氙气对新生大鼠脑白质损伤CLIC4表达的影响

发布时间：2018-06-19 17:28

本文选题：氙气 + 缺氧缺血　；参考：《青岛大学》2016年硕士论文

【摘要】：目的:通过检测脑白质损伤的新生大鼠CLIC4表达水平变化,探讨氙气对脑白质损伤的神经保护作用,为临床应用氙气治疗新生儿脑损伤提供理论基础和实验依据。方法:制作新生大鼠脑白质损伤模型:选取生后48-72小时的SD新生大鼠144只,随机分为生理盐水(NS)组(空白对照组)(n=24)、脂多糖(LPS)组(n=24)、缺氧缺血(HI)组(n=24)、脂多糖+缺氧缺血(LPS+HI)组(脑损伤对照组)(n=24),LPS组仅给予腹腔注射LPS 0.05mg/kg,HI组分离结扎右侧颈总动脉以及置于8%的氮氧混合气中1小时进行缺氧缺血处理,LPS+HI组给予腹腔注射LPS 0.05mg/kg,3小时后进行缺氧缺血处理,建立脑白质损伤模型。NS组仅给予腹腔注射等量生理盐水,不结扎不低氧处理。氙气干预处理:通过脂多糖联合缺氧缺血建立脑白质损伤模型后,根据50%氙气吸入开始时间分为A组(缺氧缺血后即刻)(n=24)、B组(缺氧缺血后2小时)(n=24),进行氙气吸入3小时。各组分别于干预处理后0小时、24小时、48小时、72小时随机各选取6只进行甲醛灌注断头取脑,予苏木素-伊红染色(HE)观察病理变化,髓鞘碱性蛋白(MBP)免疫荧光染色,并通过RT-PCR检测脑组织中CLIC4mRNA水平变化。结果:1.NS组、HI组、LPS组脑白质染色清晰,结构正常;LPS+HI组新生大鼠脑组织HE染色可见脑白质染色淡、结构疏松;神经纤维走向紊乱,神经细胞染色加深,体积缩小,核仁变小,突起减少;胶质细胞数量增多,可见细胞核固缩、胞浆疏松、细胞皱缩等凋亡改变。氙气干预组新生大鼠脑白质细胞排列紊乱,可见部分核固缩,但较LPS+HI组减轻。2.脑组织免疫荧光染色,在72小时,NS组、HI组和LPS组脑组织可见大量MBP阳性细胞;LPS+HI组MBP阳性细胞数较NS组明显减少。氙气干预组MBP阳性细胞较NS组减少、较LPS+HI组增多。3.脑损伤对照组及氙气干预组CLIC4mRNA的表达水平高于空白对照组,差异有统计学意义(P0.05),表明损伤后脑组织中CLIC4mRNA的表达水平升高;但氙气干预组CLIC4mRNA的表达水平低于脑损伤对照组,差异有统计学意义(P0.05)。24小时、48小时、72小时脑损伤对照组新生大鼠脑组织中CLIC4mRNA表达水平较空白对照组升高,氙气干预组新生大鼠脑组织CLIC4mRNA表达水平在24小时、48小时、72小时较脑损伤对照组下降,差异均有统计学意义(P0.05)。但同日龄新生大鼠氙气干预亚组中,A组与B组新生大鼠脑组织中CLIC4mRNA表达水平无明显变化,差异无统计学意义(P0.05),表明CLIC4mRNA表达水平的高低与进行氙气干预的时间点无关。结论:1.腹腔注射LPS可与缺氧缺血共同作用,导致新生大鼠脑白质损伤。2.脑白质损伤后可使脑组织中CLIC4基因表达水平升高,说明CLIC4可引起神经细胞损伤。3.氙气干预能够下调CLIC4基因表达水平,提示氙气可能对中枢神经系统存在保护作用。4.CLIC4基因表达水平的高低与脑白质损伤后氙气干预的时间点无关,本研究中尚未发现进行氙气干预的最佳时间点,提示在脑损伤后2小时内应用氙气均可发挥其神经保护作用。
[Abstract]:Objective: To explore the neuroprotective effect of xenon on brain white matter injury by detecting the changes of CLIC4 expression level in neonatal rats with brain white matter injury, and to provide theoretical basis and experimental basis for clinical application of xenon to treat neonatal brain injury. Methods: the model of brain white matter injury in newborn rats was made: 144 neonatal rats were selected for 48-72 hours after birth. Randomly divided into normal saline (NS) group (blank control group) (n=24), lipopolysaccharide (LPS) group (n=24), hypoxic-ischemic (HI) group (n=24), lipopolysaccharide + hypoxic-ischemic (LPS+HI) group (n=24), LPS group only given LPS 0.05mg/kg intraperitoneal injection, ligation of the right common carotid artery in the HI group and 1 hours of oxygen deficiency in 8% nitrogen oxygen mixture. Blood processing, group LPS+HI was given an intraperitoneal injection of LPS 0.05mg/kg, after 3 hours of hypoxic and ischemic treatment, the brain white matter injury model was established in group.NS only by intraperitoneal injection of equal amount of saline, no ligation and no oxygen treatment. Xenon intervention treatment: after the establishment of the brain white matter injury model by lipopolysaccharide combined with hypoxia ischemia, it began with the onset of 50% xenon inhalation. Group A (n=24), group B (2 hours after hypoxic ischemia) (n=24), xenon inhalation for 3 hours. Each group was randomly selected for 0 hours, 24 hours, 48 hours, 72 hours and 6 were randomly selected to perform formaldehyde perfusion to take the brain, and to observe pathological changes with hematoxylin eosin staining (HE) and myelin basic protein (MBP) immunofluorescence The changes of CLIC4mRNA level in brain tissue were detected by RT-PCR. Results: the white matter in group 1.NS, group HI and LPS group was clearly stained and the structure was normal. HE staining of brain tissue in group LPS+HI of neonatal rats showed that white matter was dyed light, structure loose, nerve fiber trend disorder, nerve cell staining deepened, volume narrowed, nucleolus became smaller, protuberance reduced; The number of cells increased, and the apoptotic changes were seen in nuclear condensation, cytoplasm loosening, and cell shrinkage. The brain white matter cells of neonatal rats in xenon intervention group were arranged in disorder, and partial nucleus retraction was visible, but a large number of MBP positive cells in group NS, HI group and LPS group were seen in group NS, HI group and LPS group, and LPS+HI group MBP positive in the group of LPS+HI. The number of cells in the xenon group was significantly lower than that in the NS group. The MBP positive cells in the xenon group were less than that in the NS group. The expression level of CLIC4mRNA in the.3. brain injury control group and the xenon group was higher than that in the xenon group. The difference was statistically significant (P0.05), indicating that the level of CLIC4mRNA expression in the brain tissue was increased after the injury, but the xenon intervention group was in CLIC4mRNA. The expression level was lower than that of the brain injury control group, the difference was statistically significant (P0.05).24 hours, 48 hours, 72 hours brain injury control group brain tissue CLIC4mRNA expression level increased compared with the blank control group, xenon group neonatal rats brain tissue CLIC4mRNA expression level at 24 hours, 48 hours, 72 hours compared with brain injury control group decreased. The difference was statistically significant (P0.05). However, there was no significant change in the expression of CLIC4mRNA in the brain tissue of A and B group rats in the xenon group of the same day old neonatal rats. The difference was not statistically significant (P0.05), indicating that the level of CLIC4mRNA expression was not related to the time point of xenon drying. Conclusion: 1. the intraperitoneal injection of LPS can be associated with hypoxia. The joint action of ischemia leads to the damage of white matter in neonatal rats with white matter damage, which can increase the level of CLIC4 gene expression in the brain tissue, indicating that CLIC4 can cause nerve cell damage and.3. xenon intervention can down regulate the expression of CLIC4 gene, suggesting that xenon may have a protective effect on the expression level of the.4.CLIC4 gene in the central nervous system. It is not related to the time point of xenon intervention after the injury of brain white matter. The best time point for xenon intervention has not been found in this study, suggesting that the application of xenon can play its neuroprotective effect within 2 hours after brain injury.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R742

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