AQP4基因沉默在新生猪HIBD模型中的DWI影像与病理对照研究

发布时间：2018-01-30 14:12

本文关键词： 缺血缺氧 AQP4基因沉默磁共振成像血气生化　出处：《大连医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的：新生儿缺氧缺血性脑病(hypoxic-ischemic encephalopathy，HIE)或称为缺氧缺血性脑损伤(HIBD)主要是由围产期窒息缺氧所引致的新生儿脑损伤疾病，窒息所致的早期缺血缺氧性脑水肿是本病的主要发病机制。临床发病较为常见，凡能引起新生儿窒息的因素，如胎儿宫内窘迫，高位产钳术，产伤等，都可引起HIE，该病是造成新生儿伤残的主要疾病。根据病情分为轻、中、重型，轻者预后良好，无后遗症残留；重者由于中枢神经系统严重缺血缺氧，可导致新生儿早期死亡或产生永久性的神经损害。本研究以注射过AQP4siRNA干扰和对照序列的新生猪为实验对象，模拟早期新生儿HIBD模型，通过观察比较两组动物在临床症状体征、影像学表现和病理学表现的差异，来探讨AQP4基因沉默技术在早期新生猪缺血缺氧性脑损伤中的潜在价值，以期为新生儿HIBD疾病的诊断和治疗提供新的理论依据和方向。方法：将32只健康清洁的新生约克种猪随机分为实验组和对照组，通过脑脊液循环途径给药，分别鞘注等量的AQP4siRNA干扰序列和对照序列混合溶液。经过24h的脑内转染后，对实验组和对照组新生猪进行相同程度的缺血缺氧处理，模拟早期新生儿HIBD模型。通过对比两组动物缺血缺氧后各时点在早期神经行为学、血气生化酶学指标、MR分子影像学和脑神经组织的早期病理学表现的差异，运用柱状图或堆积条图对两组动物的神经行为学评分和血气生化酶学指标进行直观的比较，并采用T检验或非参数检验对各时点评分和血气生化指标进行两两比较，采用重复测量方差分析和T检验的方法对两组动物各时点DWI图像的ADC值进行比较，同时采用免疫组织化学法（SP法）检测两组新生猪脑内AQP4蛋白的表达水平并进行阳性细胞计数，判断两组的差异是否具有显著性（P0.05）。所用数据均采用SPSS16.0统计软件包进行统计分析。结果：实验组（即注射AQP4siRNA干扰序列组）和对照组（即注射AQP4siRNA对照序列组）新生猪在自主呼吸、皮肤黏膜颜色和神经系统症状上均出现不同程度的异常，但实验组在自主规律呼吸和正常皮肤黏膜颜色的恢复更快，在意识、颅神经、反射、动作及合作协调等神经功能的反应表现更佳；且对照组在HI后2h、3h、6h的神经行为学评分分别为7.200±0.789、10.000±1.700、13.400±1.174，明显低于实验组。实验组HI后12h的PCO2、PO2、LDH分别为34.77±9.40mmHg、85.90±18.93mmHg、586.22±111.58U/L，对照组该时点各指标值分别为45.94±12.52mmHg、65.40±23.13mmHg、813.10±325.30U/L，两者的差异具有统计学意义（P0.05）。在DWI图像上，随着时间延长，对照组各时点异常高信号区出现范围和强度较实验组明显，两组各时点ADC值存在显著性差异（P0.05）；将两组动物的海马、额顶区、侧脑室旁区的石蜡切片进行SP免疫组化染色并采用DAB显色后，两组的水通道蛋白4被不同程度着色，实验组各时点AQP4蛋白表达阳性的细胞数明显较对照组少（P0.05或0.01），且海马、额顶区的AQP4蛋白表达比侧脑室旁区多。两组动物AQP4蛋白表达的变化趋势大致相似，且与ADC值和DWI图像各时点的变化情况相对应。结论：①AQP4基因沉默技术能显著降低HIBD模型中新生猪脑组织早期水通道蛋白4的表达，，缓解临床缺氧症状和体征，减轻脑组织水肿的程度，提示在新生儿缺血缺氧性脑损伤疾病中，水通道蛋白4可作为一个潜在的治疗靶点。②DWI结合ADC图/值可以较好的评价HIE/HIBD脑水肿的程度。
[Abstract]:Objective: neonatal hypoxic ischemic encephalopathy (hypoxic-ischemic, encephalopathy, HIE) or hypoxic ischemic brain damage (HIBD) is the main disease of neonatal brain injury caused by perinatal asphyxia and hypoxia, early hypoxic ischemic brain edema caused by asphyxia is the main pathogenesis of the disease. Clinical disease is common, which can cause factors of neonatal asphyxia, such as fetal distress, high forceps, birth trauma, can cause HIE, the disease is the main disease causing neonatal disability. According to the condition is divided into light, heavy, light, good prognosis, no residual sequelae; severe ischemia due to severe CNS hypoxia, can lead to nerve damage in early neonatal death or have permanent. In this study, neonatal pigs injected AQP4siRNA interference and the control sequence as the experimental object, the simulation of early neonatal HIBD model, through observation and comparison The difference between two groups of animals in clinical symptoms and signs, imaging findings and pathological findings is to explore the potential value of AQP4 gene silencing in early neonatal swine hypoxic ischemic brain damage, so as to provide new theoretical basis and direction for the diagnosis and treatment of neonatal HIBD disease.
Methods: 32 healthy and clean York newborn pigs were divided into experimental group and control group, administered through the cerebrospinal fluid circulation pathways, respectively AQP4siRNA interference sequence of intrathecal equivalent and the control sequence mixed solution. After 24h in the brain after transfection, the experimental group and the control group of neonatal pigs with the same degree of ischemia and hypoxia simulation, early neonatal HIBD model. By comparing the two groups of animal after hypoxic ischemia at each time point in the early neurological behavior, blood biochemical index, pathological differences in early MR molecular imaging and brain imaging, using the histogram or stacked bar chart on neurobehavioral scores of the two groups of animal and blood biochemistry index for direct comparison, and the comparison of 22 T test or non parametric test on the score and blood biochemical index at each time point, by using repeated measures ANOVA and T test method On the two animal groups at each time point DWI image ADC value, at the same time by immunohistochemical method (SP method) for detection of AQP4 protein positive cells of two groups of newborn piglets in expression level, to determine whether the difference between the two groups was significant (P0.05). The data were collected for statistical analysis by SPSS16.0 the statistical software package.
Results: the experimental group (i.e. injection AQP4siRNA interference sequence group) and control group (i.e. control group injected with AQP4siRNA sequence) of newborn pigs in spontaneous breathing, skin and mucosa color and neurological symptoms were abnormal in different degrees, breathing in the independent law and normal skin mucosa color but the experimental group recovered faster, in consciousness. Cranial nerve, reflection, action and coordination etc. neural function response to better performance; and the control group at HI after 2h, 3h, 6h, neurobehavioral scores were 7.200 + 0.789,10.000 + 1.700,13.400 + 1.174, significantly lower than the experimental group. The experimental group PCO2, 12h HI PO2, LDH = 34.77 + 9.40mmHg, 85.90 + 18.93mmHg, 586.22 + 111.58U/L group, the time of each index = 45.94 + 12.52mmHg control, 65.40 + 23.13mmHg, 813.10 + 325.30U/L, the difference was statistically significant (P0.05). In the DWI image, with the prolongation of time, The control group at each time point of abnormal high signal area range and intensity was significantly higher than the experimental group, there were significant differences between the two groups at each time point ADC value (P0.05); the two group animal hippocampus, frontal and parietal areas, paraffin periventricular area for immunohistochemical staining of SP and the DAB color, water channel protein two group of 4 different degrees of coloring, experimental group AQP4 protein expression positive cell number was significantly less than that of the control group (P0.05 or 0.01), and the hippocampus, frontal and parietal areas, the expression of AQP4 protein in the periventricular region. More than two groups of animal protein AQP4 expression showed similar changing trends, changes and with ADC and DWI images corresponding to each time point.
Conclusion: the expression of AQP4 gene silencing technology can significantly reduce the early water channel protein in neonatal brain tissue in HIBD model 4, relieve the clinical symptoms and signs of hypoxia, reduce brain edema, suggesting that in neonatal hypoxic ischemic brain injury disease, aquaporin 4 can be used as a potential therapeutic target. DWI combined with ADC map / value can better evaluate HIE/HIBD brain edema.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R-332;R722.1

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