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目的探讨早产、氧浓度异常对胆红素的神经细胞毒性的影响及其可能的发生机制。方法 7 d龄新生足月和早产大鼠各36只,完全随机分为常氧+生理盐水组、常氧+胆红素组、低氧+生理盐水组、低氧+胆红素组、高氧+生理盐水组、高氧+胆红素组。常氧+生理盐水组,经小脑延髓池注射0.02 ml生理盐水;常氧+胆红素组,经小脑延髓池注射胆红素20μg/g;低氧+生理盐水组,在氧浓度8%的密闭箱中暴露3 h,经小脑延髓池注射0.02 ml生理盐水;高氧+生理盐水组,在氧浓度95%的密闭箱中暴露3 h,经小脑延髓池注射0.02 ml生理盐水;低氧+胆红素组,氧浓度8%的密闭箱中暴露3 h后经小脑延髓池注射胆红素20μg/g;高氧+胆红素组,氧浓度95%的密闭箱中暴露3 h后经小脑延髓池注射胆红素20μg/g。各组均于24 h断头取脑。HE染色及TUNEL显示海马神经细胞凋亡,免疫组织化学染色法检测iNOS和TNF-α表达。结果①注射胆红素的大鼠均出现俯伏、转圈、四肢颤动、翻滚、活动减少等异常神经行为,早产组最明显。②HE染色观察胆红素+低氧组海马神经细胞坏死、凋亡最明显。③TUNEL显示,早产胆红素组海马神经细胞凋亡数(30.17±3.76)较足月组(23.33±4.89)明显升高(P<0.05);足月和早产低氧+胆红素组的海马神经细胞凋亡率[分别为(27.67±3.01)和(34.33±3.01)]较胆红素组[分别为(23.33±4.89)和(30.17±3.76)]明显升高(P<0.05),而高氧+胆红素组海马神经细胞凋亡率与胆红素组相似。④早产胆红素组海马神经细胞TNF-α释放(0.425±0.014)较足月组(0.388±0.020)显著增多(P<0.05),两组iNOS表达水平相似;低氧+胆红素组海马神经细胞TNF-α和iNOS表达[(0.437±0.024)和(0.440±0.001)]较胆红素组[(0.253±0.010)和(0.388±0.020)]显著增加(P<0.05);高氧+胆红素组海马神经细胞TNF-α释放(0.416±0.011)显著增多(P<0.05),iNOS表达无明显升高。结论胆红素可诱导TNF-α等炎症因子释放增多;低氧、早产增加TNF-α和/或iNOS等炎症因子的释放和表达,增强胆红素的神经毒性。减少围产期窒息、早产,控制炎症因子释放,对防治胆红素脑病有重要意义。
Objective To investigate the effects of premature labor and abnormal oxygen concentration on the neurotoxicity of bilirubin and its possible mechanism. Methods Thirty-six neonates with 7-day-old newborn and preterm rats were randomly divided into normoxia + saline group, normoxia + bilirubin group, hypoxia + saline group, hypoxia + bilirubin group, Oxygen + saline group, hyperoxia + bilirubin group. Normoxia + saline group, injected into the cisterna magna with 0.02 ml normal saline; normoxia + bilirubin group, injected bilirubin 20μg / g into the cisterna magna; hypoxic + saline group, Exposed to a closed box for 3 hours, then injected into the cisterna magna with 0.02 ml of saline; hyperoxia + saline group exposed for 3 hours in a closed box with oxygen concentration of 95% and injected with 0.02 ml of normal saline through the cisterna magna; hypoxic + Bilirubin group, the oxygen concentration of 8% of the closed box exposed 3 h after injection of bilirubin in the cerebellar bulbar cistern 20μg / g; hyperoxia + bilirubin group, oxygen concentration of 95% Cerebellum cisterna injection bilirubin 20μg / g. Each group was decapitated at 24 h brain. The apoptosis of hippocampus neurons was detected by HE staining and TUNEL staining. The expressions of iNOS and TNF-α were detected by immunohistochemical staining. Results ① Bilirubin-injected rats showed abnormal neurological behaviors such as depression, circling, limb flutter, tumbling, and decreased activity, most notably in preterm birth. ② HE staining observed bilirubin + hypoxic group of hippocampal neuronal necrosis, the most obvious apoptosis. (3) TUNEL showed hippocampal neuron apoptosis (30.17 ± 3.76) in premature bilirubin group was significantly higher than that of full-term group (23.33 ± 4.89) (P <0.05); hippocampus of term and preterm hypoxic + bilirubin group Compared with bilirubin group [(23.33 ± 4.89) and (30.17 ± 3.76) and (30.17 ± 3.76, respectively]), the apoptotic rate of neurocytes was significantly higher (27.67 ± 3.01 and 34.33 ± 3.01, P <0.05) The apoptosis rate of hippocampal neurons in hyperoxia + bilirubin group was similar to that in bilirubin group. (4) TNF-α release in hippocampal neurons of preterm bilirubin group (0.425 ± 0.014) was significantly higher than that of full-term group (0.388 ± 0.020) (P <0.05), and iNOS expression levels in both groups were similar; hippocampus Compared with bilirubin group [(0.253 ± 0.010) and (0.388 ± 0.020)], the expression of TNF-α and iNOS in nerve cells significantly increased (P <0.05) In the bilirubin group, the release of TNF-αin the hippocampus was significantly increased (0.416 ± 0.011) (P <0.05), and the expression of iNOS was not significantly increased. Conclusion Bilirubin can induce the release of inflammatory cytokines such as TNF-α and so on. Hypoxia and premature labor increase the release and expression of inflammatory factors such as TNF-α and / or iNOS and enhance the neurotoxicity of bilirubin. Reduce perinatal asphyxia, premature delivery, control the release of inflammatory cytokines, prevention and treatment of bilirubin encephalopathy is of great significance.