大鼠ZnT1基因siRNA慢病毒载体构建及筛选
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摘要
目的:构建并筛选针对SD大鼠锌离子转运体1(ZnT1)的siRNA慢病毒载体。并检测其在大鼠神经元中的作用。方法:设计并合成针对ZnT1基因的3条(A,B,C)siRNA序列与1条阴性对照序列,将以上序列退火与pFU-GW-iRNA质粒相连接,通过测序鉴定后,分别与慢病毒包装质粒共感染293T细胞,检测收获病毒滴度后感染体外培养的SD大鼠脑皮层神经细胞,设置感染复数(MOI)分别为1,3,6,8,于转染后72 h计算转染效率。在最佳MOI值下,设置未经病毒感染组(CON组)、阴性对照病毒感染组(NC组)和慢病毒阳性干扰组(ZnT1-siR-NA-A组,ZnT1-siRNA-B组,ZnT1-siRNA-C组),72 h后利用Western blot技术检测ZnT1的表达情况,确定对ZnT1蛋白的最有效的干扰序列与抑制效率。结果:测序证实目的干扰序列已被准确克隆到pFU-GW-iRNA质粒,收获的慢病毒颗粒滴度为8×108TU/ml,其在MOI=6时对神经细胞的转染效率最高(93%),并保持神经细胞良好的生存状态。转染后ZnT1的表达被不同程度的抑制,A,B,C三种干扰序列对ZnT1的抑制率分别为47.74%±1.40%,81.19%±1.36%,94.10%±2.41%。结论:成功构建了ZnT1-siRNA慢病毒载体,其在MOI=6时对神经细胞具有最佳的转染效率,并有效沉默神经细胞中ZnT1蛋白的表达。
Objective: To construct and screening lentiviral vectors of RNA interference(RNAi) targeting ZnT1 gene of SD rats and detect the effect of the vector in rat neurons.Methods: Three target sequences of ZnT1(A,B,C) and a negative control sequence were developed and cloned into the pFU-GW-iRNA vector,then the results were confirmed by DNA sequencing.Recombinant lentivirus vectors were respectively transfected into 293T cells by cotransfection of packaging plasmids and then the virus titer of harvested lentivirus was determined.The cerebral cortical neurons of SD rats were transfected under the different MOI conditions(MOI=1,3,6,8) for 72 h to detect the transfection efficiency.Under the best MOI,the transfected neurons were divided into cells without transfection group(CON group),negative control group(NC group) and cells with positive transfection group(ZnT1-siRNA-A group,ZnT1-siRNA-B group,ZnT1-siRNA-C group).72 h after transfected,the changes of ZnT1 protein was detected by Western blot to detect the most efficient RNAi sequence and the inhibition efficiency.Results: The result showed that target sequences were accurately cloned into pFU-GW-iRNA plasmid.The titer of harvested lentivirus was 8×108 TU/ml,the transfection efficiency was best at MOI=6(93%) and the neurons were at a good condition as well.The ZnT1 expression was inhibited to different degrees by A,B,C sequences after transfection,the inhibition effects were about 47.74%±1.40%,81.19%±1.36%,94.10%±2.41% respectively.Conclusion: ZnT1-siRNA lentivirus vector has been constructed successfully,MOI=6 is the best transfect condition in neurons and it can effectively silence the ZnT1 expression in neurons.
Objective: To construct and screening lentiviral vectors of RNA interference(RNAi) targeting ZnT1 gene of SD rats and detect the effect of the vector in rat neurons.Methods: Three target sequences of ZnT1(A,B,C) and a negative control sequence were developed and cloned into the pFU-GW-iRNA vector,then the results were confirmed by DNA sequencing.Recombinant lentivirus vectors were respectively transfected into 293T cells by cotransfection of packaging plasmids and then the virus titer of harvested lentivirus was determined.The cerebral cortical neurons of SD rats were transfected under the different MOI conditions(MOI=1,3,6,8) for 72 h to detect the transfection efficiency.Under the best MOI,the transfected neurons were divided into cells without transfection group(CON group),negative control group(NC group) and cells with positive transfection group(ZnT1-siRNA-A group,ZnT1-siRNA-B group,ZnT1-siRNA-C group).72 h after transfected,the changes of ZnT1 protein was detected by Western blot to detect the most efficient RNAi sequence and the inhibition efficiency.Results: The result showed that target sequences were accurately cloned into pFU-GW-iRNA plasmid.The titer of harvested lentivirus was 8×108 TU/ml,the transfection efficiency was best at MOI=6(93%) and the neurons were at a good condition as well.The ZnT1 expression was inhibited to different degrees by A,B,C sequences after transfection,the inhibition effects were about 47.74%±1.40%,81.19%±1.36%,94.10%±2.41% respectively.Conclusion: ZnT1-siRNA lentivirus vector has been constructed successfully,MOI=6 is the best transfect condition in neurons and it can effectively silence the ZnT1 expression in neurons.
引文
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[17]Nowak G,Legutko B,Szewczyk B,et al.Zinc treatment inducescortical brain-derived neurotrophic factor gene expression[J].Eur JPharmacol,2004,492:57-59.
[18]Hwang JJ,Park MH,Choi SY,et al.Activation of the Trk signa-ling pathway by extracellular zinc.Role of metalloproteinases[J].JBiol Chem,2005,280:11995-12001.
[19]Jayaraman AK,Jayaraman S.Increased level of exogenous zinc in-duces 137 cytotoxicity and up-regulates the expression of the ZnT-1zinc transporter 138 gene in pancreatic cancer cells[J].J Nutr Bio-chem,2011,22:79-88.
[2]Koh JY,Suh SW,Gwag BJ,et al.The role of zinc in selective neu-ronal death after transient global cerebral ischemia[J].Science,1996,272:1013-1016.
[3]Wang Y,Me X,Zhang L,et al.Supplement moderate zinc as aneffective treatment for spinal cord injury[J].Med Hypotheses,2011,77:589-590.
[4]Wang Y,Mei X,Zhang L,et al.The correlation among the dynam-ic change of Zn(2+),ZnT-1,and brain-derived neurotrophic fac-tor after acute spinal cord injury in rats[J].Biol Trace Elem Res,2011,143:351-358.
[5]温丽君,滕军放,赵莘瑜,等.APP695-siRNA慢病毒载体构建及其对SH-SY5Y细胞中APP695基因的沉默作用[J].郑州大学学报(医学版),2011,46:520-523.
[6]孙丹丹,李昌,李太元等.HIV-1包膜蛋白ENV慢病毒载体的构建及表达[J].中国免疫学杂志,20l2,28:440-443.
[7]赖建明,林建华,林文平等.猪TGF-β1基因重组慢病毒载体的构建及其在BMSCs中的表达[J].中国修复重建外科杂志,2012,26:849-854.
[8]Cao S,Wu C,Yang Y,et al.Lentiviral vector-mediated stable ex-pression of sTNFR-Fc in human macrophage and neuronal cells asapotential therapy for neuroAIDS[J].J Neuroinflammation,2011,8:48.
[9]Anesti AM,Simpson GR,Price T,et al.Expression of RNA inter-ference triggers from an oncolytic herpes simplex virus results in spe-cific silencing in tumour cells in vitro and tumours in vivo[J].BMCCancer,2010,10:486.
[10]曾东风,陈幸华,孔佩艳,等.PINC-1基因RNAi慢病毒载体的构建及鉴定[J].重庆医学,2011,40:3029-3031.
[11]Altar CA,Cai N,Bliven T,et al.Anterograde transport of brain-derived neurotrophic factor and its role in the brain[J].Nature,1997,389:856-860.
[12]Garraway SM,Turtle JD,Huie JR,et al.Intermittent noxious stim-ulation following spinal cord contusion injury impairs locomotor re-covery and reduces spinal brain-derived neurotrophic factor-tropomy-osin-receptor kinase signaling in adult rats[J].Neuroscience,2011,199:86-102.
[13]Yi H,Hu J,Qian J,et al.Expression of brain-derived neurotro-phic factor is regulated by the Wnt signaling pathway[J].Neurore-port,2012,23:189-194.
[14]Sharma HS.Selected combination of neurotrophins potentiate neuro-protection and functional recovery following spinal cord injury in therat[J].Acta Neurochir Suppl,2010,106:295-300.
[15]Guo L,Hu X,Xu T,et al.Over-expression of Zip-13 mRNA inkidney and lung during dietary zinc deficiency in Wistar rats[J].Mol Biol Rep,2011,38:1869-1874.
[16]Fang HY,Jiang YG,Liu J,et al.Effects of zinc deficiency on thecAMP/PKA-CREB-BDNF signaling pathway of hippocampus andcortex in rats[J].Acta Nutrimenta Sinica,2008,30:153-156.
[17]Nowak G,Legutko B,Szewczyk B,et al.Zinc treatment inducescortical brain-derived neurotrophic factor gene expression[J].Eur JPharmacol,2004,492:57-59.
[18]Hwang JJ,Park MH,Choi SY,et al.Activation of the Trk signa-ling pathway by extracellular zinc.Role of metalloproteinases[J].JBiol Chem,2005,280:11995-12001.
[19]Jayaraman AK,Jayaraman S.Increased level of exogenous zinc in-duces 137 cytotoxicity and up-regulates the expression of the ZnT-1zinc transporter 138 gene in pancreatic cancer cells[J].J Nutr Bio-chem,2011,22:79-88.