罗格列酮防止β淀粉样蛋白寡聚体引起的突触发育损伤的作用及机制研究

发布时间：2018-06-22 00:24

本文选题：PPARγ + 罗格列酮　；参考：《宁波大学》2012年硕士论文

【摘要】：目的1.研究可溶性β淀粉样蛋白寡聚体42（soluble amyloid-β protein42oligomers, SO Aβ42）处理对体外培养大鼠海马神经元树突丝的密度、运动速度和长度以及树突棘密度的潜在损伤效应，以了解SO Aβ42对海马神经元突触发育的影响。2.研究过氧化物酶体增殖物激活受体（peroxisome proliferator-activatedreceptor gamma, PPAR）激动剂罗格列酮预处理对SO Aβ介导的海马神经元突触发育损伤的影响及其量效关系，并探讨PPARγ在罗格列酮发挥功能中的作用。3.探寻罗格列酮在以上研究中发挥潜在作用的可能性机制。最终为进一步了解阿尔茨海默氏病（Alzheimer’s disease, AD）的发病机制以及探寻AD的治疗靶点和治疗药物提供实验依据。方法取新生第1天Wistar大鼠的海马回做原代神经元培养。在体外培养第5天（day5in vitro, DIV5），用定位在膜上的绿色荧光蛋白（F-GFP）和GFP标记的肌动蛋白（GFP-actin）对神经元进行共转染，以显示神经元树突及其突起的详细形态学细节；在DIV7和DIV15，实验组给予神经元500nM SO Aβ42孵育3h，干预组在给予SO Aβ42孵育前对神经元分别给予0.1μM、0.5μM，5μM罗格列酮或5μM罗格列酮+5μM GW9662预孵育24h。通过活细胞成像方法观察各试验组DIV7神经元树突丝的密度、运动速度和长度以及DIV15神经元树突棘密度的情况。其次，在DIV5，用F-GFP、GFP-actin和靶向线粒体表达的红色荧光蛋白（Mitochondrially targeted red fluorescent protein, MitoRed）对神经元进行共转染，以显示神经元树突和其突起以及神经元线粒体的形态学细节；在DIV15，实验组给予神经元500nM SO Aβ42孵育3h，干预组在给予SO Aβ42孵育前对神经元给予5μM罗格列酮预孵育24h，通过活细胞成像方法观察相应试验组DIV15神经元树突中线粒体分布的情况。结果1.单独500nM SO Aβ42处理3h导致DIV7大鼠海马神经元树突丝的密度明显下降；罗格列酮预处理24h以剂量依赖的方式逆转SO Aβ42介导的树突丝密度减小。SO Aβ42和罗格列酮处理均不引起树突丝的运动速度和长度发生改变。2.单独500nM SO Aβ42处理3h引起DIV15大鼠海马神经元树突棘密度显著性减小；罗格列酮预处理24h以剂量依赖的方式逆转SO Aβ42介导的树突棘密度下降。3.5μM PPARγ特异性拮抗剂GW9662和5μM罗格列酮对神经元共同预处理有效阻断5μM罗格列酮逆转SO Aβ42介导的神经元树突丝或树突棘密度下降。4.单独500nM SO Aβ42处理3h降低了DIV15大鼠海马神经元树突中线粒体总长度与对应树突干总长度的比值、树突中离散线粒体的密度以及含线粒体的树突棘数目与总树突棘数目的比例，SO Aβ42处理引起的该现象在给予神经元5μM罗格列酮预处理24h后被有效逆转。SO Aβ42和罗格列酮处理均不改变树突中线粒体的长度。结论SO Aβ42处理对体外培养大鼠海马神经元的突触发育存在损伤作用，，导致神经元的突触数目下降；罗格列酮预处理可以浓度依赖地逆转SO Aβ42介导的神经元突触发育损伤，该效应通过罗格列酮激活PPARγ受体起作用。罗格列酮的保护效应机制可能与其可以逆转SO Aβ42介导的神经元树突中线粒体分布异常，包括树突中线粒体数目减少以及含线粒体的树突棘比例下降有关。
[Abstract]:Objective 1. to study the effects of soluble beta amyloid oligomer 42 (soluble amyloid- beta protein42oligomers, SO A beta 42) on the density, movement speed, length and dendrite density of dendritic spines in cultured rat hippocampal neurons in vitro, in order to understand the effect of SO A beta 42 on the synaptic development of hippocampal neurons by.2. peroxidation The effect of rosiglitazone preconditioning with peroxisome proliferator-activatedreceptor gamma (PPAR) agonist roglitazone on the synaptic development damage of hippocampal neurons mediated by SO A beta and its quantitative effect relationship, and to explore the potential of PPAR gamma in the function of rosiglitazone to explore the potential of rosiglitazone to play potential in the above study. The possible mechanism of the action can provide an experimental basis for further understanding the pathogenesis of Alzheimer 's disease (AD) and the target of the treatment of AD and the treatment of drugs.
Methods the primary cultured hippocampal neurons of Wistar rats were cultured for first days. The neurons were cultured in vitro for fifth days (day5in vitro, DIV5). The neurons were co transfected with the green fluorescent protein (F-GFP) and the GFP labeled actin (GFP-actin), in order to show the detailed morphological details of the neuron dendrites and their protrusions; in DIV7 and DIV15, the experimental group was incubated with 500nM SO A beta 42 to incubate 3h, and the intervention group was given 0.1 mu M, 0.5 micron, 5 u M rosiglitazone or 5 mu M rosiglitazone before incubating SO A beta. Second, in DIV5, the neurons were co transfected with F-GFP, GFP-actin and Mitochondrially targeted red fluorescent protein (MitoRed) expressed in the target mitochondria in order to show the morphological details of the neuron dendrites and its protuberances and the mitochondria of the divinity. In DIV15, the experimental group was given. The neurons were incubated with 500nM SO A beta 42 for 3h, and the intervention group was incubated with 5 u M rosiglitazone before SO A beta 42 incubated to incubate 24h, and the distribution of mitochondria in the dendrites of the experimental group was observed by living cell imaging.
Results 1. 500nM SO A beta 42 treated 3H resulted in a significant decrease in the density of the dendrites of the hippocampal neurons in DIV7 rats; rosiglitazone pretreated 24h in a dose-dependent manner to reverse the dendrite density mediated by SO A beta 42, which reduced the.SO A beta 42 and the rosiglitazone treatment without causing the change of the kinetic speed and length of the dendrites. The density of dendritic spines in hippocampal neurons of DIV15 rats was significantly reduced by 3H treatment with beta 42; rosiglitazone pretreated 24h in a dose dependent manner to reverse the density of dendritic spines mediated by SO A beta 42,.3.5 mu M PPAR gamma specific antagonist GW9662 and 5 micron M rosiglitazone, which effectively blocked 5 micron M rosiglitazone to reverse SO 42. The density of neuron dendrites or dendrites decreased.4. alone 500nM SO A beta 42 treatment 3H reduced the ratio of the total length of mitochondria to the total length of the dendrites of the hippocampal neurons of DIV15 rats, the density of the discrete mitochondria in the dendrites and the proportion of the number of dendritic spines with the mitochondrial dendrites and the number of the total dendritic spines, SO A beta 42 treatment caused the 3H This phenomenon was reversed effectively after giving neurons 5 micron M rosiglitazone pretreatment 24h..SO A A 42 and rosiglitazone did not change the length of mitochondria in dendrites.
Conclusion SO A beta 42 treatment can damage the synaptic development of hippocampal neurons in vitro, and lead to a decrease in the number of synapses. Rosiglitazone preconditioning can reverse the synaptic development damage induced by SO A beta 42. This effect is mediated by rosiglitazone activation of PPAR gamma receptor. The protection of rosiglitazone The effect mechanism may reverse the abnormal mitochondrial distribution in the neuron dendrites mediated by SO A beta 42, including the decrease in the number of mitochondria in the dendrites and the decrease in the proportion of the dendritic spines containing the mitochondria.
【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R749.16

【共引文献】