国际脑血管病杂志
國際腦血管病雜誌
국제뇌혈관병잡지
INTERNATIONAL JOURNAL OF CEREBROVASCULAR DISEASES
2013年
1期
41-46
,共6页
张赛%杨颖%李明月%陶玉倩
張賽%楊穎%李明月%陶玉倩
장새%양영%리명월%도옥천
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
神經元%細胞凋亡%信號素3A%燐脂酰肌醇3-激酶類%原癌基因蛋白質c-akt%原癌基因蛋白質c-bcl-2%細胞,培養的%大鼠
신경원%세포조망%신호소3A%린지선기순3-격매류%원암기인단백질c-akt%원암기인단백질c-bcl-2%세포,배양적%대서
Neurons%Apoptosis%Semaphorin-3A%Phosphatidy linositol 3-Kinases%Proto-Oncogene Proteins c-akt%Proto-Oncogene Proteins c-bcl-2%Cells,Cultured%Rats
目的 探讨外源性信号素3A(semaphorin 3A,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,PI3K)/丝氨酸-苏氨酸蛋白激酶(serine/threonine kinase,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、1 000和2 000 μg/ml)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、1 000、2 000 μg/ml Sema3A组细胞存活率分另为对照组的(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、1 000、2 000μg/ml Sema3A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.ooo).TUNEL染色显示,正常对照组及500、1 000、2 000 μg/ml Sema3A组神经元凋亡率分别为(23.9±1.1)％、(31.9±1.0)％、(40.1±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-Akt(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.
目的 探討外源性信號素3A(semaphorin 3A,Sema3A)對原代培養大鼠皮質神經元凋亡的影響和燐脂酰肌醇-3-激酶(phosphoinositide-3-kinase,PI3K)/絲氨痠-囌氨痠蛋白激酶(serine/threonine kinase,Akt)通路在Sema3A誘導凋亡中的作用.方法 體外培養新生Sprague-Dawley大鼠皮質神經元,併用微管相關蛋白-2免疫熒光染色鑒定.培養大鼠皮質神經元隨機分為正常對照組和不同濃度Sema3A(500、1 000和2 000 μg/ml)組.CCK8法檢測神經元存活率,Hoechst33342染色和TUNEL染色觀察神經元凋亡,蛋白質印跡法檢測P-Akt、Akt和Bcl-2錶達.結果 培養的皮質神經元純度達95％以上.CCK8檢測顯示,500、1 000、2 000 μg/ml Sema3A組細胞存活率分另為對照組的(80.9±5.3)％、(67.5±3.9)％和(50.2±4.4)％(F=165.042,P=0.000).Hoechst33342染色顯示,正常對照組及500、1 000、2 000μg/ml Sema3A組神經元凋亡率分彆為(22.4±1.2)％、(34.0±1.2)％、(39.3±1.4)％和(47.3 ±2.3)％ (F=103.237,P=0.ooo).TUNEL染色顯示,正常對照組及500、1 000、2 000 μg/ml Sema3A組神經元凋亡率分彆為(23.9±1.1)％、(31.9±1.0)％、(40.1±1.5)％和(51.4±3.4)％ (F=103.118,P=0.000).蛋白質印跡法顯示,隨著Sema3A濃度的增高,P-Akt(F=15.959,P=0.001)和Bcl-2(F=18.776,P=0.001)錶達逐漸下調;而Akt錶達無顯著改變(F=0.590,P=0.639).結論 Sema3A主要通過誘導神經元凋亡而降低培養皮質神經元存活率,其機製可能與下調P-Akt和Bcl-2錶達有關.
목적 탐토외원성신호소3A(semaphorin 3A,Sema3A)대원대배양대서피질신경원조망적영향화린지선기순-3-격매(phosphoinositide-3-kinase,PI3K)/사안산-소안산단백격매(serine/threonine kinase,Akt)통로재Sema3A유도조망중적작용.방법 체외배양신생Sprague-Dawley대서피질신경원,병용미관상관단백-2면역형광염색감정.배양대서피질신경원수궤분위정상대조조화불동농도Sema3A(500、1 000화2 000 μg/ml)조.CCK8법검측신경원존활솔,Hoechst33342염색화TUNEL염색관찰신경원조망,단백질인적법검측P-Akt、Akt화Bcl-2표체.결과 배양적피질신경원순도체95％이상.CCK8검측현시,500、1 000、2 000 μg/ml Sema3A조세포존활솔분령위대조조적(80.9±5.3)％、(67.5±3.9)％화(50.2±4.4)％(F=165.042,P=0.000).Hoechst33342염색현시,정상대조조급500、1 000、2 000μg/ml Sema3A조신경원조망솔분별위(22.4±1.2)％、(34.0±1.2)％、(39.3±1.4)％화(47.3 ±2.3)％ (F=103.237,P=0.ooo).TUNEL염색현시,정상대조조급500、1 000、2 000 μg/ml Sema3A조신경원조망솔분별위(23.9±1.1)％、(31.9±1.0)％、(40.1±1.5)％화(51.4±3.4)％ (F=103.118,P=0.000).단백질인적법현시,수착Sema3A농도적증고,P-Akt(F=15.959,P=0.001)화Bcl-2(F=18.776,P=0.001)표체축점하조;이Akt표체무현저개변(F=0.590,P=0.639).결론 Sema3A주요통과유도신경원조망이강저배양피질신경원존활솔,기궤제가능여하조P-Akt화Bcl-2표체유관.
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 Sprague-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 48hours.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 groups 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 group,respectively (F =165.042,P =0.000).Hoechst33342 staining showed that the apoptosis rate in the normal control group and the groups of 500,1 000and 2 000 μg/ml Sema3A were 22.4％ ± 1.2％,34.0％ ± 1.2％,39.3％ ± 1.4％ and 47.3％ ±2.3％,respectively (F =103.237,P =0.000).TUNEL staining showed that the apoptosis rate in the normal control group and the groups 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 gradually,while the expression of Akt had no significant 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-regulation of expressions of P-Akt and Bcl-2.
