miR-132在异丙酚所致术后认知功能障碍中参与调控海马成年神经再生的分子机制研究

Propofol is the most frequently used intravenous sedative anesthetic in clinical anesthesia and ICU, the effect of propofol in central nervous system are still disputing, and the molecular mechanism has not been elucidated. Studies have shown that propofol has a protective effect in brain assaulted by ischemic injury; however, other studies also found propofol has a deleterious effect in the development of newborn neurons and hippocampal related learning and memory. The molecular mechanisms of paradoxical effect of propofol in central nervous system are also unclear.In previous study, we have found that the expression level of miR-132 changed when treat with propofol. According this, we hypothesis that the change of miR-132 expression level induced by propofol may regulate the expression level of its target genes and proteins which are probably in the neurodevelopmental related pathways, and eventually affect the synaptic plasticity and hippocampal related learning and memory. In this study,firstly, we will analyze and identify the target mRNA of miR-132 through bioinformatics and molecular biological techniques. After identified the target gene, we will try to elucidate the probable molecular mechanisms of the propofol related miR-132 in synapse development and hippocampus related learning and memory through molecular biological, immunological, cellular biological, neurobiological techniques in vitro and in vivo. Furthermore, the study would provide a theoretical basis of the effect of propofol in synapse development and hippocampus related learning and memory.

异丙酚是临床应用最广泛的麻醉药物，其对神经发育的影响是临床安全用药的重要问题，寻找新的调控靶点是其研究热点。本课题拟采用大鼠为研究对象，对前期应用microRNA microarray筛选出异丙酚麻醉后大鼠海马差异表达明显的miR-132进行生物信息学分析，进一步采用Northern Blot法及qRT-PCR法加以验证。应用原位杂交和神经元免疫组化双标记法，鉴定miR-132对异丙酚调控神经发育的影响。通过敲除或过表达miR-132，进一步阐明异丙酚在体内调控神经发育的机制。应用细胞模型观察miR-132对其靶基因的作用，阐明其作用机制；应用动物模型进一步从功能上对异丙酚影响miR-132靶基因调节神经发育的作用加以验证。本研究旨在从体外和体内实验探讨miR-132在异丙酚在神经发育的作用和机制，揭示异丙酚影响神经发育的新分子机制，为发现异丙酚影响神经发育作用防治新靶点提供理论依据。

异丙酚以其起效快、蓄积少、苏醒快、副作用少等优点而广泛应用于儿科手术全身麻醉的诱导和维持。近年的临床前研究表明异丙酚对于发育期大脑具有神经毒性。发育期脑内神经干细胞的数量和功能对大脑功能的发育非常重要。我们的研究发现异丙酚可影响大鼠神经干细胞增殖和分化方向。我们随后应用miRNA测序技术筛选并验证出异丙酚作用后神经干细胞多种microRNA的变化，并对它们的靶基因做出预测并验证。过往研究显示其中miR-124-3p与神经元的分化有密切联系，我们发现异丙酚使miR-124表达下调，而过表达miR-124可逆转异丙酚对神经干细胞分化方向的影响。我们通过Luciferase实验表明miR-124-3p可靶定预测的转录因子Sp1（Sp1 transcription factor），异丙酚使神经干细胞Sp1表达上调。通过干扰Sp1表达，验证了异丙酚通过miR-124-3p/Sp1途径影响神经干细胞分化的方向。最后通过ChIP（染色质共沉淀）发现Sp1可与下游的Cdkn1b启动子结合，且异丙酚增强Sp1在该启动序列的富集。根据上述系列研究得出，异丙酚通过调控神经干细胞miR-124-3p/Sp1/Cdkn1b轴影响其分化的方向。该研究阐述了异丙酚对神经干细胞的一个作用机制。为今后探讨异丙酚发育期大脑影响的临床前及临床研究提供了重要方向。