本文選題：LPS + 白介素-1β��； 參考：《南方醫(yī)科大學(xué)》2017年博士論文

【摘要】：新生兒膿毒癥長(zhǎng)期嚴(yán)重威脅著兒童生存及健康,可引起患兒長(zhǎng)期的神經(jīng)功能障礙。因圍生期腦處于發(fā)育階段、血腦屏障的不完整,全身嚴(yán)重的感染可波及中樞,引起腦內(nèi)的炎癥反應(yīng),導(dǎo)致神經(jīng)元軸突、突觸的病變,這可能是神經(jīng)功能障礙的原因之一,但其具體分子機(jī)制不清。小膠質(zhì)細(xì)胞是腦內(nèi)主要炎癥細(xì)胞,嚴(yán)重的感染、應(yīng)激時(shí)在急性期處于激活狀態(tài),大量增殖并釋放過(guò)量的炎癥因子,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與眾多神經(jīng)系統(tǒng)病變有關(guān)。而p38-MAPK是與炎癥反應(yīng)密切相關(guān)的信號(hào)通路,且白介素-1β能激活這條信號(hào)通路導(dǎo)致下游蛋白的表達(dá)異常。因此在本研究中提出假設(shè):新生兒膿毒癥可出現(xiàn)腦內(nèi)小膠質(zhì)細(xì)胞的激活,釋放大量的白介素-1β,通過(guò)激活神經(jīng)元的p38-MAPK信號(hào)通路,導(dǎo)致神經(jīng)元、突觸相關(guān)重要蛋白的異常表達(dá),可能是導(dǎo)致新生兒膿毒癥神經(jīng)軸突、突觸損傷的機(jī)制之一。目的:證實(shí)新生膿毒癥大鼠模型可出現(xiàn)神經(jīng)軸突、突觸的損傷、以及腦內(nèi)小膠質(zhì)細(xì)胞的激活和白介素-1β的大量表達(dá),體外實(shí)驗(yàn)證實(shí)白介素-1β可通過(guò)激活p38-MAPK導(dǎo)致神經(jīng)元、突觸相關(guān)重要蛋白的異常表達(dá)。方法:1.給予新生SD大鼠腹腔注射LPS,模擬膿毒癥模型,與腹腔注射PBS的對(duì)照組對(duì)比,蛋白印跡及免疫熒光注射后7天、14天、28天檢測(cè)軸突、突觸以及髓鞘相關(guān)的重要蛋白(PLP、NFL、NFM、NFH、Synaptophysin、PSD95)的表達(dá),電鏡檢查腦內(nèi)超微結(jié)構(gòu)的變化,通過(guò)免疫熒光和尼氏染色明確神經(jīng)元的凋亡情況。2.給予新生SD大鼠腹腔注射LPS后通過(guò)蛋白印跡、免疫熒光觀察腦內(nèi)小膠質(zhì)細(xì)胞是否激活、表達(dá)白介素-1β的量變化以及其受體的神經(jīng)元軸突上的表達(dá)情況。3.摸索理想濃度的白介素-1β干預(yù)原代培養(yǎng)的神經(jīng)元,通過(guò)蛋白印跡和免疫熒光檢測(cè)軸突、突觸相關(guān)重要蛋白的表達(dá)。4.白介素-1β干預(yù)原代培養(yǎng)的神經(jīng)元,檢測(cè)p38-MAPK信號(hào)通路的激活情況。使用p38-MAPK信號(hào)通路的抑制劑SB203580,將原代培養(yǎng)神經(jīng)元分為4個(gè)組:對(duì)照組、白介素-1β組、白介素-1β+SB203580組、SB203580組,蛋白印跡檢測(cè)軸突、突觸相關(guān)蛋白的表達(dá),以明確p38-MAPK介導(dǎo)了白介素-1β對(duì)于神經(jīng)元軸突、突觸蛋白表達(dá)的調(diào)控。結(jié)果:1.給予新生SD大鼠腹腔注射LPS后7天、14天、28天檢測(cè)到軸突相關(guān)蛋白(NFL、NFM、NFH)、突觸相關(guān)蛋白(Synaptophysin)、髓鞘相關(guān)蛋白(PLP)的表達(dá)均較對(duì)照組明顯下降(P0.05);電鏡下可見(jiàn)28天LPS組的白質(zhì)區(qū)軸突的稀疏、髓鞘、郎飛結(jié)、軸突的破壞,對(duì)軸突的直徑統(tǒng)計(jì)發(fā)現(xiàn)LPS組的軸突的直徑較對(duì)照組明顯變小(P0.05),28天LPS組皮層區(qū)的部分神經(jīng)元、樹(shù)突呈“發(fā)黑”的表現(xiàn),突觸囊泡腫脹、內(nèi)質(zhì)網(wǎng)及線粒體嵴的破壞;而尼氏染色和凋亡的檢測(cè)上在7天、14天、28天皮層神經(jīng)元的凋亡在LPS和對(duì)照組之間無(wú)明顯差異。2.腹腔注射LPS后6小時(shí)、2天、4天、6天發(fā)現(xiàn)胼胝體區(qū)小膠質(zhì)細(xì)胞呈圓形激活狀,數(shù)目有明顯增加,大量的表達(dá)白介素-1β,與相應(yīng)的對(duì)照組相比,有顯著差異(P0.05);與此同時(shí),相應(yīng)受體IL-1R1在神經(jīng)元的軸突上的表達(dá)也有明顯上調(diào)(P0.05)。3.原代神經(jīng)元培養(yǎng)后給予白介素-1β的干預(yù),發(fā)現(xiàn)軸突(NFL、NFM)、突觸相關(guān)蛋白(Synaptophysin)的表達(dá)相對(duì)于對(duì)照組有明顯的下調(diào)(P().05),而使用IL-1R1的拮抗劑能拮抗白介素-1β的下調(diào)作用(P0.05)。4.白介素干預(yù)原代培養(yǎng)的神經(jīng)元可在0.5小時(shí)即明顯上調(diào)p38-MAPK的磷酸化(P0.05);p38-MAPK信號(hào)通路的抑制劑的干預(yù)可明顯減少白介素-1β對(duì)于軸突(NFL、NFM)、突觸相關(guān)蛋白(Synaptophysin)的表達(dá)的下調(diào)作用(P0.05)。結(jié)論:小膠質(zhì)細(xì)胞來(lái)源的白介素-1β可以通過(guò)激活p38-MAPK抑制神經(jīng)元軸突、突觸相關(guān)蛋白的表達(dá),可能是新生兒膿毒癥導(dǎo)致神經(jīng)軸突、突觸損傷的部分機(jī)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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R720.597

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