腺相关病毒介导Parkin对帕金森模型大鼠的治疗作用

Adenovirus-mediated therapeutic effect of Parkin on Parkinson’s model rats

目的探讨腺相关病毒介导Parkin对帕金森模型大鼠的治疗作用。方法 60只SD大鼠随机分为对照组、模型组和Parkin组, 模型组和Parkin组大鼠采用颈背部皮下注射鱼滕酮乳化液建立帕金森(PD)模型。Parkin组大鼠在建模后于左侧黑质定向注射携带Parkin基因的腺相关病毒, 对照组和模型组经左侧黑质定向注射携带空载体的阴性腺相关病毒。在注射21 d后, 对3组大鼠进行行为学分析;并处死大鼠, 采用原位缺口末端标记法(TUNEL)分析3组大鼠黑质细胞凋亡指数;采用超氧化物歧化酶(SOD)和谷胱甘肽(GSH)-px试剂盒检测SOD和GSH-px水平;采用蛋白质印迹法(Western blot)分析凋亡相关蛋白半胱氨酰天冬氨酸特异性蛋白酶-3(Caspase-3)、B细胞淋巴瘤/白血病-2(bcl-2)和bcl-2相关X蛋白(bax)的水平。不同处理比较采用方差分析, 组间比较采用LSD法。结果 Parkin组大鼠脑组织黑质区Parkin蛋白表达水平(1.54±0.18)明显高于对照组和模型组(0.81±0.11、0.89±0.12), 差异有统计学意义(t=9.257、8.014, P<0.05)。Parkin组大鼠10 min内旋转圈数[(4.87±0.85)圈]明显低于模型组[(12.25±3.21)圈], 差异有统计学意义(t=4.264, P<0.05)。Parkin组大鼠脑组织黑质区细胞凋亡指数[(10.67±4.91)%]明显低于模型组[(34.59±8.32)%], 差异有统计学意义(t=3.698, P<0.05)。Parkin组大鼠外周血清SOD和GSH-px酶活性[(3.87±0.61)、(7.98±1.27) U/ml]明显高于模型组[(2.18±0.51)、(4.10±1.09) U/ml], 差异有统计学意义(t=6.594、7.251, P<0.05)。Parkin组大鼠脑组织Caspase-3和bax水平(1.12±0.08、1.23±0.06)明显低于模型组大鼠脑组织Caspase-3和bax水平(1.58±0.08、1.69±0.11), 差异有统计学意义(t=8.174、9.241, P<0.05)。Parkin组大鼠大鼠脑组织bcl-2水平(1.38±0.10)高于模型组(1.02±0.08), 差异均有统计学意义(t=5.141, P<0.05)。结论采用腺相关病毒介导Parkin蛋白在帕金森大鼠黑质表达, 可以显著抑制细胞凋亡, 显著改善大鼠的行为。

Objective To explore the therapeutic effect of adeno-associated virus-mediated Parkin on Parkinson’s model rats.Methods A total of 60 SD rats were randomly divided into a control group,a model group,and a Parkin group.Parkinson’s disease(PD)model was established by subcutaneous injection of fisetin emulsion into the neck and back in the model group and Parkin group.After modeling,the rats in the Parkin group were injected with adeno-associated viruses carrying the Parkin gene in the left substantia nigra.The rats in the control and model groups were injected with negative adeno-associated viruses carrying empty vectors in the left substantia nigra.The behavioral analysis was done on three groups of rats after 21 days of injection.The rats were euthanized,and the apoptosis index of substantia nigra cells in the three groups was analyzed using terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL).The superoxide dismutase(SOD)and glutathione(GSH)-px levels were analyzed using SOD and GSH-px assay kits.The levels of apoptosis related proteins[cysteinyl aspartate-specific protease-3(Caspase-3),B cell lymphoma/leukemia-2(bcl-2),and bcl-2 associated X protein(bax)]were detected by Western blotting.Different treatments were compared using analysis of variance,and inter-group comparisons were made using LSD.Results The expression level of Parkin protein in the substantia nigra of rats in the Parkin group(1.54±0.18)was significantly higher than that in the control group and model group(0.81±0.11,0.89±0.12,t=9.257,8.014,P<0.05).The number of rotations within 10 min in the Parkin group[(4.87±0.85)circles]was significantly less than that in the model group[(12.25±3.21)circles,t=4.264,P<0.05].The apoptosis index of substantia nigra cells in the Parkin group[(10.67±4.91)%]was significantly less than that in the model group[(34.59±8.32)%,t=3.698,P<0.05].The peripheral serum SOD and GSH-px enzyme activities in the Parkin group[(3.87±0.61),(7.98±1.27)U/ml]were significantly higher than those in the model group[(2.18±0.51),(4.10±1.09)U/ml,t=6.594,7.251,P<0.05].The levels of Caspase-3 and bax in the brain tissue of Parkin group rats(1.12±0.08,1.23±0.06)were significantly lower than those in the model group rats(1.58±0.08,1.69±0.11,t=8.174,9.241,P<0.05).The level of bcl-2 in the brain tissue of Parkin group rats(1.38±0.10)was higher than that of the model group(1.02±0.08,t=5.141,P<0.05).Conclusion Adenovirus-mediated expression of Parkin protein in the substantia nigra of Parkinson’s rats can significantly inhibit cell apoptosis,and significantly improve rat behavior.