mNGF联合康复训练对痉挛型脑性瘫痪仔鼠脑组织BDNF、Caspase-3表达的影响

关键词：鼠神经生长因子  痉挛型脑性瘫痪  康复训练  仔鼠

Keywords:mouse nerve growth factor  spastic cerebral palsy  rehabilitation training  neonatal rat

摘要：

      摘要 目的：分析鼠神经生长因子联合康复训练对痉挛型脑瘫仔鼠脑组织BDNF、Caspase-3表达的影响。 方法：7日龄清洁级SD大鼠150只采用随机数字表法分为对照组（NS组，n=40）和模型组（T组，n=110）。T组于7日龄左侧颅内锥体束定向注射无水乙醇（1μl/g），NS组左侧颅内锥体束定向注射等量生理盐水。各组均在注射24h后（8日龄），采用改良李晓捷等脑瘫仔鼠模型判定标准结合Longa评分法判定痉挛型脑瘫仔鼠模型，将造模成功的T组SD大鼠采用随机数字表法选取90只，并分为A1、A2、A3组，每组30只。造模当日记为实验第0天，于实验第1天（8日龄）开始干预，A1组给予肌肉注射mNGF（30ng/g/d），A2组给予肌肉注射等量mNGF并联合康复训练，A3组给予肌肉注射等量生理盐水（2.27ml/kg/d）。干预至21日龄。NS组给予肌肉注射等量生理盐水。于8、14、21日龄进行HE染色动态观察脑组织内囊区细胞形态学的改变，TUNEL染色动态观察脑组织内囊区细胞凋亡率的改变，Western Bolt检测动态观察脑组织海马区BDNF及Caspase-3蛋白表达量的改变。 结果：模型制备：采用改良李晓捷等脑瘫仔鼠模型判定标准结合Longa评分法，8日龄T组仔鼠共判定痉挛型脑瘫仔鼠97只，NS组无脑瘫仔鼠。TUNEL染色：A1、A2、A3组各日龄仔鼠细胞凋亡率明显高于NS组（P<0.01），14、21日龄A3组明显高于A1、A2组（P<0.01），且A1组明显高于A2组（P<0.01）。Western Blot：14日龄A1、A2、A3组各日龄仔鼠BDNF、Caspase-3蛋白相对表达量均高于NS组（P<0.05），A1、A2组BDNF蛋白相对表达量高于A3组（P<0.05），A2组高于A1组（P<0.05），21日龄A2组与A1组比较无显著性差异（P>0.05）；A1、A3组Caspase-3蛋白相对表达量高于A2组（P<0.05），A3组明显高于A1组（P<0.05）。 结论：推测mNGF及联合康复训练均可通过促进BDNF蛋白表达降低Caspase-3的活性而抑制神经细胞凋亡， mNGF联合康复训练的效果优于单独应用mNGF。

Abstract:

      Abstract Objective：To analyze the effect of mouse nerve growth factor （mNGF） combined with rehabilitation training on the expression of BDNF and Caspase-3 in brain tissue of newborn rats with spastic cerebral palsy（SCP）. Method：A total of 150 clean-grade SD rats aged 7days were randomly divided into the control group （group NS， n=40） and the model group （group T， n=110）using a random number table. Group T received directional injection of absolute ethanol （1μl/g） into the left intracranial pyramidal tract at the age of 7 days， while the NS group received an equal volume of saline. 24 hours after injection （at 8 days old）， the spastic cerebral palsy rat model was determined by modified Li Xiaojie et al.’s criteria combined with Longa scoring method. Ninety successfully modeled SD rats in group T were randomly divided into group A1， A2 and A3, with 30 each. The intervention began on the 1st day of the experiment （8 days old）. Group A1 was given intramuscular injection of mNGF （30ng/g）， group A2 was given intramuscular injection of an equal dose of mNGF plus rehabilitation training， and group A3 was given intramuscular injection of an equal volume of saline（2.27ml/kg）. The group NS was given intramuscular injection of the equal volume of normal saline. Intervention lasted until 21 days of age. At 8， 14 and 21 days of age， HE staining was used to observe the dynamic changes in the morphology of cells in the internal capsule area of brain， TUNEL staining to observe the dynamic changes in the apoptosis rate of cells in the internal capsule area of brain， and Western Blot to monitor dynamic changes in the expression of BDNF and Caspase-3 proteins in the hippocampus. Result：Model preparation showed that a total of 97 spastic cerebral palsy litters were identified in group T at 8 days old, while no rats with cerebral palsy in group NS. TUNEL staining revealed that the apoptosis rates of the rats in group A1， A2 and A3 were significantly higher than in group NS at each age （P<0.01）， and were significantly higher in group A3 than in group A1 and A2 at 14 and 21 days of age （P<0.01）， with group A1 significantly higher than group A2 （P<0.01）. Western Blot showed that the relative expression of BDNF and Caspase-3 protein in group A1， A2 and A3 at 14 days old were higher than that in group NS（P<0.05）. The relative expression of BDNF protein in group A1 and A2 was higher than that in group A3（P<0.05）， with group A2 was higher than that in group A1（P<0.05）. There was no significant difference between group A2 and A1 at 21 days old（P>0.05）. The relative expression of Caspase-3 protein in group A1 and A3 was higher than that in group A2（P<0.05）. and group A3 was significantly higher than that in A1 group（P<0.05）. Conclusion：It is speculated that both mNGF and combined rehabilitation training can inhibit neuronal apoptosis by promoting BDNF protein expression and reducing the activity of Caspase-3. The effect of mNGF combined with rehabilitation training is better than that of mNGF alone.