cells were divided into control group （0 mmol＂ L-1 dbcAMP）, 0.3 mmol· L-1 dbcAMP group, 0.6 mmol·L-1 dbcAMP group, 1.0 mmol· L-1 dbcAMP group and 2.0 mmol · L-1 dbcAMP group; the morphological changes of SH-SYSY cells were observed.The Image-Pro Plus 5.0 software was used to measure the length of neurites of the neuron-like cells, and the percentage of the SH-SY5Y cells with neurites longer than 30 ±m was calculated. The immunofluoreseence cytochemistry technique was utilized to test GAD65 positive cells, and the positive rate of immune response was calculated. Results The results of light microscope observation showed that the cells in control group were polygonal, circular or shuttle type with smooth membrane and clear boundaries. With the increasing of the concentrations of dbcAMP and the prolongation of time, the morphology of SH-SY5Y cells＇ bodies became smaller with longer processes in dbcAMP groups. The cells interwined each other and showed mature neuron phenotype in 1.0 mmol · L-1 dbcAMP group. Compared with control group（31.4%±4.2%）, the percentages of the cell with neuriteslonger than 30 /tin in 0.3, 0.6, 1.0, and 2.0 dbcAMP groups （40. 1% ± 5.7%, 47.5% ± 6.2%, 73.1%±3.2%, and 74. 3% ± 6.1%） 72 h after induction were significantly increased（P〈0. 05 or P〈0.01）. Compared with control group（10.2% ± 2.1%）, the GAD65 positive expression rates in 0.3, 0. 6, 1.0, and 2.0mmol-L-1, dbcAMP groups（22.1% _--±- 2. 4% , 46.9%=k3.2%, 70.7%=[=3.4%, and 72.3%±3.7%）72 h after induction were significantly increased （P〈 0.05 or P〈 0. 01）. Oonclusion The SH-SY5Y cells have the poten

关 键 词：双丁酰环磷腺苷 人神经母细胞瘤细胞 诱导 分化 

分 类 号：R741[医药卫生—神经病学与精神病学]