高胆红素血症新生大鼠脑组织Tau蛋白的变化与细胞凋亡的关系

Relationship of Expression of Microtubule-Associated Protein Tau and Neurocyte Apoptosis in Brain Tissue of Neonatal Rats with Hyperbilirubinemia

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 observe the expression of microtubule-associated protein Tau in the brain tissue of neonatal rats with hyperbilirubinemia and its relationship with neurocyte apoptosis,and provide experimental gist of the pathogenesis of central nervous system injury and the valid prevention and treatment for the further study of hyperbilirubinemia.Methods Total 90 seven-day-old SD rats were randomly assigned to the normal control group（group C,n=10）and the experimental group（group T,n=80）.The rats of group T were medially subdivided into 2 groups：group T1 and group T2（40 cases in each group）according to the increasing bilirubin injected intraperitoneally.Bilirubin solutions were injected into the abdominal cavity of the rats of group T1（100 μg·g-1）and group T2（200 μg·g-1）,and 1 mL physiological saline was injected in the rats of group C.The specimens were collected at each time point（4,8,12,24 hours）after the bilirubin injection.The behavior disorders of each group at each time point were observed.The pathological changes in hippocampus were observed with HE staining and the expressions of Tau and p-Tau were observed by immunohistochemistry staining under the light microscope.The apoptosis in hippocampus was detected by terminal deoxynucleotidy tranferase-mediated 2-deoxyuridine 5＇-riphosphate-biotin nick end labeling（TUNEL）staining.Results The neurons in group C had complete structure and lined up in order,the neurons in group T shrank obviously,and normal structure and arrangement disappeared.The expressions of Tau and p-Tau were not significantly different between group C and group T at 4 hours.The expressions of Tau and p-Tau in T1,T2 group increased respectively at the time points after the bilirubin injection at 8 h,12 h and 24 h.The TUNEL positive rate and the expression of Tau and p-Tau had the same current.Conclusions The phenomena of neurocyte apoptpsis and necrosis exist in the models of bilirubin encephalopathy.The expressions of Tau and p-Tau increase in neonatal rats with hyperbilirubinemia.It indicates that Tau and p-Tau may play important roles in the pathogenesis of brain injury in neonatal rats caused by hyperbilirubinemia.Tau may be used as a sensitive marker for hyperbilirubinemia and brain injury.