SK2通道外化在七氟醚致幼鼠学习记忆损害中的作用及其转运机制的研究

The effect of general anesthetics on the developing brain is still unsettled at present and its mechanism need to be further clarified. Small-conductance Ca2+-activated potassium channels (SKs) are widely expressed throughout the central nervous system, SK2 channel is one subtype of SKs channels and it is largely expressed in hippocampus, its distribution is changed at the different phases of developmental brain. Whether Sevoflurane could affect SK2 channel in CA1 area of hippocampus and further affect developing brain is not clear. In our preliminary experiments, we found that Sevoflurane could increase the expression of SK2 channels in postsynaptic membrane of the hippocampal CA1 area in post-natal day 14 mice; it also could inhibit SK channel endocytosis of HA-tagged SK2 channel transfected COS-7 cells. We postulate that Sevoflurane could increase the number of SK2 channel in the cell membrane by inhibiting its endocytosis and further affect the development of developing brain, but its mechanisms and its effect on brain development need to be further elucidated. So we will investigate the role of SK2 channel in the effect of Sevoflurane on learning and memory ability in neonatal mice from the perspectives of cell, tissue and animal experiments by cell transfection skill, hemagglutinin (HA)-tagged skill and confocal laser scaning microscope (CLSM) scanning technology and so on. Our research will investigate the effect of sevoflurane on learning and memory disability from the perspective of SK2 channels.

全麻药对发育中神经系统影响的机制有待于进一步阐明；小电导钙激活钾通道(SK)广泛分布在神经元上，SK2通道属于SK通道的一种亚型，在海马区分布最多、表达最强，其在海马区神经元上的分布变化随着神经系统发育阶段的不同而改变；七氟醚是否通过海马区SK2通道影响神经系统的发育，目前尚无相关研究。我们的预实验发现七氟醚增加幼鼠海马区神经元突触后膜SK2通道的表达，抑制COS-7细胞膜SK2通道的內吞作用；据此推测七氟醚通过抑制SK2通道的內吞作用，增加其在细胞膜中的数量，进而对神经系统的发育产生一定的影响，但其确切机制及对神经发育的影响有待进一步验证。为此，我们将通过离体实验和动物实验，采用细胞转染、生物素标记、激光共聚焦显微镜扫描等技术手段，从细胞、组织及动物整体水平多方面探讨SK2通道在幼鼠七氟醚暴露相关的后期学习记忆影响中的作用。

全麻药对发育中神经系统影响的机制有待于进一步阐明；我们构建SD幼鼠七氟醚暴露动物模型，通过水迷宫行为学实验、电生理实验、生物素标记技术和Western blot等实验方法，研究发现：1，幼鼠七氟醚暴露导致后期长时程增强(long term potentiation, LTP)阈电位升高；2，幼鼠七氟醚暴露利于后期长时程抑制(long term depression, LTD)的形成；3，幼鼠七氟醚暴露抑制SK2通道的内吞作用，使得SK2通道在细胞膜中的表达增加；4，SK2通道蛋白阻滞剂Apamin能够逆转幼鼠七氟醚暴露后所致的AMPA受体内向整流的增加；5，阻断SK2通道蛋白或者GluR2缺乏的AMPA受体能够改善幼鼠七氟醚暴露所致的学习缺陷。我们的研究揭示了暴露于七氟醚中的幼鼠出现后期学习能力的障碍与SK2通道内吞作用被抑制，同时突触处Glu2缺乏的AMPA受体表达增加，SK2通道阻滞剂Apamin或者AMPA受体拮抗剂1-Napthylacetyl spermine (NASPM)能够改善幼鼠七氟醚暴露所致的后期学习记忆能力障碍。本课题从幼鼠七氟醚暴露抑制海马神经元细胞内吞作用，进而影响神经元突触可塑性（LTP的阈值增加和LTD的易化），造成幼鼠后期学习记忆功能障碍这一全新的视角，探索麻醉药对发育脑远期学习记忆的影响。我们的研究还发现幼鼠七氟醚暴露后神经突触缺乏Glu2的AMPA受体表达增加，SK2通道抑制剂Apamin可以逆转能够使AMPA受体相关的内向整流作用，表明SK2通道和AMPA受体之间存在着交互作用。以上发现为我们接下来从离子通道这一视角探索麻醉药对发育脑远期学习记忆的影响提供了新思路，为临床工作安全优质化医疗提供了理论基础。