TAT-NBD抑制NF-κB活化在胆红素诱导大鼠皮层星形胶质细胞炎症反应中的作用

发布时间：2018-03-29 02:30

本文选题：胆红素脑损伤　切入点：皮层星形胶质细胞　出处：《重庆医科大学》2015年硕士论文

【摘要】：目的：观察反式激活蛋白-NEMO结合域(TAT-NBD)对胆红素诱导的大鼠皮层星形胶质细胞核因子-κB(NF-κB)活化及炎症反应的作用。初步探究腹腔内注射TAT-NBD治疗胆红素脑病新生鼠对星形胶质细胞核因子-κB (NF-κB)活化的影响,能否改善运动、学习记忆能力。方法：分离、培养、鉴定SD新生鼠大脑皮层2代星形胶质细胞,随机分为三组：对照组,胆红素组,TAT-NBD干预组。免疫荧光染色法观察细胞形态,改良MTT法检测细胞相对存活率,AnnexinV-FITC/PI双染法观察凋亡情况,Western blot检测NF-KBp65表达,EMSA检测NF-κB的入核、活化。ELISA检测上清中炎症因子IL-1β、TNF-α、IL-6的水平。用5日龄SD新生鼠随机分组后造模成三组：对照组,胆红素组,TAT-NBD干预组。细胞免疫荧光观测建模后24h各组脑组织切片中细胞形态和NF-κBp65表达；建模时及建模后3d记录新生鼠每天体重变化,记录建模后各组新生鼠30d以内存活状态；取28日龄各组SD大鼠进行转棒测试和避暗实验检测平衡运动、学习记忆能力。结果：胆红素组的NF-KBp65蛋白灰度比值显著高于对照组(p0.01),2h、12h达高峰(p0.05); TAT-NBD干预使NF-κBp65蛋白表达明显低于胆红素组(p0.01)。IL-1β、TNF-α、IL-6的释放与胆红素诱导的NF-KBp65表达有时间相关性,于12h达高峰,且TAT-NBD干预2h、12h的IL-1β、TNF-α、IL-6浓度均低于胆红素组(p0.01)。星形胶质细胞的存活率与胆红素作用时间负相关,TAT-NBD干预2h、12h的相对存活率均高于胆红素组(p0.05)。TAT-NBD能维持细胞正常形态,减轻细胞凋亡和损伤。建模后24h,胆红素模型鼠脑组织中GFAP、NF-KBp65的表达及NF-κB入核、活化较对照组明显增多,TAT-NBD干预可减少胆红素组脑组织中GFAP、NF-KBp65的表达和NF-κB的入核、活化。TAT-NBD干预组SD新生鼠建模后体重增长趋势连续3d高于胆红素组但无明显差异(p0.05),显著低于对照组(p0.01)；近期死亡率显著低于胆红素组(p0.01)；远期生存状态较胆红素组好转差异无统计学意义。平衡运动和避暗逃避实验中,TAT-NBD干预组在转棒上停留时间及进入黑箱的潜伏期均显著短于对照组(p0.01),长于胆红素组(p0.05)。结论：胆红素可诱导原代培养的大鼠皮层星形胶质细胞NF-κB激活、过表达,TAT-NBD抑制NF-κB的活化高峰、减少炎症因子释放发挥抗炎保护作用,可用于防治胆红素脑损伤。腹腔注射TAT-NBD在急性期可显著减轻胆红素脑病新生鼠脑组织内GFAP、NF-κB的激活和过表达,减少NF-κB的入核、活化；显著提升胆红素脑病新生鼠觅食能力,明显降低其近期死亡率；远期可适当改善其生存状态,显著提升其平衡运动、学习记忆能力。
[Abstract]:Objective: to observe the effects of transactivator protein Nemo binding domain (TAT-NBDD) on activation and inflammatory response of rat cortical astrocyte nuclear factor- 魏 B (NF- 魏 B) induced by bilirubin, and to explore the effect of intraperitoneal injection of TAT-NBD in the treatment of bilirubin encephalopathy in neonatal rats with bilirubin encephalopathy. Effects of glial nuclear factor-魏 B on the activation of NF- 魏 B, Methods: two generations of astrocytes were isolated, cultured and identified in the cerebral cortex of neonatal SD rats. They were randomly divided into three groups: control group, TAT-NBD intervention group, and immunofluorescence staining method. The relative survival rate of cells was detected by modified MTT method and the apoptosis was observed by Annexin V-FITC / Pi double staining. The expression of NF-KBp65 was detected by Western blot and NF- 魏 B was detected by EMSA. Activation. Elisa was used to detect the level of IL-1 尾 TNF- 伪 and IL-6 in the supernatant. Five day old SD newborn rats were randomly divided into three groups: the control group, the control group, and the control group. The cell morphology and NF- 魏 Bp65 expression in brain slices of each group were observed 24 hours after modeling, the changes of body weight of newborn rats were recorded during modeling and 3 days after modeling, and the survival status of newborn rats within 30 days after modeling was recorded. SD rats of 28 days old were tested by rotation test and dark avoidance test to detect the balance movement. Results: the gray level ratio of NF-KBp65 protein in bilirubin group was significantly higher than that in control group (P 0.01 / 12 h), and the expression of NF- 魏 Bp65 protein in bilirubin group was significantly lower than that in bilirubin group (P 0.01). IL-1 尾 TNF- 伪 IL-6 release was correlated with NF-KBp65 expression induced by bilirubin. The concentration of IL-1 尾 TNF- 伪 IL-6 was significantly lower than that of bilirubin group (P 0.01). The survival rate of astrocytes was negatively correlated with the time of bilirubin action. The relative survival rate of TAT-NBD for 2 h was higher than that of bilirubin group for 2 h. TAT-NBD could maintain the normal morphology of the cells. The expression of NF-KBp65 and the entry of NF- 魏 B into the brain tissue of bilirubin model were significantly increased compared with the control group. TAT-NBD intervention decreased the expression of GFAPNF-KBp65 and the entry of NF- 魏 B in the brain tissue of bilirubin group. The trend of body weight gain in the activated. TAT-NBD intervention group after modeling was higher than that in the bilirubin group for 3 days, but there was no significant difference between the two groups, which was significantly lower than that in the control group (p0.01), the short-term mortality was significantly lower than that in the bilirubin group (P 0.01), and the long-term survival status was better than that in the bilirubin group. There was no significant difference between the two groups. In the balanced exercise and avoidance experiments, the time of staying on the rotating rod and the latency of entering the black box in TAT-NBD intervention group were significantly shorter than those in the control group (p0.01) and longer than that in the bilirubin group. Conclusion: bilirubin can induce primary culture. NF- 魏 B was activated in cultured rat cortical astrocytes. Overexpression of TAT-NBD inhibits the peak of NF- 魏 B activation, reduces the release of inflammatory factors, plays an anti-inflammatory and protective role, and can be used to prevent and treat bilirubin brain injury. Intraperitoneal injection of TAT-NBD can significantly reduce the activation and overexpression of GFAP- 魏 B in the brain tissue of newborn rats with bilirubin encephalopathy at acute stage. Reduce NF- 魏 B entry into the nucleus, activation; significantly improve the feeding ability of neonatal rats with bilirubin encephalopathy, significantly reduce their short-term mortality; in the long run, can improve their survival status, significantly improve their balanced movement, learning and memory ability.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R722.1

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