摘要
目的 观察Notch1信号通路对缺血再灌注损伤后神经元细胞增殖与凋亡的影响.方法 将海马神经元细胞分成神经元组（cell组）、空载组为对照组（NC组）、Notch1干扰表达组（si组）和过表达NICD组（Notch1组）.细胞计数试剂盒（CCK-8）法检测各组缺血再灌注损伤前、后24、48和72 h的Notch1基因敏感性CCK-8值,绘制增殖率和抑制率曲线;流式细胞技术检测各组细胞缺血再灌注损伤后的凋亡率;Western blot检测各组缺血再灌注损伤后半胱氨酰天冬氨酸特异性蛋白酶-3（Caspase-3）、B细胞淋巴瘤/白血病-2（bcl-2）、bcl-2相关X蛋白（bax）蛋白表达.结果 缺血再灌注后24、48和72 h后,Notch1组CCK-8值（0.40±0.00、0.46±0.01、0.59±0.01）高于NC组（0.38±0.01、0.42±0.01、0.52±0.01,P＜0.05）与cell组（0.37±0.01、0.41±0.00、0.52±0.01,P＜0.05）;缺血再灌注后24、48和72 h后,si组CCK-8值（0.34±0.00、0.34±0.01、0.45±0.01,P ＜0.05）低于cell、NC组;Notch1、cell和对照组细胞增殖率呈上升趋势,而si组呈下降趋势;Notch1组细胞抑制率呈下降趋势,而si组呈上升趋势;与空载组（4.38±0.31）比较,Notch1组（0.92±0.11,P＜0.05）晚期凋亡细胞和死亡细胞较少;si组凋亡细胞、晚期凋亡细胞和死亡细胞（16.05±1.03、9.23±0.56）比空载组（10.50±1.08、4.38±0.31,P＜0.05）增多,si组（68.97±5.13）活细胞较空载组（81.65±5.62,P＜ 0.05）减少;与空载组（4 474.313±208.000、2406.723±111.000、4 215.677 ±226.000）比较,Notch1组bcl-2（8 623.742±301.000,P＜0.05）蛋白表达增加而Caspase-3、bax（277.0034±25.000、709.949±66.000,P＜0.05）蛋白表达减少;si组与空载对照组比较,bcl-2（1 855.511±166.000,P＜0.05）蛋白表达减少而Caspase-3、bax（5 159.642±296.000、7 143.668±283.000,P＜0.05）蛋白表达增加.结论 神经元细胞缺血再灌注损伤后,Notch1表达与神经元细胞增殖呈正相关,而与神经元细胞凋亡呈负相关.
Objective To investigate the effect of Notch1 signal pathway on the proliferation and apoptosis of neurons induced by ischemia-reperfusion injury.Methods Primary hippocampal neurons were divided into four groups ： neuron cells group （cell group）, no-live load group （NC group）, interference expression of Notch1 group （si group）, and over-expression of NICD group （Notch1 group）.The cell counting kit 8 （CCK-8） was used to detect the sensitivity of Notch1 gene in each group before, and at 24, 48 and 72 h after ischemia-reperfusion injury, respectively.The apoptosis rate in each group after ischemia-reperfusion injury was detected by flow cytometry.The expression of Caspase-3, B cell lymphoma/leukemia-2 （bcl-2） and bcl-2 associated X protein （bax） proteins was detected in each group by Western blotting after ischemia-reperfusion injury.Results The CCK-8 value in Notch1 group at 24, 48 and 72 h after ischemia-reperfusion injury was 0.40 ±0.00, 0.46 ±0.01, and 0.59 ±0.01 respectively, which was significantly higher than that in NC group （0.38 ±0.01,0.42 ±0.01, and 0.52 ± 0.01 ,P 〈0.05） and cell group （0.37 ±0.01,0.41 ±.0.00, 0.52 ±0.01 ,P 〈0.05）.The CCK-8 value in si group at 24, 48 and 72 h after ischemia-reperfusion injury was 0.34 ± 0.00, 0.34 ± 0.01 , and 0.45 ±0.01 respectively, which was significantly lower than that of NC group and cell group （P 〉0.05）.Cell proliferation rate in Notch1, cell, and control groups was increased after the ischemia-reperfusion injury to neurons.However, cell proliferation rate in the si group was decreased.The inhibition rate of cellx in Notch1 group was decreased, but increased in si group after the ischemia-reperfusion injury to neurons.Apoptotic and dead cells in Notch1 group in the later period was less than those of control group （P 〈0.05）.Apoptotic cells, apoptotic cells in the later period, and dead cells in si group were more than those of control group （P 〈 0.05）.However, the living cells in si group were more than those of control group （P 〈 0.05）.The expression of bcl-2 in Notch l group was higher than that of control group （P 〈 0.05）.However, the expression of Caspase-3 and bax in Notch1 group was lower than that of control group （P 〈 0.05）.The expression of bcl-2 in si group was lower than that of control group （P 〈 0.05）.However, the expression of Caspase-3 and bax in si group was higher than that of control group （P 〈 0.05）.Conclusion The expression of Notch1 decreases with the decrease in the proliferation of neurons and the increase of apoptosis after the ischemia-reperfusion injury to neurons.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2015年第11期2735-2738,共4页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目（81260202）
江西省自然科学基金资助项目（20114BAB205062）
江西省教育厅科学技术研究项目（GJJ13688）
“江西省普通本科高校中青年教师发展计划访问学者专项资金”资助项目
