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目的观察微管相关蛋白Tau蛋白在高胆红素血症新生大鼠脑组织中的变化与神经细胞凋亡的关系,探讨高胆红素血症中枢神经损伤的发生机制,为高胆红素血症中枢神经系统损伤提供较为特异的早期检测指标。方法新生7日龄SD大鼠90只。随机分为正常对照组(C组,n=10)和实验组(T组,n=80),T组又根据腹腔注射胆红素剂量的不同分为T1组和T2组,每组40只。C组大鼠腹腔注射9g.L-1盐水1.0mL,T1组腹腔注射胆红素100μg.g-1,T2组腹腔注射胆红素200μg.g-1。各实验组根据造模结束后时间点不同(4h、8h、12h、24h)分为4组,每组10只。观察各组动物不同时间点的神经行为异常如抽搐、翻滚、俯伏、对外界刺激的逃避反应减弱等,取各组动物脑组织常规苏木精-伊红(HE)染色,光镜下观察其脑组织病理改变,免疫组织化学法动态观察其海马区Tau蛋白及p-Tau的表达,脱氧核糖核酸末端转移酶介导的缺口末端标记法检测其海马区神经细胞凋亡率。结果 HE染色可见C组大鼠海马区神经元排列整齐、结构完整,未见胆红素沉积;T组大鼠脑组织内可见不等量的胆红素沉积及神经元细胞数量减少、排列紊乱等。C组大鼠造模结束后未见明显的神经行为异常,T1组大鼠于造模结束后12h出现翻滚、震颤、俯伏及对外界刺激的逃避反应减弱等表现,T2组大鼠行为异常表现较T1组明显。T组海马区神经细胞出现Tau蛋白表达增加及其过度磷酸化,并表现出时间-剂量依赖性;神经细胞凋亡率随Tau蛋白及p-Tau表达的增加而升高。结论高胆红素血症时神经细胞发生凋亡增加,Tau蛋白的过度表达及过度磷酸化参与胆红素诱导的神经细胞凋亡,Tau蛋白可作为高胆红素血症早期脑损伤较为特异的检测指标之一,也可将阻止Tau蛋白的过度表达及其磷酸化作为胆红素脑病的治疗靶点之一。
Objective To investigate the relationship between the changes of microtubule-associated protein Tau protein and brain cell apoptosis in neonatal rats with hyperbilirubinemia and explore the mechanism of central nervous system injury in hyperbilirubinemia. Serum CNS injury provides more specific early detection indicators. Methods Ninety newborn SD rats aged 7 days were enrolled. The rats in group T were randomly divided into control group (group C, n = 10) and experimental group (group T, n = 80). Group T was divided into group T1 and group T2 according to the dose of intraperitoneal injection of bilirubin . Rats in group C were intraperitoneally injected with 1.0 mL of 9 g · L-1 saline, T1 was intraperitoneally injected with 100 μg.g-1 of bilirubin, and T2 was intraperitoneally injected with 200 μg.g-1 of bilirubin. The experimental groups were divided into 4 groups with 10 rats in each group according to different time points (4h, 8h, 12h, 24h) after modeling. Each group of animals were observed at different time points of neurological behavioral abnormalities such as convulsions, tumbling, prostration, to avoid the stimulation of the external response weakened, and so on each group of animals brain tissue conventional hematoxylin-eosin (HE) staining, light microscopy The histopathological changes and the expression of Tau protein and p-Tau in hippocampus were observed by immunohistochemical method. The apoptosis rate of hippocampal neurons in hippocampus was detected by DNA nick end labeling (TdT). Results HE staining showed that neurons in hippocampus of rats in group C were arranged neatly with complete structure without bilirubin deposition. In group T, unequal amounts of bilirubin deposition and neuronal cell number were seen in brain tissues of rats in group T, Wait. There was no obvious neurobehavioral abnormality in the rats in group C at the end of modeling, and the rats in group T1 showed tumbling, tremor and depression as well as weakened evasion response to external stimuli 12h after the model was established. The abnormal behavior of rats in group T2 More obvious than T1 group. In T group, the expression of Tau protein in hippocampal neurons increased and hyperphosphorylated in a time-and dose-dependent manner. The apoptosis rate of neurons increased with the increase of Tau protein and p-Tau expression. Conclusions Hyperbilirubinemia is associated with increased apoptosis of neuronal cells, over-expression of Tau protein and hyperphosphorylation of bilirubin-induced neuronal apoptosis. Tau protein may be more specific as early brain injury in hyperbilirubinemia One of the indicators of detection, but also can prevent the overexpression of Tau protein and phosphorylation as a therapeutic target of bilirubin encephalopathy.