腺相关病毒介导神经生长因子对大鼠脑损伤的保护作用

The protective effect of adeno-associated virus mediated nerve growth factor on brain injury in rats

目的探讨腺相关病毒介导神经生长因子(NGF)对大鼠脑损伤的保护作用及其机制。方法2018年11月到2019年11月,将购自河南省实验动物中心的45只SD随机分为对照组、模型组和NGF组,每组15只。采用自由落体法建立大脑急性脑损伤大鼠模型,对照组和模型组大鼠建模后经颅内注射携带空载体的腺相关病毒1×10^10 vg,NGF组大鼠建模后经颅内注射携带NGF的腺相关病毒1×10^10 vg,3组在治疗2周后,采用神经行为学评分评价大鼠神经功能症状,采用Morris水迷宫实验评估3组大鼠空间学习记忆能力;采用原位缺口末端标记法(TUNEL)染色分析3组大鼠脑组织凋亡,采用蛋白质印迹法(Western blot)分析3组大鼠脑组织中半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-3和B细胞淋巴瘤/白血病-2相关X蛋白(bax)蛋白表达水平。组间比较采用单因素方差分析。结果与模型组神经损伤程度评分[(11.24±3.99)分]比较,NGF组大鼠神经损伤程度评分[(6.09±4.11)分]显著下降,差异有统计学意义(t=3.011,P<0.05)。与模型组逃避潜伏期[(29.55±5.94)s]比较,NGF组大鼠逃避潜伏期[(17.19±3.84)s]显著下降,差异有统计学意义(t=3.415,P<0.05)。与模型组穿台指数(1.54±1.09)比较,NGF组大鼠穿台指数(3.58±2.10)显著下降,差异有统计学意义(t=3.001,P<0.05)。与模型组大鼠脑细胞凋亡率[(31.58±6.04)%]比较,NGF组大鼠大鼠脑细胞凋亡率[(15.87±4.28)%]显著下降,差异有统计学意义(t=3.841,P<0.05)。与对照组大鼠脑组织Caspase-3和bax表达水平(0.25±0.09、0.15±0.08)比较,模型组脑组织Caspase-3和bax表达水平显著增加(0.94±0.15、1.18±0.18),差异有统计学意义(t=3.491、2.192,P<0.05)。与模型组大鼠脑组织Caspase-3和bax表达水平(0.94±0.15、1.18±0.18)比较,NGF组大鼠脑组织Caspase-3和bax表达水平显著下降(0.39±0.12、0.28±0.14),差异有统计学意义(t=3.718、2.004,P<0.05)。结论腺相关病毒介导NGF在通过抑制脑组织细胞凋亡,可显著改善脑组织神经功能,提高学习和记忆能力。

Objective To observe the protective effect and molecular mechanism of adeno-associated virus mediated nerve growth factor(NGF)on brain injury in rats.Methods 45 SD rats from Henan experimental animal center during November 2018 to November 2019 were randomly divided into control group,model group and NGF group.After modeling,the rats in control group and model group were injected with 1×10^10 vg of adeno-associated virus carrying empty vector,and the rats in NGF group were injected with 1×10^10 vg of adeno-associated virus carrying NGF.After treatment for two weeks,the neurobehavioral score was used to evaluate the neurological symptoms in the three groups.The spatial learning and memory ability of the three groups were analyzed by Morris water maze test.The apoptosis of the three groups were analyzed by terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL)staining.The expression levels of cysteinyl aspartate-specific protease(Caspase)-3 and B cell lymphoma/leukemia-2 associated X protein(bax)protein in the three groups were analyzed by Western blotting.One-way ANOVA was used to compare the data between groups,and P<0.05 was statistically significant.Results Compared with the nerve injury degree score(11.24±3.99)in the model group,the nerve injury degree score in the NGF group(6.09±4.11)significantly decreased(t=3.011,P<0.05).Compared with the escape latency[(29.55±5.94)s]in the model group,the escape latency in the NGF group[(17.19±3.84)s]significantly decreased(t=3.415,P<0.05).Compared with index of crossing the platform in the model group(1.54±1.09),index of crossing the platform in the NGF group(3.58±2.10)significantly decreased(t=3.001,P<0.05).Compared with the model group[(31.58±6.04)%],The apoptosis rate in the NGF group[(15.87±4.28)%]significantly decreased(t=3.841,P<0.05).Compared with the expression level of Caspase-3 and bax(0.25±0.09,0.15±0.08),the expression level of Caspase-3 and bax in the model group(0.94±0.15,1.18±0.18)significantly increased(t=3.491,2.192,P<0.05).Compared with the expression level of Caspase-3 and bax in model group(0.94±0.15,1.18±0.18),the expression level of Caspase-3 and bax in NGF group(0.39±0.12,0.28±0.14)significantly decreased(t=3.718,2.004,P<0.05).Conclusion Adeno-associated virus mediated NGF can significantly improve the neural function of brain tissue and improve the ability of learning and memory by inhibiting the apoptosis of brain cells.