Exendin-4对成年小鼠脑室下区神经干细胞分化作用的体外研究*

doi:10.12047/j.cjap.5848.2019.055

中国应用生理学杂志 ›› 2019, Vol. 35 ›› Issue (3): 262-267.doi: 10.12047/j.cjap.5848.2019.055

Exendin-4对成年小鼠脑室下区神经干细胞分化作用的体外研究^*

赵飞^{1, 2}, 徐会友², 马柯², 江继鹏², 张健², 代晨², 靳颖², 李平², 孙洪涛², 王振国^2△, 陈旭义^2△

1. 锦州医科大学研究生院, 辽宁锦州 121000;
2. 武警特色医学中心脑科中心, 武警部队脑创伤与神经疾病研究所, 天津市神经创伤修复重点实验室, 天津 300162

出版日期:2019-05-28 发布日期:2019-06-28
通讯作者: Tel: 18920677110; E-mail: chenxuyi1979@sina.com.cn, wangxinwaike@163.com
基金资助:
国家重点研发计划 (2016YFC1101500);国家自然科学基金(11672332,11102235);天津市科技支撑重点项目(17YFZCSY00620);天津市自然科学基金项目(15JCYBJC28600, 17JCDJC5400);天津市救援医学临床中心基金(15ZXLCSY00040)

Effects of Exendin-4 on the differentiation of neural stem cells from subventricular zone of adult mice in vitro

ZHAO Fei^1,2, XU Hui-you², MA Ke², JIANG Ji-peng², ZHANG Jian², DAI Chen², JIN Ying², LI Ping², SUN Hong-tao², WANG Zhen-guo^2△, CHEN Xu-yi^2△

1. Graduate School of Jinzhou Medical University, Jinzhou 121000;
2. Center for Neurology and Neurosurgery of People’s Armed Police Forces Medical Center, Tianjin 300162, China

Online:2019-05-28 Published:2019-06-28

摘要/Abstract

摘要： 目的:研究艾塞那肽(Ex-4)对成年小鼠脑室下区(SVZ)神经干细胞(NSCs)分化的影响及机制。方法:提取5周龄C57BL/6J小鼠SVZ的NSCs,100 nmol/L Ex-4处理分化14 d观察细胞形态,用免疫荧光检测巢蛋白(nestin)和胰高糖素样肽-1受体(GLP-1R)的表达。用shRNA敲低GLP-1R,将研究分为四组:对照组,Ex-4组,GLP-1R敲低组,GLP-1R敲低+Ex-4组。100 nmol/L Ex-4处理14 d后免疫荧光标记β-微管蛋白3(β-tublin III)和胶质纤维酸性蛋白(GFAP)并统计β-tublin III阳性细胞比例,Western blot检测环磷腺苷效应元件结合蛋白(CREB)的活化。为进一步研究Ex-4对MAPK和PI3K通路的影响,分别以丝裂原活化蛋白激酶(MAPK)抑制剂U0126 0.07 μmol/L预处理细胞30 min、磷脂酰肌醇-3激酶(PI3K)抑制剂LY294002 50 μmol/预处理细胞2 h,将研究分为: 对照组,Ex-4组,U0126组,U0126+Ex-4组,LY294002组,LY294002+Ex-4组,Western blot检测各组CREB的活化,各组实验独立重复三次。结果:成功从C57BL/6J小鼠SVZ提取NSCs,免疫荧光提示NSCs中nestin以及GLP-1R阳性。相对于对照组,Ex-4组分化为神经元的比例更高。GLP-1R敲低+Ex-4组中神经元比例与对照组基本一致(P＜0.01),β-tublin III阳性的细胞显示出GLP-1R以及CREB活化阳性。Western blot显示Ex-4组中CREB显著活化,GLP-1R敲低+Ex-4组的CERB活化与对照组基本一致(P＜0.01)。U0126+Ex-4组与Ex-4组CERB活化水平一致,LY294002+Ex-4组与对照组CERB活化水平一致(P＜0.01)。 结论:Ex-4通过GLP-1R受体促进成年小鼠SVZ中NSCs分化为神经元,这一作用可能通过PI3K/ CREB通路来实现。

关键词: 脑室下区, 神经干细胞, 艾塞那肽, 细胞分化, PI3K信号通路, C57BL/6J小鼠

Abstract: Objective: To study the effect of exendin-4(Ex-4) on the differentiation of neural stem cells(NSCs) in adult mouse subventricular zone(SVZ)and its mechanism . Methods: NSCs in the SVZ were derived from 5-week C57BL/6J mice and the expression of nestin was detected by immunofluorescence. The cell morphology was observed after the cells treatmed with 100 nmol/L Ex-4 for 14 days.The expressions of nestin and glucagon-like peptide-1 receptor (GLP-1R) were detected by immunofluorescence. GLP-1R was knocked down by using shRNA and the study was divided into four groups: control group, Ex-4 group, GLP-1R knockdown group, GLP-1R knockdown + Ex-4 group. After treatment with 100 nmol/L Ex-4 for 14 d, β-tublin III and glial fibrillary acidic protein (GFAP) were labeled by immunofluorescence and then the proportion of β-tublin III positive cells were counted. Western blot was used to detect the activation of cAMP-response element binding protein (CREB) in NSCs. In order to further study the effects of Ex-4 on mitogen-activated protein kinase(MAPK) and phosphatidylinositol 3-hydroxy kinase (PI3K) pathways, the cells were pretreated with MAPK inhibitor U0126 at a concentration of 0.07 μmol/L for 30 min or PI3K inhibitor LY294002 at 50 μmol for 2 h, respectively. The study was divided into six groups: control group, Ex-4 group, U0126 group, U0126 + Ex-4 group, LY294002 group, LY294002 + Ex-4 group. The activation of CREB in each group was detected by Western blot. The experiment was repeated three times independently. Results: NSCs were successfully extracted from SVZ of C57BL/6J mice. Immunofluorescence showed that nestin and GLP-1R were positive in NSCs. Compared with the control group, the proportion of neurons differentiated from Ex-4 group was higher. The percentage of neurons in GLP-1R knockdown + Ex-4 group was basically the same as that in control group (P＜0.01). The positive cells of beta-tublin III showed positive activation of GLP-1R and CREB. Western blot showed that CREB was significantly activated in the Ex-4 group, and knockdown of GLP-1R abolished its activation (P＜0.01). U0126 did not affect Ex-4-mediated CERB activation, and LY294002 significantly reduced Ex-4-mediated CREB activation (P＜0.01). Conclusion: Ex-4 promotes the differentiation of NSCs into neurons in SVZ of adult mice through GLP-1R receptor, which may be achieved through PI3K/CREB pathway.

Key words: subventricular zone, neural stem cells, exendin-4, cell differentiation, PI3K signaling pathway, C57BL/6J mice

赵飞, 徐会友, 马柯, 江继鹏, 张健, 代晨, 靳颖, 李平, 孙洪涛, 王振国, 陈旭义. Exendin-4对成年小鼠脑室下区神经干细胞分化作用的体外研究^*[J]. 中国应用生理学杂志, 2019, 35(3): 262-267.

ZHAO Fei, XU Hui-you, MA Ke, JIANG Ji-peng, ZHANG Jian, DAI Chen, JIN Ying, LI Ping, SUN Hong-tao, WANG Zhen-guo, CHEN Xu-yi. Effects of Exendin-4 on the differentiation of neural stem cells from subventricular zone of adult mice in vitro[J]. CJAP, 2019, 35(3): 262-267.

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Exendin-4对成年小鼠脑室下区神经干细胞分化作用的体外研究^*

Effects of Exendin-4 on the differentiation of neural stem cells from subventricular zone of adult mice in vitro

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