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目的确定细胞周期依赖性蛋白激酶-5(cyclin dependent kinases-5,cdk5)在C型尼曼-皮克病(Niemann-Pickdisease type C,NPC)神经元变性过程中的作用,为NPC的临床治疗提供可能的药物作用靶点。方法将针对cdk5基因的si RNA序列克隆入rAAV-2载体,对4周龄npc-/-小鼠进行小脑rAAV-cdk5-si RNA-GFP注射(n=8),以空载体病毒(rAAV-GFP)注射组及非手术的同龄npc小鼠为对照(n=8)。在干预后4周连续观察小鼠脑内cdk5水平及小鼠神经生化和病理的改变。结果注射后1周可见针道附近、小脑深部核团及浦肯野细胞出现绿色荧光蛋白表达。与空载体病毒组相比,注射4周后小鼠脑内cdk5的表达水平降低34.17%(P<0.05,n=6);轴突球状体数量减少30.76%(P<0.05,n=6),存活浦肯野细胞数增加300%(P<0.05,n=8);细胞骨架蛋白磷酸化程度降低16.32%(P<0.05,n=6)。结论rAAV2-cdk5-si RNA小脑注射,能显著减轻npc小鼠神经生化和病理改变。cdk5的激活在NPC神经元变性过程中起到了关键性的作用,有可能成为NPC治疗的新靶标。
Objective To determine the role of cdk5 in the degeneration of neurons in C-type Niemann-Pick disease type C (NPC) Provide a potential drug target. Methods The si RNA sequence of cdk5 gene was cloned into rAAV-2 vector and rAAV-cdk5-si RNA-GFP (n = 8) was injected into 4 weeks old npc - / - GFP injection group and non-surgical npc mice of the same age as control (n = 8). The level of cdk5 in mice brain and the changes of neurobiochemical and pathological changes were observed 4 weeks after the intervention. Results One week after injection, green fluorescent protein (EGFP) expression appeared in the vicinity of the needle path, deep nuclei of cerebellum and Purkinje cells. Compared with control group, the expression of cdk5 in mouse brain decreased 34.17% (P <0.05, n = 6) 4 weeks after injection; the number of axonal spheroids decreased 30.76% (P <0.05, n = 6) (P <0.05, n = 8). The phosphorylation of cytoskeletal protein decreased by 16.32% (P <0.05, n = 6). Conclusions Cerebellar injection of rAAV2-cdk5-si RNA can significantly reduce the neurobiochemical and pathological changes of npc mice. The activation of cdk5 plays a crucial role in the degeneration of NPC neurons and may become a new target for the treatment of NPC.