七氟烷对新生大鼠神经系统影响的实验研究
发布时间：2018-08-09 19:56
【摘要】：背景每年全世界数百万婴幼儿接受全身麻醉和手术,但全身麻醉药对神经系统处于突触爆发期的儿童智力是否有影响,还不清楚。新生动物使用全身麻醉药麻醉后会出现多个脑区神经细胞凋亡、突触发育的异常和神经退行性变,有些在成年后还会遗留神经认知或社交方面的障碍,其具体机制还不清楚。目的通过实验研究七氟烷对核糖体蛋白S6的影响及其对其上下游通路的信号分子的影响,以研究七氟烷对神经系统作用的机制。方法建立新生动物七氟烷吸入麻醉模型。选用P8的SD大鼠随机分为两组,麻醉组和氧气对照组。以70%氧气空气混合气为载气,麻醉组大鼠给予七氟烷麻醉,分别在0.5h,1h,2h,4h取样,当麻醉持续4h后停止麻醉,将鼠仔放回母亲身边,并于放回后0.5h和2h分别取样。另一组是氧气对照组,直接使用70%空气氧气混合气吸入。并在相同时间点取样。一方面,取出新生大鼠的海马和皮层组织,抽提蛋白行蛋白印迹实验,进行Caspase-3,rp S6,p-rp S6,AKT,pAKT,m TOR,p-m TOR,ERK,p-ERK,JNK,p-JNK,P38,p-P38检测,以actin为内参。另一方面将新生大鼠从左心室灌流,取脑切片,行免疫组化实验,检测Caspase-3和p-rp S6的表达分部状况。此外,七氟烷麻醉4h和氧气对照4h的新生大鼠剥取海马和皮层,抽提RNA后送样进行基因芯片检测,观察七氟烷新生动物麻醉对海马和皮层基因表达的影响。结果七氟烷新生动物麻醉4h后,在海马和皮层均可见Caspase-3的表达上调;给予新生大鼠七氟烷持续麻醉,会造成大脑皮层和CA1区明显时间依赖性rp S6磷酸化的抑制;七氟烷能明显抑制大脑皮层和海马区的AKT活性,但对AKT的下游的m TOR没有影响;七氟烷对海马和大脑皮层的ERK活性产生了比较矛盾的效应;七氟烷增加了皮层和海马的PP1的活性;基因芯片提示七氟烷麻醉对多种基因的表达产生了上调或下调的效应,这些基因中许多是由于神经系统发育,认知记忆密切相关的基因。结论七氟烷麻醉新生动物确实会引起中枢神经系统的细胞凋亡等神经损伤的表现。这一损伤可能是由于七氟烷作用于核糖体蛋白S6使其磷酸化受到抑制所介导的,这一抑制作用又是通过对不依赖于m TOR的AKT活性抑制和对PP1活性增强实现的。基因芯片的结果提示,七氟烷对新生动物的神经系统发育相关的多个基因产生强烈的抑制或激活作用,他们也可能是导致细胞退变的原因之一。
[Abstract]:Background millions of infants around the world are subjected to general anesthesia and surgery every year, but it is not clear whether general anesthesia has an effect on the intelligence of children whose nervous system is in synaptic outbreak. After anesthetized with general anesthetic, the newborn animals may have several neuronal apoptosis, abnormal synaptic development and neurodegenerative changes, and some will leave behind cognitive or social obstacles in adulthood, the specific mechanism is not clear. Aim to investigate the effects of sevoflurane on ribosomal protein S6 and signal molecules in its upstream and downstream pathways in order to study the mechanism of the effects of sevoflurane on nervous system. Methods the sevoflurane inhalation anesthesia model of newborn animals was established. SD rats with P 8 were randomly divided into two groups: anesthesia group and oxygen control group. The anesthetized rats were anesthetized by sevoflurane with 70% oxygen air mixture as carrier. The anesthetized rats were taken at 0.5 h for 1 h and 2 h for 4 h respectively. The anesthetic was stopped after 4 h of anesthesia, and the rats were put back to their mothers. The rats were sampled at 0.5 h and 2 h after the anesthesia was put back. The other group was oxygen control group, which was inhaled directly with 70% air oxygen mixture. And sampling at the same time. On the one hand, the hippocampal and cortical tissues of newborn rats were taken out, and the protein Bank Western blot test was carried out. The detection of p-ERKP38 was carried out by using actin as the internal control. On the other hand, the neonatal rats were perfused from the left ventricle, the brain sections were taken and the expression of Caspase-3 and p-rp S6 were detected by immunohistochemistry. In addition, sevoflurane anesthetized newborn rats were anesthetized for 4 h and oxygen control group for 4 h. The hippocampus and cortex of newborn rats were stripped. After RNA was extracted, the gene expression in hippocampus and cortex was detected by gene chip analysis. The effect of sevoflurane neonate anesthesia on gene expression in hippocampus and cortex was observed. Results the expression of Caspase-3 was up-regulated in hippocampus and cortex 4 hours after sevoflurane anesthesia in neonatal rats, and the inhibition of rpS6 phosphorylation in cerebral cortex and CA1 area was induced by continuous sevoflurane anesthesia in neonatal rats. Sevoflurane inhibited AKT activity in cerebral cortex and hippocampus, but had no effect on m TOR downstream of AKT. Sevoflurane had a contradictory effect on ERK activity in hippocampus and cerebral cortex. Sevoflurane increased the activity of PP1 in cortex and hippocampus, and gene chip suggested that sevoflurane anesthesia could up-regulate or down-regulate the expression of many genes, many of which were related to the development of nervous system and cognitive memory. Conclusion sevoflurane anesthetized newborn animals can induce neuronal damage such as apoptosis in the central nervous system. The damage may be mediated by the inhibition of phosphorylation of ribosomal protein S6 by sevoflurane, which is mediated by inhibition of AKT activity independent of m TOR and enhancement of PP1 activity. The microarray results suggest that sevoflurane strongly inhibits or activates multiple genes associated with the development of the nervous system in newborn animals, and they may also be one of the causes of cell degeneration.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R726.1
[Abstract]:Background millions of infants around the world are subjected to general anesthesia and surgery every year, but it is not clear whether general anesthesia has an effect on the intelligence of children whose nervous system is in synaptic outbreak. After anesthetized with general anesthetic, the newborn animals may have several neuronal apoptosis, abnormal synaptic development and neurodegenerative changes, and some will leave behind cognitive or social obstacles in adulthood, the specific mechanism is not clear. Aim to investigate the effects of sevoflurane on ribosomal protein S6 and signal molecules in its upstream and downstream pathways in order to study the mechanism of the effects of sevoflurane on nervous system. Methods the sevoflurane inhalation anesthesia model of newborn animals was established. SD rats with P 8 were randomly divided into two groups: anesthesia group and oxygen control group. The anesthetized rats were anesthetized by sevoflurane with 70% oxygen air mixture as carrier. The anesthetized rats were taken at 0.5 h for 1 h and 2 h for 4 h respectively. The anesthetic was stopped after 4 h of anesthesia, and the rats were put back to their mothers. The rats were sampled at 0.5 h and 2 h after the anesthesia was put back. The other group was oxygen control group, which was inhaled directly with 70% air oxygen mixture. And sampling at the same time. On the one hand, the hippocampal and cortical tissues of newborn rats were taken out, and the protein Bank Western blot test was carried out. The detection of p-ERKP38 was carried out by using actin as the internal control. On the other hand, the neonatal rats were perfused from the left ventricle, the brain sections were taken and the expression of Caspase-3 and p-rp S6 were detected by immunohistochemistry. In addition, sevoflurane anesthetized newborn rats were anesthetized for 4 h and oxygen control group for 4 h. The hippocampus and cortex of newborn rats were stripped. After RNA was extracted, the gene expression in hippocampus and cortex was detected by gene chip analysis. The effect of sevoflurane neonate anesthesia on gene expression in hippocampus and cortex was observed. Results the expression of Caspase-3 was up-regulated in hippocampus and cortex 4 hours after sevoflurane anesthesia in neonatal rats, and the inhibition of rpS6 phosphorylation in cerebral cortex and CA1 area was induced by continuous sevoflurane anesthesia in neonatal rats. Sevoflurane inhibited AKT activity in cerebral cortex and hippocampus, but had no effect on m TOR downstream of AKT. Sevoflurane had a contradictory effect on ERK activity in hippocampus and cerebral cortex. Sevoflurane increased the activity of PP1 in cortex and hippocampus, and gene chip suggested that sevoflurane anesthesia could up-regulate or down-regulate the expression of many genes, many of which were related to the development of nervous system and cognitive memory. Conclusion sevoflurane anesthetized newborn animals can induce neuronal damage such as apoptosis in the central nervous system. The damage may be mediated by the inhibition of phosphorylation of ribosomal protein S6 by sevoflurane, which is mediated by inhibition of AKT activity independent of m TOR and enhancement of PP1 activity. The microarray results suggest that sevoflurane strongly inhibits or activates multiple genes associated with the development of the nervous system in newborn animals, and they may also be one of the causes of cell degeneration.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R726.1
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