神经束蛋白155高表达少突胶质细胞模型的构建
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摘要
目的构建神经束蛋白155(neurofascin155,NF155)高表达的少突胶质细胞模型。方法 RT-PCR法提取NF155 c DNA,经T-A克隆及双酶切将其与p UM-T simple重组,构建NF155慢病毒载体质粒,NheⅠ、AgeⅠ双酶切后,片段大小及基因测序均显示NF155慢病毒载体质粒构建成功。取对数生长期少突胶质细胞分为空白对照组、阴性慢病毒感染组、NF155慢病毒感染组,空白对照组不做任何处理,阴性慢病毒感染组予10μL空载体慢病毒+100μL培养基,NF155慢病毒感染组予10μL慢病毒载体质粒+100μL培养基。培养12 h采用荧光定量PCR法检测NF155 mRNA,采用Western blotting法检测NF155蛋白。结果 NF155慢病毒感染组、空白对照组、阴性慢病毒感染组NF155 mRNA相对表达量分别为3. 25±0. 11、1. 00±0. 06、1. 08±0. 01,与空白对照组、阴性慢病毒感染组比较,NF155慢病毒感染组NF155 mRNA相对表达量升高(P均<0. 05)。NF155慢病毒感染组、空白对照组、阴性慢病毒感染组NF155蛋白相对表达量分别为1. 57±0. 08、0. 51±0. 12、0. 50±0. 06,与空白对照组、阴性慢病毒感染组比较,NF155慢病毒感染组NF155蛋白相对表达量升高(P均<0. 05)。结论成功构建NF155高表达的少突胶质细胞瞬转细胞模型。
Objective To constructthe oligodendrocytes model with high expression of neurofascin 155( NF155).Methods NF155 c DNA was cloned by RT-PCR and inserted into p UM-T simple vector by T-A clone methodand double digestion to construct a lentivirus vector plasmid. Nhe I and Age I were used to cut off the target gene,the size of the fragment and gene sequencing showed that the lentivirus vector plasmid was successfully constructed. Oligodendrocytes in logarithmic growth phase were divided into the blank control group,negative lentivirus infection group,and NF155 lentivirus infection group. The blank control group was not treated,the negative lentivirus infection group was treated with 10 μL empty vector lentivirus + 100 μL medium,and the NF155 lentivirus infection group with 10 μL lentivirus vector plasmid +100 μL medium. The mRNA and protein expression levels of NF155 were detected by real-time PCRand Western blotting.Results The relative expression levels of NF155 mRNA in the NF155 lentivirus infection group,blank control group and negative lentivirus infection group were 3. 25 ± 0. 11,1. 00 ± 0. 06,and 1. 08 ± 0. 01,respectively. The relative expression of NF155 mRNA in the NF155 lentivirus infection group was higher than that in the blank control group and negative lentivirus infection group( P < 0. 05). The relative expression levelsof NF155 protein in the lentivirus infection group,blank control group,and negative lentivirus infection group were 1. 57 ± 0. 08,0. 51 ± 0. 12,and 0. 50 ± 0. 06,respectively. The lentivirus infection group had higher relative expression of NF155 proteinthan the blank control group and negative lentivirus infection group( P < 0. 05). Conclusion The oligodendrocytes transient cell model with high expression of NF155 is successfully constructed.
Objective To constructthe oligodendrocytes model with high expression of neurofascin 155( NF155).Methods NF155 c DNA was cloned by RT-PCR and inserted into p UM-T simple vector by T-A clone methodand double digestion to construct a lentivirus vector plasmid. Nhe I and Age I were used to cut off the target gene,the size of the fragment and gene sequencing showed that the lentivirus vector plasmid was successfully constructed. Oligodendrocytes in logarithmic growth phase were divided into the blank control group,negative lentivirus infection group,and NF155 lentivirus infection group. The blank control group was not treated,the negative lentivirus infection group was treated with 10 μL empty vector lentivirus + 100 μL medium,and the NF155 lentivirus infection group with 10 μL lentivirus vector plasmid +100 μL medium. The mRNA and protein expression levels of NF155 were detected by real-time PCRand Western blotting.Results The relative expression levels of NF155 mRNA in the NF155 lentivirus infection group,blank control group and negative lentivirus infection group were 3. 25 ± 0. 11,1. 00 ± 0. 06,and 1. 08 ± 0. 01,respectively. The relative expression of NF155 mRNA in the NF155 lentivirus infection group was higher than that in the blank control group and negative lentivirus infection group( P < 0. 05). The relative expression levelsof NF155 protein in the lentivirus infection group,blank control group,and negative lentivirus infection group were 1. 57 ± 0. 08,0. 51 ± 0. 12,and 0. 50 ± 0. 06,respectively. The lentivirus infection group had higher relative expression of NF155 proteinthan the blank control group and negative lentivirus infection group( P < 0. 05). Conclusion The oligodendrocytes transient cell model with high expression of NF155 is successfully constructed.
引文
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[10] Rathjen FG,Wolff JM,Chang S,et al. Neurofascin:a novelc chick cell-surface glycoprotein involved in neurite-neurite interactions[J]. Cell,1987,51(5):841-849.
[11]陈丽华,金伯全.免疫球蛋白超家族分子在神经系统中的分布及功能[J].细胞生物学杂志,2000,22(2):63-67.
[12]Lin HP,Ho KWD,Chuquilin M. Presence of both anti-contactin 1and anti-neurofascin 140 antibodies in a case of chronic inflammatory demyelinating polyneuropathy[J]. e NeurologicalSci,2018,13:38-39.
[13]Kira JI,Yamasaki R,Ogata H. Anti-neurofascin autoantibody and demyelination[J]. Neurochem Int,2018,186(18):30433-30439.
[14]Zonta B,Desmazieres A,Rinaldi A,et al. A critical role for neurofascin in regulating action potential initiation through maintenance of the axon initial segment[J]. Neuron,2011,69(5):945-956.
[15]Lustig M,Zanazzi G,Sakurai T,et al. Nr-CAM and neurofascin interactions regulate ankyrin G and sodium channel clustering at the node of Ranvier[J]. Curr Biol,2001,11(23):1864-1869.
[16]Feinberg K,Eshed-Eisenbach Y,Frechter S,et al. A glial signal consisting of gliomedin and Nr CAM clusters axonal Na+channels during the formation of nodes of Ranvie[J]. Neuron,2010,65(4):490-502.
[17]Kriebel M,Wuchter J,Trinks S,et al. Neurofascin:a switch between neuronal plasticity and stability[J]. Int J Biochem Cell Biol,2012,44(5):694-697.
[18]Pomicter AD,Shroff SM,Fuss B,et al. Novel forms of neurofascin155 in the central nervous system:alterations in paranodal disruption models and multiple sclerosis[J]. Brain,2010,133(Pt2):389-405.
[19]Sherman DL,Tait S,Melrose S,et al. Neurofascins are required to establish axonal domains for saltatory conduction[J]. Neuron,2005,48(5):737-742.
[20]Mathey EK,Derfuss T,Storch MK,et al. Neurofascin as a novel target for autoantibody-mediated axonal injury[J]. J Exp Med,2007,204(10):2363-2372.
[21]Pang MS,Chen X,Lu B,et al. Lentiviral vector-mediated doxycycline-inducible i ASPP gene targeted RNA interference in hepatocellular carcinoma[J]. Chin J Cancer,2010,29(9):796-801.
[22]李文怡,范余娟,徐红,等.人SNCG基因shRNA慢病毒载体质粒的构建及鉴定[J].山东医药,2016,56(26):13-16.
[2]Roche SL,Sherman DL,Dissanayake K,et al. Loss of glial neurofascin155 delays developmental synapse elimination at the neuromuscular junction[J]. J Neurosci,2014,34(38):12904-12918.
[3]Ng JK,Malotka J,Kawakami N,et al. Neurofascin as a target for autoantibodies in peripheral neuropathies[J]. Neurology,2012,79(23):2241-2248.
[4] Tajima Y,Matsumura M,Yaguchi H,et al. Possible combined central and peripheral demyelination presenting as optic neuritis,cervical myelitis,and demyelinating polyneuropathy with marked nerve hypertrophy[J]. Intern Med,2018,57(6):867-871.
[5]Zhang YP,Huang QL,Zhao CM,et al. GM1 improves neurofascin155 association with lipid rafts and prevents rat brain myelin injury after hypoxia-ischemia[J]. Braz J Med Biol Res,2011,44(6):553-561.
[6]Purger D,Gibson EM,Monje M. Myelin plasticity in the central nervous system[J]. Neuropharmacology,2016,110(Pt B):563-573.
[7]Kawabata S,Takano M,Numasawa-Kuroiwa Y,et al. Grafted humans i PS cell-derived oligodendrocyte precursor cell contribute to robust remyelination of demyelinated axons after spinal cord injury[J]. Stem Cell Reports,2016,6(1):1-8.
[8]Cortese A,Devaux JJ,Zardini JJ,et al. Neurofascin-155 as a putative antigen in combined central and peripheral demyelination[J]. Neurol Neuroimmunol Neuroinflamm,2016,3(4):e238.
[9]张雨平,黄其林. NF155及其在脑白质损伤疾病中的研究[J].中华神经外科疾病研究杂志,2009,8(5):475-477.
[10] Rathjen FG,Wolff JM,Chang S,et al. Neurofascin:a novelc chick cell-surface glycoprotein involved in neurite-neurite interactions[J]. Cell,1987,51(5):841-849.
[11]陈丽华,金伯全.免疫球蛋白超家族分子在神经系统中的分布及功能[J].细胞生物学杂志,2000,22(2):63-67.
[12]Lin HP,Ho KWD,Chuquilin M. Presence of both anti-contactin 1and anti-neurofascin 140 antibodies in a case of chronic inflammatory demyelinating polyneuropathy[J]. e NeurologicalSci,2018,13:38-39.
[13]Kira JI,Yamasaki R,Ogata H. Anti-neurofascin autoantibody and demyelination[J]. Neurochem Int,2018,186(18):30433-30439.
[14]Zonta B,Desmazieres A,Rinaldi A,et al. A critical role for neurofascin in regulating action potential initiation through maintenance of the axon initial segment[J]. Neuron,2011,69(5):945-956.
[15]Lustig M,Zanazzi G,Sakurai T,et al. Nr-CAM and neurofascin interactions regulate ankyrin G and sodium channel clustering at the node of Ranvier[J]. Curr Biol,2001,11(23):1864-1869.
[16]Feinberg K,Eshed-Eisenbach Y,Frechter S,et al. A glial signal consisting of gliomedin and Nr CAM clusters axonal Na+channels during the formation of nodes of Ranvie[J]. Neuron,2010,65(4):490-502.
[17]Kriebel M,Wuchter J,Trinks S,et al. Neurofascin:a switch between neuronal plasticity and stability[J]. Int J Biochem Cell Biol,2012,44(5):694-697.
[18]Pomicter AD,Shroff SM,Fuss B,et al. Novel forms of neurofascin155 in the central nervous system:alterations in paranodal disruption models and multiple sclerosis[J]. Brain,2010,133(Pt2):389-405.
[19]Sherman DL,Tait S,Melrose S,et al. Neurofascins are required to establish axonal domains for saltatory conduction[J]. Neuron,2005,48(5):737-742.
[20]Mathey EK,Derfuss T,Storch MK,et al. Neurofascin as a novel target for autoantibody-mediated axonal injury[J]. J Exp Med,2007,204(10):2363-2372.
[21]Pang MS,Chen X,Lu B,et al. Lentiviral vector-mediated doxycycline-inducible i ASPP gene targeted RNA interference in hepatocellular carcinoma[J]. Chin J Cancer,2010,29(9):796-801.
[22]李文怡,范余娟,徐红,等.人SNCG基因shRNA慢病毒载体质粒的构建及鉴定[J].山东医药,2016,56(26):13-16.