沉默Cdk5对星形胶质细胞活化增殖和细胞周期的影响
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摘要
目的观察沉默细胞周期蛋白依赖性激酶5(Cdk5)对星形胶质细胞活化增殖和细胞周期的影响。方法培养及鉴定SD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),将Cdk5 siRNA转染入星形胶质细胞,通过Real-time PCR和Western blot检测沉默效率;分为对照组及Cdk5 siRNA干预组,在不同时间点(3、6、12及24 h)采用Edu染色及流式细胞术检测细胞增殖及细胞周期情况。结果成功利用Cdk5 siRNA沉默星形胶质细胞Cdk5;Edu染色显示Cdk5 siRNA干预组干预3、6、12 h Edu染色阳性率较对照组显著降低(P<0.01);流式细胞术显示Cdk5 siRNA干预组干预3、6、12 h处于S期的星形胶质细胞比例较对照组显著降低(P<0.05)。结论沉默Cdk5可抑制星形胶质细胞的增殖及细胞周期进展,提示Cdk5在星形胶质细胞增殖过程中起到重要作用。
Objective To observe the effect of Cyclin-dependent kinase5( Cdk5) konckdown by RNA interfering on astrocyte proliferation and cell cycle progression. Methods Astrocytes were cultured from SD neonate rats and the purity of astrocytes were identified by immunofluorescence staining of glial fibrillary acidic protein. Cdk5 siRNA were designed and synthesized. Those siRNAs were tansfected into astrocytes via Lipofecttamine 2000 and the silence efficiency was counted by Real-time PCR and Western blot. Astrocytes were randomly divided into two groups:control group and Cdk5 siRNA group. At different time points( 3 h,6 h,12 h and 24 h). Edu staining and flow cytometry were used to measure the proliferation and cell cycle of astrocytes. Results Specificity Cdk5 silencing on astrocytes could be established by Cdk5 siRNA sequence successfully. Edu staining showed that the percentage of Edu positive cells in Cdk5 siRNA intervention group was significantly lower than that in the control group at 3 h,6 h and 12 h after intervention( P < 0. 01). flow cytometry showed that the percentage of astrocytes in S phase in Cdk5 siRNA intervention group was lower than that in the control group at 3 h,6 h and 12 h after intervention( P < 0. 05). Conclusion Knockdown of Cdk5 could inhibite the proliferation and cell cycle progression of astrocytes. It was suggested that Cdk5 might paly important roles in the proliferation of astrocytes.
Objective To observe the effect of Cyclin-dependent kinase5( Cdk5) konckdown by RNA interfering on astrocyte proliferation and cell cycle progression. Methods Astrocytes were cultured from SD neonate rats and the purity of astrocytes were identified by immunofluorescence staining of glial fibrillary acidic protein. Cdk5 siRNA were designed and synthesized. Those siRNAs were tansfected into astrocytes via Lipofecttamine 2000 and the silence efficiency was counted by Real-time PCR and Western blot. Astrocytes were randomly divided into two groups:control group and Cdk5 siRNA group. At different time points( 3 h,6 h,12 h and 24 h). Edu staining and flow cytometry were used to measure the proliferation and cell cycle of astrocytes. Results Specificity Cdk5 silencing on astrocytes could be established by Cdk5 siRNA sequence successfully. Edu staining showed that the percentage of Edu positive cells in Cdk5 siRNA intervention group was significantly lower than that in the control group at 3 h,6 h and 12 h after intervention( P < 0. 01). flow cytometry showed that the percentage of astrocytes in S phase in Cdk5 siRNA intervention group was lower than that in the control group at 3 h,6 h and 12 h after intervention( P < 0. 05). Conclusion Knockdown of Cdk5 could inhibite the proliferation and cell cycle progression of astrocytes. It was suggested that Cdk5 might paly important roles in the proliferation of astrocytes.
引文
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[15]Zhu J.Li W and Mao Z[J].Cdk5:mediator of neuronal development,death and the response to DNA damage.Mech Ageing Dev,2011,132(8/9):389-394.
[16]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.
[17]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 U S A,2008,105(25):8772-8777.
[18]Cheung ZH.Gong K and Ip NY[J].Cyclin-dependent kinase 5supports neuronal survival through phosphorylation of Bcl-2.J Neurosci,2008,28(19):4872-4877.
[19]Zhang J.Li H and herrup K[J].Cdk5 nuclear localization is p27-dependent in nerve cells:implications for cell cycle suppression and caspase-3 activation.J Biol Chem,2010,285(18):14052-14061.
[20]Glab N,Labidi B,Qin LX,et al.Olomoucine,an inhibitor of the CDC2/CDK2 kinases activity,blocks plant-cells at the G1 to S and G2 to M cell-cycle transitions[J].FEBS Lett,1994,353(2):207-211.
[21]Meijer L,Borgne A,Mulner O,et al.Biochemical and cellular effects of roscovitine,a potent and selective inhibitor of the cyclindependent kinases cdc2,cdk2 and cdk5[J].Eur J Biochem,1997,243(1/2):527-536.
[22]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.
[23]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.
[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 U S A,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 P34(CDC2)[J].J Biol Chem,1992,267(36):25922-25926.
[4]Dhavan R,Tsai LH.A decade of CDK5[J].Nat Rev Mol Cell Biol,1994,2(10):51-52.
[5]Shelton SB,Johnson GV.Cyclin-dependent kinase-5 in neurodegeneration[J].J Neurochem,1994,88(6):51-52.
[6]Lagace DC,Benavides DR,Kansy JW,et al.Cdk5 is essential for adult hippocampal neurogenesis[J].Proc Natl Acad Sci U S A,2008,105(47):18567-18571.
[7]Lai KO,Ip NY.Recent advances in understanding the roles of Cdk5 in synaptic plasticity[J].Biochim Biophys Acta,1994,1792(8):51-52.
[8]Di Giovanni S,Knoblach SM,Brandoli C,et al.Gene profiling in spinal cord injury shows role of cell cycle neuronal death[J].Ann Neurol,2003,53(4):454-468.
[9]Liu DX,Greene LA.Regulation of neuronal survival and death by E2F-dependent gene repression and derepression[J].Neuron,1994,32(3):51-52.
[10]Pekny M,Nilsson M.Astrocyte activation and reactive gliosis[J].Glia,2005,50(4,SI):427-434.
[11]Wang W,Bu BT,Xie MJ,et al.Neural cell cycle dysregulation and central nervous system diseases[J].Prog Neurobiol,2009,89(1):1-17.
[12]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].Journal of Clinical Neuroscience,2008,15(3):278-285.
[13]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.
[14]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.
[15]Zhu J.Li W and Mao Z[J].Cdk5:mediator of neuronal development,death and the response to DNA damage.Mech Ageing Dev,2011,132(8/9):389-394.
[16]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.
[17]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 U S A,2008,105(25):8772-8777.
[18]Cheung ZH.Gong K and Ip NY[J].Cyclin-dependent kinase 5supports neuronal survival through phosphorylation of Bcl-2.J Neurosci,2008,28(19):4872-4877.
[19]Zhang J.Li H and herrup K[J].Cdk5 nuclear localization is p27-dependent in nerve cells:implications for cell cycle suppression and caspase-3 activation.J Biol Chem,2010,285(18):14052-14061.
[20]Glab N,Labidi B,Qin LX,et al.Olomoucine,an inhibitor of the CDC2/CDK2 kinases activity,blocks plant-cells at the G1 to S and G2 to M cell-cycle transitions[J].FEBS Lett,1994,353(2):207-211.
[21]Meijer L,Borgne A,Mulner O,et al.Biochemical and cellular effects of roscovitine,a potent and selective inhibitor of the cyclindependent kinases cdc2,cdk2 and cdk5[J].Eur J Biochem,1997,243(1/2):527-536.
[22]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.
[23]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.