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

作　　者：赵飞 徐会友 马柯 江继鹏 张健 代晨 靳颖[2] 李平[2] 孙洪涛[2] 王振国 陈旭义[2] 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(Graduate School of Jinzhou Medical University,Jinzhou 121000;Center for Neurology and Neurosurgery of People’sArmed Police Forces Medical Center,Tianjin 300162,China)

机构地区：[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　　处：《中国应用生理学杂志》2019年第3期262-267,I0001,I0002,共8页Chinese Journal of Applied Physiology

基　　金：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)

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,100nmol/LEx-4处理分化14d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。100nmol/LEx-4处理14d后免疫荧光标记β-微管蛋白3(β-tublinIII)和胶质纤维酸性蛋白(GFAP)并统计β-tublinIII阳性细胞比例,Westernblot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U01260.07μmol/L预处理细胞30min、磷脂酰肌醇-3激酶(PI3K)抑制剂LY29400250μmol/预处理细胞2h,将研究分为:对照组,Ex-4组,U0126组,U0126+Ex-4组,LY294002组,LY294002+Ex-4组,Westernblot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-tublinIII阳性的细胞显示出GLP-1R以及CREB活化阳性。Westernblot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。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 C57 BL/6 J 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 for14 days.The expressions of nestin and glucagon-like peptide-1 receptor( GLP-1 R) were detected by immunofluorescence. GLP-1 R was knocked down by using shRNA and the study was divided into four groups: control group,Ex-4 group,GLP-1 R knockdown group,GLP-1 R 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 c AMP-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( PI3 K) pathways,the cells were pretreated with MAPK inhibitor U0126 at a concentration of 0.07 μmol/L for 30 min or PI3 K 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 C57 BL/6 J mice. Immunofluorescence showed that nestin and GLP-1 R 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-1 R knockdown + Ex-4 group was basically the same as that in control group( P<0.01). The positive cells of betatublin III showed positive activation of GLP-1 R and CREB. Western blot showed that CREB was significantly activated in the Ex

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 

分 类 号：R34[医药卫生—基础医学]