小分子RNA干扰鞘氨醇激酶1表达对APP/PS1小鼠海马细胞凋亡的影响

Effects of Small Interfering RNA Targeting Sphingosine Kinase-1 Gene On Apoptosis of Hippocampal Neural Cells in APP/PS1 Mice

目的:观察腺病毒介导的鞘氨醇激酶1(sphk1)低表达对AD小鼠海马细胞凋亡的影响。方法:构建siRNA-Sphk1腺病毒载体,立体定位法将携带siRNA-Sphk1的腺病毒载体(siRNA-Sphk1组)或等量生理盐水(saline组)注射至APP/PS1双转基因AD模型鼠海马齿状回;30 d后,荧光显微镜下观察各组小鼠海马绿色荧光蛋白的表达,westen blot法观察海马Sphk1及caspase-3蛋白的表达,Tunel法观察海马齿状回细胞凋亡的情况。结果:siRNA-Sphk1组海马齿状回可见绿色荧光的表达,saline组无绿色荧光表达;siRNA-Sphk1组海马Sphk1蛋白的表达较saline组下降(P<0.05),caspase-3蛋白较saline组的表达升高(P<0.05);siRNA-sphk1组小鼠海马组织的细胞凋亡率较saline组和阴性对照组升高(P<0.05)。结论:成功构建siRNA-sphk1的重组腺病毒,移植后,可抑制AD小鼠海马Sphk1蛋白的表达,促进海马齿状回细胞的凋亡。

Objective：To observe the effects of the recombinant adenovirus vectors that expressed small interfering RNA （siRNA） against the sphingosine kinasel （sphkl） gene （siRNA-Sphkl） on the apoptosis of hippocampal neural cells in APP/PS1 double transgenic mice. Methods： The recombinant adenovirus vectors were constructed to express siRNA against the sphkl gene （siRNA-Sphkl）. The recombinant adenovirus vectors siRNA-Sphkl （siRNA-Sphkl group） or equal volume of saline （saline group） were stereotactic injected into the dentate gyrus of APP/PS1 double transgenic race. Thirty days later, the expression of green fluorescent protein （GFP）, sphkl protein and caspase-3 protein and the neuronal apoptosis in the dentate gyrus were examined. Results：The GFP expression was observed in the dentate gyrus of mice in siRNA-Sphkl group but not in the saline group. Compared with those in the saline group, the sphkl expression was significantly down-regulated and the caspase-3 expression was significantly up-regulated in siRNA-Sphkl group （P〈0.05）. The apoptosis rate in DG in the siRNA-Sphkl group was significantly higher than those in the saline and negative control groups （P〈0.05）. Conclusion： The recombinant adenovirus vectors siRNA-Sphkl were successfully constructed to silence SPhK1 gene and increase the apoptosis ofhippocampus in vivo.