RNA干扰技术沉默星形胶质细胞Cdk5模型的建立
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摘要
目的建立星形胶质细胞细胞周期蛋白依赖性激酶5(Cdk5)沉默模型。方法新生24 h内无特定病原(SPF)级SD雄性大鼠乳鼠240只。培养及鉴定SD雄性大鼠星形胶质细胞。设计合成针对星形胶质细胞Cdk5的小干扰RNA(siRNA)3种(Si-r-Cdk5-001、Si-r-Cdk5-002及Si-r-Cdk5-003),将3种siRNA分为SiT1组、SiT2组、SiT3组,在Lipofectamine 2000介导下转染星形胶质细胞;阴性对照组加入阴性对照si RNA。应用荧光染色观察转染效率,通过实时聚合酶链反应(PCR)和Western blot检测沉默效率。结果成功将Cdk5 siRNA转染入星形胶质细胞,转染效率为85%。与阴性对照组相比,SiT1组、SiT2组及SiT3组Cdk5 mRNA及蛋白表达均降低(P<0.01),其中SiT3组沉默效果最佳,分子水平沉默效率为68%,蛋白水平沉默效率为79%。结论成功通过RNA干扰技术建立星形胶质细胞Cdk5特异性沉默模型。
Objective To establish a model of cyclin-dependent kinase 5(Cdk5) silence in astrocytes by RNA interference method. Methods A total of 240 24-hour newborn male SD rats with specific pathogen free(SPF) were enrolled, the astrocytes were cultured and identified. Three Cdk5 small interference RNA(siRNA) of Si-r-Cdk5-001(SiT1 group), Si-r-Cdk5-002(SiT2 group) and Si-r-Cdk5-003(SiT3 group) were designed and synthesized, and then transfected into astrocytes via Lipofecttamine2000. The negative control group was added negative control si RNA. The transfection effect was tested by fluorescence micro-scope and the silence efficiency was counted by real-time polymerase chain reaction(PCR) and Western blot. Results The Cdk5 siRNA sequence was transfected into astrocytes successfully, and the transfection efficiency was 85 %. The expression level of Cdk5 m RNA and protein in SiT1 group, SiT2 group and SiT3 group were significantly lower than that in control group(P < 0.01). The best efficiency of silence was appeared in Si T3 group, the molecular level silence efficiency was 68 % and protein level silence efficiency was 79 %. Conclusion It is demonstrated that the specific Cdk5 silencing model can be established by RNA interference technique.
Objective To establish a model of cyclin-dependent kinase 5(Cdk5) silence in astrocytes by RNA interference method. Methods A total of 240 24-hour newborn male SD rats with specific pathogen free(SPF) were enrolled, the astrocytes were cultured and identified. Three Cdk5 small interference RNA(siRNA) of Si-r-Cdk5-001(SiT1 group), Si-r-Cdk5-002(SiT2 group) and Si-r-Cdk5-003(SiT3 group) were designed and synthesized, and then transfected into astrocytes via Lipofecttamine2000. The negative control group was added negative control si RNA. The transfection effect was tested by fluorescence micro-scope and the silence efficiency was counted by real-time polymerase chain reaction(PCR) and Western blot. Results The Cdk5 siRNA sequence was transfected into astrocytes successfully, and the transfection efficiency was 85 %. The expression level of Cdk5 m RNA and protein in SiT1 group, SiT2 group and SiT3 group were significantly lower than that in control group(P < 0.01). The best efficiency of silence was appeared in Si T3 group, the molecular level silence efficiency was 68 % and protein level silence efficiency was 79 %. Conclusion It is demonstrated that the specific Cdk5 silencing model can be established by RNA interference technique.
引文
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[2]Hellmich MR,Pant HC,Wada E,et al.Neuronal cdc2-like kinase:a cdc2-related protein kinase with predominantly neuronal expression[J].Proc Natl Acad Sci USA,1992,89(22):10867-10871.
[3]Lew J,Winkfein RJ,Paudel HK,et al.Brain proline-directed protein kinase is a neurofilament kinase which displays high sequence homology to p34cdc2[J].J Biol Chem,1992,267(36):25922-25926.
[4]Mita N,He X,Sasamoto K,et al.Cyclin-dependent kinase 5regulates dendritic spine formation and maintenance of cortical neuron in the mouse brain[J].Cereb Cortex,2016,26(3):967-976.
[5]O’Hare MJ,Kushwaha N,Zhang Y,et al.Differential roles of nuclear and cytoplasmic cyclin-dependent kinase 5 in apoptotic and excitotoxic neuronal death[J].J Neurosci,2005,25(39):8954-8966.
[6]Zhang J,Cicero SA,Wang L,et al.Nuclear localization of Cdk5 is a key determinant in the postmitotic state of neurons[J].Proc Natl Acad Sci USA,2008,105(25):8772-8777.
[7]Liu Z,Chopp M.Astrocytes,therapeutic targets for neuroprotection and neurorestoration in ischemic stroke[J].Prog Neurobiol,2016,144:103-120.
[8]Wang W,Redecker C,Yu ZY,et al.Rat focal cerebral ischemia induced astrocyte proliferation and delayed neuronal death are attenuated by cyclin-dependent kinase inhibition[J].J Clin Neurosci,2008,15(3):278-285.
[9]Zhu Z,Zhang Q,Yu Z,et al.Inhibiting cell cycle progression reduces reactive astrogliosis initiated by scratch injury in vitro and by cerebral ischemia in vivo[J].Glia,2007,55(5):546-558.
[10]Ko J,Humbert S,Bronson RT,et al.p35 and p39 are essential for cyclin-dependent kinase 5 function during neurodevelopment[J].J Neurosci,2001,21(17):6758-6771.
[11]Shupp A,Casimiro MC,Pestell RG.Biological functions of CDK5 and potential CDK5 targeted clinical treatments[J].Oncotarget,2017,8(10):17373-17382.
[12]Ino H,Chiba T.Intracellular localization of cyclin-dependent kinase 5(CDK5)in mouse neuron:CDK5 is located in both nucleus and cytoplasm[J].Brain Res,1996,732(1-2):179-185.
[13]Shah K,Lahiri DK.Cdk5 activity in the brain-multiple paths of regulation[J].J Cell Sci,2014,127(Pt 11):2391-2400.
[14]Miyamoto Y,Yamauchi J,Chan JR,et al.Cdk5 regulates differentiation of oligodendrocyte precursor cells through the direct phosphorylation of paxillin[J].J Cell Sci,2007,120(Pt24):4355-4366.
[15]Luo F,Zhang J,Burke K,et al.The activators of cyclin-dependent kinase 5 p35 and p39 are essential for oligodendrocyte maturation,process formation,and myelination[J].J Neurosci,2016,36(10):3024-3037.
[16]Fang-Hu,Zhang HH,Yang BX,et al.Cdk5 contributes to inflammation-induced thermal hyperalgesia mediated by the p38 MAPK pathway in microglia[J].Brain Res,2015,1619:166-175.
[17]Glab N,Labidi B,Qin LX,et al.Olomoucine,an inhibitor of the cdc2/cdk2 kinases activity,blocks plant cells at the G1to S and G2 to M cell cycle transitions[J].FEBS Lett,1994,353(2):207-211.
[18]Meijer L,Borgne A,Mulner O,et al.Biochemical and cellular effects of roscovitine,a potent and selective inhibitor of the cyclin-dependent kinases cdc2,cdk2 and cdk5[J].Eur J Biochem,1997,243(1-2):527-236.
[19]Glicksman MA,Cuny GD,Liu M,et al.New approaches to the discovery of cdk5 inhibitors[J].Curr Alzheimer Res,2007,4(5):547-549.
[20]Paddison PJ,Silva JM,Conklin DS,et al.A resource for large-scale RNA-interference-based screens in mammals[J].Nature,2004,428(6981):427-431.
[21]Kumar V,Khare T,Shriram V,et al.Plant small RNAs:the essential epigenetic regulators of gene expression for saltstress responses and tolerance[J].Plant Cell Rep,2017 Sep 26.doi:10.1007/s00299-017-2210-4.