信号素3A诱导培养大鼠皮质神经元凋亡与PI3K／Akt通路有关  

Involvement of PDICAkt pathway in apoptosis induced by semaphorin 3A in cultured rat cortical neurons

作　　者：张赛[1] 杨颖[2] 李明月[1] 陶玉倩[1] 

机构地区：[1]中山大学附属第一医院神经内科,510080 [2]西安交通大学第二附属医院干部病房,710005

出　　处：《国际脑血管病杂志》2013年第1期41-46,共6页International Journal of Cerebrovascular Diseases

基　　金：广东省自然科学基金（52011010002574）;广东省科技计划（20128031800106）

摘　　要：目的探讨外源性信号素3A（semap3A，Sema3A）对原代培养大鼠皮质神经元凋亡的影响和磷脂酰肌醇-3-激酶（phosphoinositide-3-kinase，P13K）／丝氨酸-苏氨酸蛋白激酶（serine／threoninekinase，Akt）通路在Sema3A诱导凋亡中的作用。方法体外培养新生Sprague—Dawley大鼠皮质神经元，并用微管相关蛋白-2免疫荧光染色鉴定。培养大鼠皮质神经元随机分为正常对照组和不同浓度Sema3A（500、1000和2000μ倒）组。CCK8法检测神经元存活率，Hoechst33342染色和TUNEL染色观察神经元凋亡，蛋白质印迹法检测P—Akt、Akt和Bcl-2表达。结果培养的皮质神经元纯度达95％以上。CCK8检测显示，500、1000、2000g／mlSema3A组细胞存活率分别为对照组的（80．9±5．3）％、（67．5±3．9）％和（50．2±4．4）％（F=165．042，P=0．000）。Hoechst33342染色显示，正常对照组及500、1000、2000Sema3A组神经元凋亡率分别为（22．4±1．2）％、（34．0±1．2）％、（39．3±1．4）％和（47．3±2．3）％（F=103．237，P=0．000）。TUNEL染色显示，正常对照组及500、1000、2000Sema3A组神经元凋亡率分别为（23．9±1．1）％、（31．9±1．0）％、（40．14-1．5）％和（51．4±3．4）％（F=103．118，P=0．000）。蛋白质印迹法显示，随着Sema3A浓度的增高，P—Ala（F=15．959，P=0．001）和Bcl-2（F=18．776，P=0．001）表达逐渐下调；而Akt表达无显著改变（F=0．590．P=0．639）。结论Sema3A主要通过诱导神经元凋亡而降低培养皮质神经元存活率，其机制可能与下调P—Akt和Bcl-2表达有关。Objective To investigate the effect of exogenous semaphorin 3A （Sema3A） on apoptosis in primary cultured rat cortical neurons and the roles of phosphoinositide 3-kinase （PI3K）/serine-threonine kinase （Akt） pathway in apoptosis induced by Sema3A. Methods Newborn Spmgue-Dawley rat cortical neurons were cultured in vitro and they were identified by microtubule associated protein-2 （MAP-2） staining The cultured cortical neurons were treated with various concentrations of Sema3A （0, 500, 1 000, and 2 000 μg/ml） for 48 hours. Neuronal survival rate was detected with CCK8 assay. Neuronal apoptosis was detected with Hoechst33342 staining and TUNEL staining. The expressions of P-Akt, Akt and Bcl-2 in cortical neurons were determined with Western blotting Results The purity of cortical neurons culture was more than 95%. CCK8 assay showed that the survival rates of cortical neurons in the oups of 500, 1 000and 2 000 μg/ml Sema3A were 80. 9% ±5. 3%, 67. 5% ±3.9% and 50. 2% ±4. 4% of the control ffoup, respectively （F= 165. 042,P =0. 000）. Hoechst33342 staining showed that the apoptosis rate in the normal control ffoup and the goups ofS00, 1 000and2 000 μg/ml Sema3A were22. 4% ± 1.2%,34.0% ±1.2%,39.3% ±1.4% and47. 3% ± 2. 3%, respectively （F = 103.237 ,P =0. 000）. TUNEL staining showed that the apoptosis rate in the normal control goup and the ffoups of 500, 1 000and 2 000 μg/ml Sema3A were 23.9% ± 1.1%, 31.9% ± 1.0%, 40. 1% ± 1.5% and 51.4% ± 3.4%, respectively （F = 103. 118 ,P = 0. 000）. Western blotting showed that the expressions of P-Akt （F = 15. 959, P =0. 001） and Bcl-2 （F = 18. 776,P =0. 001） decreased gaduaUy, while the expression of Akt had no sifffificant changes （F = 0. 590, P = 0. 639）. Conclusions Sema3A can decrease the survival rate of the cultured cortical neurons, mainly by inducing apoptosis, and the mechanism of which might be related to the down-realation of expressions of P-Akt and Bcl-2.

关 键 词：神经元 细胞凋亡 信号素3A 磷脂酰肌醇3-激酶类 原癌基因蛋白质c-akt 原癌基因蛋白质C-BCL-2 细胞 培养的 大鼠 

分 类 号：R74[医药卫生—神经病学与精神病学]