摘要
目的 评价自噬在氧糖缺失早期诱发大鼠皮质神经元凋亡中的作用.方法 选择孕16- 18 d的SD雌鼠,体重350-400 g,分离培养胎鼠原代皮质神经元,接种于培养板上,采用随机数字表法将其分为4组（n=7）：对照组（C组）常规培养;氧糖缺失（OGD） 30 min组、OGD 1 h组、OGD 2 h组、OGD 3 h组将皮质神经元置于含有5％CO2-95％N2的无糖DMEM培养基中分别孵育30 min、1、2和3h;OGD＋自噬诱导剂雷帕霉素组（OGD＋R组）和OGD＋自噬抑制剂3-甲基腺嘌呤组（OGD＋ 3-MA组）分别在无糖DMEM培养基中预加入雷帕霉素（终浓度100 nmol/L）、3-甲基腺嘌呤（终浓度10mmol/L）孵育1h.处理结束后采用CCK-8法测定皮质神经元存活情况,Western blot法检测LC3Ⅱ和caspase-3的表达水平.结果 与C组比较,其余各组皮质神经元存活率降低,OGD 30 min组、OGD 1 h组、OGD 2 h组和OGD 3h组神经元LC3Ⅱ表达上调（P＜0.05）,OGD＋R组皮质神经元caspase-3表达差异无统计学意义（P＞0.05）,其余组皮质神经元caspase-3表达上调（P＜0.05）;与OGD 1 h组比较,OGD 2 h组、OGD 3 h组和OGD＋ 3-MA组皮质神经元存活率降低,OGD＋R组皮质神经元存活率升高,caspase-3表达下调,OGD＋ 3-MA组皮质神经元caspase-3表达上调,OGD 2 h组和OGD 3 h组皮质神经元LC3Ⅱ表达下调（P＜0.05）.结论 大鼠皮质神经元氧糖缺失早期启动自噬后可抑制神经元凋亡,产生内源性神经保护作用.
Objective To evaluate the role of autophagy in apoptosis in cortical neurons of rats in the early phase of oxygen-glucose deprivation （OGD）.Methods Female Sprague-Dawley rats at 16-18 days of gestation,weighing 350-400 g,were sacrificed after being anesthetized with pentobarbital sodium.The fetal rats were obtained and the primary cortical neurons were isolated and seeded in culture plates.The neurons were then divided into 4 groups （n =7 each） using a random number table：control group （group C）,OGD 30 min group,OGD 1 h group,OGD 2 h group,and OGD 3 h group,OGD ＋ autophagy inducer rapamycin group （OGD ＋ R group）,and OGD ＋ autophagy inhibitor 3-methyladenine （3-MA） group （OGD ＋ 3-MA group）.The neurons were incubated in sugar-free DMEM medium containing 5％ CO2 and 95％ N2 for 30 min,1,2 and 3 h in OGD 30 min,1 h,2 h,and 3 h groups,respectively.Rapamycin （final concentration of 100 nmol/L） and 3-MA （final concentration of 10 mmol/L） were added to sugar-free DMEM medium in OGD ＋ R and OGD ＋ 3-MA groups,respectively,and the neurons were then incubated for 1 h.The CCK-8 assay was used to detect the neuronal viability after the end of OGD.The expression of caspase-3 and LC3 Ⅱ was measured by Western blot.Results Compared with C group,the survival rates of cortical neurons were significantly decreased in the other groups,the expression of LC3 Ⅱ was up-regulated in OGD 30 min,1 h,2 h and 3 h groups,no significant change was found in caspase-3 expression in OGD ＋ R group,and the expression of caspase-3 was up-regulated in the other groups.Compared with OGD 1 h group,the survival rates of cortical neurons were significantly decreased in OGD2 h,OGD3 h and OGD ＋ 3-MA groups,the survival rates of cortical neurons were increased,and the expression of caspase-3 was down-regulated in OGD ＋ R group,the expression of LC3 Ⅱ was up-regulated in OGD ＋ 3-MA group,and the expression of LC3 Ⅱ was down-regulated in OGD2 h and 3 h groups.Conclusion Autophagy initiated in the early phase of OGD can inhibit apoptosis in cortical neurons of fetal rats.
出处
《中华麻醉学杂志》
CAS
CSCD
北大核心
2014年第12期1492-1494,共3页
Chinese Journal of Anesthesiology
关键词
自噬
脑缺血
神经元
细胞凋亡
Autophagy
Brain ischemia
Neurons
Apoptosis
