缺氧缺血新生大鼠神经元自噬基因表达节律及调控机制  

Expression rhythm of autophagic gene in neurons of neonatal rats with hypoxia/ischemia and its regulatory mechanism

作　　者：李世平[1] 朱将虎[1] 赵凤艳[1] 郑臻[1] 母得志[1] 屈艺[1] 

机构地区：[1]四川大学华西第二医院儿科/出生缺陷与相关妇儿疾病教育部重点实验室,四川成都610041

出　　处：《中国当代儿科杂志》2017年第8期938-944,共7页Chinese Journal of Contemporary Pediatrics

摘　　要：目的通过新生大鼠缺氧缺血脑损伤后神经元自噬基因和节律基因的表达,探讨缺氧缺血引起神经损伤的新机制。方法将12只Sprague-Dawley大鼠随机分为缺氧缺血组和假手术组,每组6只。采用结扎并切断大鼠右侧颈总动脉并给予低氧处理的方法建立体内缺氧缺血脑损伤模型。Western blot检测两组大鼠大脑皮层和海马组织节律蛋白Clock表达情况。体外培养大鼠神经元细胞,随机分为氧糖剥夺(OGD)组和对照组,OGD组加入无糖无血清DMEM培养基模拟细胞缺血状态,并给予低氧处理建立体外缺氧缺血脑损伤模型。采用Western blot检测两组不同时间点自噬相关蛋白Beclin1和LC3,以及节律基因Clock蛋白表达情况。应用si RNA技术抑制神经元Clock蛋白表达后,检测Beclin1和LC3的表达变化。结果体外培养神经元Beclin1和LC3Ⅱ的表达呈现节律性波动;OGD处理后,体外培养神经元Beclin1和LC3Ⅱ的表达随着时间的延长逐渐升高,不再呈现节律性波动;与假手术组相比,缺氧缺血引起大鼠皮层和海马组织Clock表达降低(P<0.05);体外培养神经元经OGD处理后,Clock表达也较对照组显著降低(P<0.05);与阴性对照组相比,抑制神经元节律基因Clock表达后,自噬相关蛋白Beclin1和LC3Ⅱ的表达均显著下降(P<0.05)。结论缺氧缺血引起神经元Beclin1和LC3Ⅱ表达节律紊乱,其机制可能与Clock参与调控Beclin1和LC3Ⅱ的表达有关。Objective To investigate the expression of autophagic gene and circadian gene in the neurons of neonatal rats after hypoxic-ischemic brain damage and the mechanism of nerve injury induced by hypoxia/ischemia.Methods Twelve Sprague-Dawley（SD） rats were randomly divided into hypoxic-ischemic（HI） group and shamoperation group,with 6 rats in each group.Ligation of the right common carotid artery and hypoxic treatment were performed to establish a model of hypoxic-ischemic brain damage.Western blot was used to measure the expression of the circadian protein Clock in the cortex and hippocampus.The neurons of the rats were cultured in vitro and randomly divided into oxygen glucose deprivation（OGD） group and control group.The neurons in the OGD group were treated with DMEM medium without glucose or serum to simulate ischemic state,and hypoxic treatment was performed to establish an in vitro model of hypoxic-ischemic brain damage.Western blot was used to measure the expression of autophagy-related proteins Beclin1 and LC3 and Clock protein at different time points.The changes in the expression of Beclin1 and LC3 were measured after the expression of Clock protein in neurons was inhibited by small interfering RNA technique.Results The expression of autophagy-related proteins Beclin1 and LC3 Ⅱ in neurons cultured in vitro displayed a rhythmic fluctuation; after OGD treatment,the expression of Beclin1 and LC3 Ⅱ gradually increased over the time of treatment and no longer had a rhythmic fluctuation.Compared with the sham-operation group,the HI group had a significant reduction in the expression of Clock protein in the cortex and hippocampus（P〈0.05）.After OGD treatment,the neurons cultured in vitro had a significant reduction in the expression of Clock protein（P〈0.05）.Compared with the negative control group,the Clock gene inhibition group had significant reductions in the expression of Beclin1 and LC3 Ⅱ（P〈0.05）.Conclusions Hypoxia/ischemia induces the disorder in the expression rhythm of au

关 键 词：缺氧缺血 自噬 节律基因 CLOCK BECLIN1 LC3 神经元 新生大鼠 

分 类 号：R742[医药卫生—神经病学与精神病学]