Nogo-A及NgR在墨西哥钝口螈脑组织中的表达及意义
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摘要
目的探讨勿动蛋白A(Nogo-A)及勿动蛋白受体(NgR)在正常墨西哥钝口螈及其中脑胆碱能神经元损伤后脑组织中的表达。方法以正常墨西哥钝口螈脑组织和中脑胆碱能神经元损伤后第3、7、14、21天脑组织作为研究对象,采用免疫荧光技术观察正常墨西哥钝口螈脑组织中Nogo-A及NgR的表达;分别用荧光定量PCR和蛋白质印迹法(WB法)检测正常墨西哥钝口螈和损伤组脑组织中Nogo-A及NgR的mRNA含量和蛋白含量的变化。结果正常墨西哥钝口螈脑组织中存在Nogo-A及NgR的表达,在墨西哥钝口螈中脑胆碱能神经元损伤后(注入AF64A)第3天,中脑乙酰胆碱转移酶(ChAT)阳性细胞数明显减少,与正常组比较差异有统计学意义(P<0.01),损伤后第7、14、21天均有ChAT、5-溴脱氧尿嘧啶核苷(BrdU)双阳性细胞,且随时间的延长而增加。中脑胆碱能神经元损伤后第3、7、14、21天脑组织中,Nogo-A的mRNA和蛋白水平的表达量与正常组比较差异均无统计学意义;而NgR的mRNA和蛋白水平的表达量均下降,与正常组比较差异有统计学意义(P<0.05)。结论中枢神经系统损伤后的再生可能与NgR表达量的下降有关。
Objective To explore the expression of Nogo-A and NgR in Ambystoma mexicanum brain tissue and the relationship between Nogo-A, NgR, and regeneration after central nervous system damage injury. Method The normal Ambystoma mexicanum brain tissue and the brain at 3, 7, 14, and 21 d after midbrain cholinergic neurons injury were collected to determine Nogo-A and NgR expression using immunofluorescence, western blot analysis, and Real-time Quantitative PCR(RT-PCR). Results Nogo-A and NgR had abundant expression in the normal ambystoma mexicanum brain. Three days after AF64A injection, we observed the significant loss of ChAT+ cells in the ambystoma mexicanum mesencephalic regions, as compared with the controls(P<0.01). At 7, 14 and 21 days, ChAT+BrdU+ cells were observed in the lesioned animals and increased with time course. Nogo-A in the brain tissue had no significant changes after midbrain cholinergic neurons injury in mRNA and protein expression. However, a marked decrease of NgR expression both in mRNA and protein levels was found after midbrain cholinergic neurons injury compared with normal controls. Conclusion The study also demonstrated that the ability of regeneration in Ambystoma mexicanum central nervous system might correlate with the decreases of expression of the NgR after injury.
Objective To explore the expression of Nogo-A and NgR in Ambystoma mexicanum brain tissue and the relationship between Nogo-A, NgR, and regeneration after central nervous system damage injury. Method The normal Ambystoma mexicanum brain tissue and the brain at 3, 7, 14, and 21 d after midbrain cholinergic neurons injury were collected to determine Nogo-A and NgR expression using immunofluorescence, western blot analysis, and Real-time Quantitative PCR(RT-PCR). Results Nogo-A and NgR had abundant expression in the normal ambystoma mexicanum brain. Three days after AF64A injection, we observed the significant loss of ChAT+ cells in the ambystoma mexicanum mesencephalic regions, as compared with the controls(P<0.01). At 7, 14 and 21 days, ChAT+BrdU+ cells were observed in the lesioned animals and increased with time course. Nogo-A in the brain tissue had no significant changes after midbrain cholinergic neurons injury in mRNA and protein expression. However, a marked decrease of NgR expression both in mRNA and protein levels was found after midbrain cholinergic neurons injury compared with normal controls. Conclusion The study also demonstrated that the ability of regeneration in Ambystoma mexicanum central nervous system might correlate with the decreases of expression of the NgR after injury.
引文
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[11]Funahashi S,Hasegawa T,Nagano A,et al.Differential expression patterns of messenger RNAs encoding Nogo receptors and their ligands in the rat central nervous system[J].J Comp Neurol,2008,506(1):141-160.
[12]Wang B,Xiao Z,Chen B,et al.Nogo-66 Promotes the Differentiation of Neural Progenitors into Astroglial Lineage Cells through mTOR-STAT3 Pathway[J].PLoS One,2008,3(3):e1856.
[13]Berg DA,Kirkham M,Wang H,et al.Dopamine Controls Neurogenesis in the Adult Salamander Midbrain in Homeostasis and during Regeneration of Dopamine Neurons[J].Cell Stem Cell,2011,8(4):426-433.
[14]Borrie SC,Baeumer BE,Bandtlow CE.The Nogo-66 receptor family in the intact and diseased CNS[J].Cell Tissue Res,2012,349(1):105-117.
[2]Puttagunta R,Schmandke A,Floriddia E,et al.RA-RAR-βcounteracts myelin-dependent-inhibitionof neurite outgrowth via Lingo-1 repression[J].J Cell Biol,2011,193(7):1147-1156.
[3]Duffy P,Schmandke A,Schmandke A,et al.Rho-associated kinase II(ROCKII)limits axonal growth after trauma within the adultmouse spinal cord[J].J Neurosci,2009,29(48):15266-15276.
[4]Hui SP,Monaghan JR,Voss SR,et al.Expression Pattern of Nogo-A,MAG,and NgR in Regenerating Urodele Spinal Cord[J].Dev Dyn,2013,242(7):847-860.
[5]Borrie SC,Baeumer BE,Bandtlow CE.The Nogo-66 receptor family in the intact and diseased CNS[J].Cell Tissue Res,2012,349(1):105-117.
[6]Wang T,Xiong JQ,Ren XB,et al.The role of Nogo-A in neuroregeneration:a review[J].Brain Res Bull,2012,87(6):499-503.
[7]Gonzenbach RR,Schwab ME.Disinhibition of neurite growth to repair the injured adult CNS:focusing on Nogo[J].Cell Mol Life Sci,2008,65(1):161-176.
[8]Brockes J,Kumar A.Newts[J].Curr Biol,2005,15(2):R42-R44.
[9]Monaghan JR,Walker JA,Page RB,et al.Early gene expression during natural spinal cord regeneration in the salamander Ambystoma mexicanum[J].J Neurochem,2007,101(1):27-40.
[10]Abdesselem H,Shypitsyna A,Solis GP,et al.No Nogo66-and NgR-mediated inhibition of regenerating axons in the zebrafish optic nerve[J].J Neurosci,2009,29(49):15489-15498.
[11]Funahashi S,Hasegawa T,Nagano A,et al.Differential expression patterns of messenger RNAs encoding Nogo receptors and their ligands in the rat central nervous system[J].J Comp Neurol,2008,506(1):141-160.
[12]Wang B,Xiao Z,Chen B,et al.Nogo-66 Promotes the Differentiation of Neural Progenitors into Astroglial Lineage Cells through mTOR-STAT3 Pathway[J].PLoS One,2008,3(3):e1856.
[13]Berg DA,Kirkham M,Wang H,et al.Dopamine Controls Neurogenesis in the Adult Salamander Midbrain in Homeostasis and during Regeneration of Dopamine Neurons[J].Cell Stem Cell,2011,8(4):426-433.
[14]Borrie SC,Baeumer BE,Bandtlow CE.The Nogo-66 receptor family in the intact and diseased CNS[J].Cell Tissue Res,2012,349(1):105-117.