【作者】 韩丽春；

【导师】 徐礼鲜； 武胜昔；

【作者基本信息】 第四军医大学 ， 麻醉学， 2009， 博士

【摘要】 全身麻醉作用机理研究已有160多年的历史,特别是中枢作用部位,至今仍然是国内外麻醉学界十分关注而又悬而未决的研究课题。研究目的:本研究目的旨在应用分子生物学、免疫组化及免疫荧光双标等多种先进的神经科学技术,以GAD67-GFP基因敲入小鼠为研究对象,系统观察在吸入麻醉药七氟烷的作用下,脑内GABA神经元被激活的部位及其GABAA受体参与的部位及细胞内机制。为进一步阐明全身麻醉中枢作用机理提供新的证据。研究方法:第一部分:成年雄性GAD67-GFP基因敲入小鼠随机分为3组:C组(C Group)空白对照组;S1组(S1 Group):浅麻醉状态(Incomplete anesthesia state)组,即小鼠反正反射消失但不超过30秒;S2组(S2 Group):深麻醉状态(Complete anesthesia state)组,即反正反射完全消失30s以上。各组GAD67-GFP基因敲入小鼠达到预设麻醉状态后,取脑制取脑薄片(片厚30μm),分别进行Fos免疫组化染色、GABA神经元和Fos蛋白的免疫荧光双标染色,在激光共聚焦显微镜下进行观察计数;并快速取出GAD67-GFP基因敲入小鼠海马和丘脑,制备RNA,反转录合成cDNA ,设计合成c-Fos基因mRNA序列相应的特异性引物,实施荧光定量PCR检测,所有PCR产物经琼脂糖凝胶电泳鉴定。第二部分:c57野生小鼠实验分组同第一部分,设计合成GABAA受体α4亚单位特异性引物,实施GABAA受体α4亚单位荧光定量PCR检测,同时进行GABAA受体与Fos免疫荧光双标染色,在激光共聚焦显微镜下进行观察计数研究结果:第一部分:七氟烷对GAD67-GFP基因敲入小鼠脑内GABA神经元激活作用的研究1.对GAD67-GFP基因敲入小鼠脑内Fos蛋白表达的影响结果表明:吸入麻醉药七氟烷能诱导GAD67-GFP基因敲入小鼠脑内皮质(Cg1,Cg2)、海马(CA1,DG)、下丘脑背内侧核(DM)、中脑导水管周围灰质(PAG)、丘脑室旁核(PV)、外侧隔核(LS)等部位Fos的表达,与C组相比明显增多(P<0.05),随麻醉深度增加S2组比S1组Fos蛋白表达增多(P<0.05)。还发现在杏仁核中间部(MeA)、E-W核(E-W nucleus)、弓状核(Arc)和下丘脑室旁核腹侧核(PaV)等部位Fos表达仅出现在S2组。2.对GAD67-GFP基因敲入小鼠海马和丘脑Fos mRNA的影响荧光定量PCR结果表明c-fos mRNA在海马和丘脑部位S1和S2组表达与对照组相比明显上调(P<0.01),并且S2组上调更多与S1组相比有显著差异(P<0.01)。3.对GAD67-GFP基因敲入小鼠脑内GABA和Fos共存表达的影响免疫荧光双标结果表明:在外侧隔核(LS)几乎所有的Fos都存在于GABA神经元内,并且在S2组中共存于GABA神经元内的Fos与S1组相比明显增多。在海马(CA1, DG),下丘脑背内侧核(DM),皮质(Cg1, Cg2)和中脑导水管周围灰质(PAG),仅有少部分的Fos存在于GABA神经元内,大部分Fos表达于GABA神经元之外。PV有较多的Fos表达,但并没有GABA神经元存在。而在PaV、MeA、Arc及E-W这几个部位的S2组中也有Fos表达,但是在这几个神经核团中几乎没有Fos共存于GABA神经元内。第二部分:七氟烷对野生c57小鼠脑内GABAA受体作用的研究1.对野生c57小鼠脑内GABAA受体α4亚单位mRNA表达的影响.在小鼠脑干和丘脑部位GABAA受体α4亚单位mRNA表达S1和S2组与对照组相比显著升高(P<0.01),S2组与S1组相比显著升高(P<0.01)。2.对野生c57小鼠脑内GABAA受体蛋白表达的影响免疫荧光结果表明:S1和S2组在脑干和丘脑内GABAA受体蛋白与Fos蛋白共存表达与对照组相比明显增加,并且S2组与S1组相比明显增加(P<0.05)。研究结论:1.吸入麻醉药七氟烷能诱导Fos蛋白和c-fos mRNA在GAD67-GFP基因敲入小鼠脑内多个核团神经元呈阳性表达,且表达的数量随七氟烷麻醉深度增加而增多。2.吸入麻醉药七氟烷能诱导GAD67-GFP基因敲入小鼠脑内相关核团GABA神经元和Fos存在明显的共存现象,也具有部位差异及显著的浓度相关关系。3.吸入麻醉药七氟烷能诱导野生c57小鼠脑干和丘脑GABAA受体与Fos共存表达且具有浓度相关性。以上结论可证明吸入麻醉药七氟烷能够浓度相关性的激活小鼠脑内不同部位的GABA神经元,并且通过GABAA受体将抑制信号通过细胞内c-fos基因传递出来。更多还原

【Abstract】 Objective: The mechanisms underlying volatile anesthesia agents are not well elucidated. Emerging researches have focused on the participation ofγ-aminobutyric acid (GABA) neurons but there still lacks morphological evidence. To elucidate the possible activation of GABAergic neurons by sevoflurane inhalation, Fos and green fluorescent protein (GFP) double labeling were used on the brain of glutamic acid decarboxylase (GAD) 67-GFP knock-in mice after sevoflurane inhalation. Methods: Twenty GAD67-GFP knock-in mice were divided into 3 groups: S1 group: incomplete anesthesia state induced by sevoflurane; S2 group: complete anesthesia induced by sevoflurane; control(C) group. Results 1. Real-time PCR and immunohistochemistry results indicated that sevoflurane induced a significant increase of c-fos mRNA in thalamus and hippocampus and protein expression in the dorsomedial hypothalamic nucleus (DM), periaqueductal grey (PAG), hippocampus (CA1, DG), paraventricular thalamic nucleus (PV), lateral septal nucleus (LS), cingulate cortex (Cg1, Cg2) in S1 and S2 group compared with the control group. These alterations on Fos expression were dose-dependent. In S2 group, Fos was only expressed in the amygdale, Edinger-Westphal (E-W) nucleus, arcuate hypothalamic nucleus (Arc) and the ventral part of paraventricular hypothalamic nucleus (PaV). 2. Double immunofluroscent staining indicated that in LS,almost all Fos were present in GABAergic neurons. In CA1, DG, DM, cg1, cg2 and PAG, Fos was expressed as well, but only few were present in GABAergic neurons. Fos expression was very high in paraventricular thalamic nucleus, but no coexistence were found as no GABAergic neuron was detected in this area. 3.The real-time PCR results indicated that in brain stem and thalamus the GABAA receptorα4 subtype mRNA expression upregulated significantly in S1 and S2 groups compared with C group. 4. Double immunofluroscent staining indicated that in brain stem and thalamus Fos were present in neurons that GABAA receptors were expressed in S1 and S2 groups. Conclusion: Our results provided morphological evidence that GABA neurons and GABAA receptors in speific brain areas may participate in the sevoflurane-induced anesthesia.更多还原