白介素-1β介导脓毒症新生大鼠轴突和突触损伤的机制研究

发布时间：2018-06-04 07:09

本文选题：LPS + 白介素-1β　；参考：《南方医科大学》2017年博士论文

【摘要】：新生儿脓毒症长期严重威胁着儿童生存及健康,可引起患儿长期的神经功能障碍。因围生期脑处于发育阶段、血脑屏障的不完整,全身严重的感染可波及中枢,引起脑内的炎症反应,导致神经元轴突、突触的病变,这可能是神经功能障碍的原因之一,但其具体分子机制不清。小胶质细胞是脑内主要炎症细胞,严重的感染、应激时在急性期处于激活状态,大量增殖并释放过量的炎症因子,其中白介素-1β是主要的促炎因子之一。既往有研究表明白介素-1β与众多神经系统病变有关。而p38-MAPK是与炎症反应密切相关的信号通路,且白介素-1β能激活这条信号通路导致下游蛋白的表达异常。因此在本研究中提出假设:新生儿脓毒症可出现脑内小胶质细胞的激活,释放大量的白介素-1β,通过激活神经元的p38-MAPK信号通路,导致神经元、突触相关重要蛋白的异常表达,可能是导致新生儿脓毒症神经轴突、突触损伤的机制之一。目的:证实新生脓毒症大鼠模型可出现神经轴突、突触的损伤、以及脑内小胶质细胞的激活和白介素-1β的大量表达,体外实验证实白介素-1β可通过激活p38-MAPK导致神经元、突触相关重要蛋白的异常表达。方法:1.给予新生SD大鼠腹腔注射LPS,模拟脓毒症模型,与腹腔注射PBS的对照组对比,蛋白印迹及免疫荧光注射后7天、14天、28天检测轴突、突触以及髓鞘相关的重要蛋白(PLP、NFL、NFM、NFH、Synaptophysin、PSD95)的表达,电镜检查脑内超微结构的变化,通过免疫荧光和尼氏染色明确神经元的凋亡情况。2.给予新生SD大鼠腹腔注射LPS后通过蛋白印迹、免疫荧光观察脑内小胶质细胞是否激活、表达白介素-1β的量变化以及其受体的神经元轴突上的表达情况。3.摸索理想浓度的白介素-1β干预原代培养的神经元,通过蛋白印迹和免疫荧光检测轴突、突触相关重要蛋白的表达。4.白介素-1β干预原代培养的神经元,检测p38-MAPK信号通路的激活情况。使用p38-MAPK信号通路的抑制剂SB203580,将原代培养神经元分为4个组:对照组、白介素-1β组、白介素-1β+SB203580组、SB203580组,蛋白印迹检测轴突、突触相关蛋白的表达,以明确p38-MAPK介导了白介素-1β对于神经元轴突、突触蛋白表达的调控。结果:1.给予新生SD大鼠腹腔注射LPS后7天、14天、28天检测到轴突相关蛋白(NFL、NFM、NFH)、突触相关蛋白(Synaptophysin)、髓鞘相关蛋白(PLP)的表达均较对照组明显下降(P0.05);电镜下可见28天LPS组的白质区轴突的稀疏、髓鞘、郎飞结、轴突的破坏,对轴突的直径统计发现LPS组的轴突的直径较对照组明显变小(P0.05),28天LPS组皮层区的部分神经元、树突呈“发黑”的表现,突触囊泡肿胀、内质网及线粒体嵴的破坏;而尼氏染色和凋亡的检测上在7天、14天、28天皮层神经元的凋亡在LPS和对照组之间无明显差异。2.腹腔注射LPS后6小时、2天、4天、6天发现胼胝体区小胶质细胞呈圆形激活状,数目有明显增加,大量的表达白介素-1β,与相应的对照组相比,有显著差异(P0.05);与此同时,相应受体IL-1R1在神经元的轴突上的表达也有明显上调(P0.05)。3.原代神经元培养后给予白介素-1β的干预,发现轴突(NFL、NFM)、突触相关蛋白(Synaptophysin)的表达相对于对照组有明显的下调(P().05),而使用IL-1R1的拮抗剂能拮抗白介素-1β的下调作用(P0.05)。4.白介素干预原代培养的神经元可在0.5小时即明显上调p38-MAPK的磷酸化(P0.05);p38-MAPK信号通路的抑制剂的干预可明显减少白介素-1β对于轴突(NFL、NFM)、突触相关蛋白(Synaptophysin)的表达的下调作用(P0.05)。结论:小胶质细胞来源的白介素-1β可以通过激活p38-MAPK抑制神经元轴突、突触相关蛋白的表达,可能是新生儿脓毒症导致神经轴突、突触损伤的部分机制。
[Abstract]:Neonatal sepsis has long been a serious threat to the survival and health of children. It can cause long-term neurological dysfunction in children. Because the perinatal brain is at the stage of development, the blood brain barrier is incomplete, the systemic infection can spread to the center, cause inflammation in the brain, and lead to the axons and synapses of neurons, which may be neurological dysfunction. One of the reasons, but its specific molecular mechanism is not clear. Microglia is the main inflammatory cell in the brain, serious infection, stress in the acute phase of activation, a large number of proliferation and release of excessive inflammatory factors, among which interleukin -1 beta is one of the major proinflammatory factors. Previous studies have shown that interleukin -1 beta and many nervous system lesions P38-MAPK is a signaling pathway that is closely related to inflammation, and interleukin -1 beta activates the signaling pathway leading to abnormal expression of the downstream protein. Therefore, it is hypothesized in this study that neonatal sepsis can induce activation of microglia in the brain, release a large number of interleukin -1 beta, and activate the p38-MAPK letter of neurons. The abnormal expression of important synapses related to neurons and synapses may be one of the mechanisms that cause neuroaxon and synaptic damage in neonatal sepsis. Objective: to confirm that the neonatal sepsis model can appear axon, synapse damage, and the activation of microglia in the brain and the large amount of interleukin -1 beta expression in the brain. It was confirmed that interleukin -1 beta could induce abnormal expression of important synapse related proteins in neurons by activating p38-MAPK. Methods: 1. the neonatal SD rats were given LPS in the abdominal cavity, simulated sepsis model, compared with the control group with PBS in the abdominal cavity, 7 days after Western blot and immunofluorescence injection, 14 days, and 28 days to detect the axon, synapse, and myelin related weight. The expression of protein (PLP, NFL, NFM, NFH, Synaptophysin, PSD95), the ultrastructural changes in the brain were examined by electron microscopy. The apoptosis of the neurons was determined by immunofluorescence and Nissl staining..2. was given to neonatal SD rats by intraperitoneal injection of LPS to detect the activation of microglia in the brain and to express the quantitative change of interleukin -1 beta. Expression on the axon of the neuron of its receptor.3. grope for the ideal concentration of the interleukins -1 beta intervention in the primary cultured neurons, detect the axons by Western blot and immunofluorescence, and the expression of the important synapse related protein.4. interleukin -1 beta interfered in the primary cultured neurons, and detected the activation of the p38-MAPK signaling pathway. P38 -MAPK signaling pathway inhibitor SB203580, the primary cultured neurons were divided into 4 groups: the control group, the interleukin -1 beta group, the interleukin -1 beta +SB203580 group, the SB203580 group, the Western blot detection axon, and the expression of synapse related proteins, in order to clarify the regulation of the interleukins -1 beta on the neuron axon and the synaptic protein expression by p38-MAPK. Results: 1. The axon related protein (NFL, NFM, NFH), synapse related protein (Synaptophysin) and myelin related protein (PLP) expression in the newborn SD rats were detected 7 days after intraperitoneal injection of LPS, and on the 28 day, and the expression of myelin related protein (PLP) was significantly lower than that of the control group (P0.05). Under electron microscopy, the sparsity of the axons in the white matter region of the group of LPS, myelin sheath, the damage of the axon, and the diameter of the axon were observed under the electron microscope. The diameter of the axon in the LPS group was significantly smaller than that in the control group (P0.05). The neurons in the cortical area of the group LPS of 28 days were "black", the synaptic vesicles were swollen, the endoplasmic reticulum and the mitochondrial crista were destroyed, while Nissl staining and apoptosis were detected at 7 days, 14 days, and 28 days of cortical neurons between the control group and the control group. 6 hours after intraperitoneal injection of.2., 2 days, 4 days, 4 days, and 6 days, the number of microglia in the corpus callosum was found to be circular activation, and the number of the number increased significantly. A large number of expression of interleukin -1 beta, compared with the corresponding control group, was significantly different (P0.05). At the same time, the expression of the corresponding receptor IL-1R1 on the axons of the neurons was also significantly up (P0.05).3. After primary neuron culture, the intervention of interleukins -1 beta showed that the expression of NFL, NFM and Synaptophysin was significantly down regulated compared to the control group (P ().05), while IL-1R1 antagonists could antagonize the down regulation of interleukin -1 beta (P0.05).4. interleukins intervened in the primary cultured neurons for 0.5 hours. Up regulation of phosphorylation of p38-MAPK (P0.05); intervention of inhibitors of p38-MAPK signaling pathway can significantly reduce the down-regulation of interleukin -1 beta on the expression of NFL, NFM, and synaptic related protein (Synaptophysin). Conclusion: the interleukin -1 beta derived from microglia can inhibit neuronal axons and synapse related eggs by activating p38-MAPK. The expression of white may be part of the mechanism of axonal and synaptic damage in neonatal sepsis.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R720.597

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