小分子RNA干扰鞘氨醇激酶1表达对APP/PS1小鼠海马神经元的影响

Effects of small interfering RNA targeting sphingosine kinase-1 gene on the hippocampal neurons of the animal model of Alzheimer's disease

目的观察腺病毒介导的鞘氨醇激酶1(sphingosine kinase 1,sphk1)低表达对APP/PS1双转基因AD模型鼠海马超微结构的影响。方法立体定位法将携带sphk1-siRNA的腺病毒载体注射至APP/PS1双转基因AD模型鼠海马齿状回(dentate gyrus,DG)区,以1×1011pfu/ml的病毒总量分2点注射,对照组海马内注射等量生理盐水,30d后观察海马组织DG区新生神经元及超微结构的改变,检测SNAP-25蛋白表达含量及转染后1-磷酸鞘氨醇(sphingosine1-phospate,s1p)受体的改变。结果注射sphk1-siRNA的小鼠30d后海马DG区转染后电镜结果显示:与生理盐水组相比,siRNA组小鼠有髓神经纤维减少,细胞空泡变性,核质边缘化明显加重。且共聚焦结果显示阴性对照尚有部分新生神经元形成,Western-blot结果显示siRNA组SNAP-25蛋白表达含量明显低于对照组。Real-time PCR结果显示转染后siRNA组s1pr1表达明显低于其他两组,而s1pr3表达则明显高于其他两组。结论上述结果表明,sphk1-siRNA转染后,促进了APP/PS1双转基因AD模型鼠海马DG区的神经元的脱髓鞘变性,抑制了新生神经元的形成。

Objective To observe the effects of the recombinant adenovirus vector that expressed small interfering RNA （siRNA）against the sphkl gene （sphkl-siRNA）on he hippocampal neurons of APP/PS1 double transgenie mouse. Methods After the recombinant adenovirus vector containing the sphkl gene was successfully injected into the hippocam- pus,then effects of the vector that expressed small interfering RNA （siRNA） against the sphkl gene （ siRNA-sphkl ） on the hippoeampal of APP/PS1 double transgenic mouse 30 days after teansfection were examined. Results Compared with the two control group, there were an increase expression of SNAP-25 and the receptor 1 of S1P. Meanwhile,the degeneration degree of hippocampus in the siRNA-sphkl group was severer than other groups and no obvious newborn neurons can be seen in the dentate gyrus（DG）of hippocampus. Conclusions The results show that the recombinant adenovirus vector containing the sphklgene was successfully constructed,which can silence sphkl gene and increase the apoptosis of hippocampus in vivo.