芬戈莫德对神经干细胞和星形胶质细胞增殖及迁移的影响及其对小鼠脊髓损伤的保护机制
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摘要
目的:探讨芬戈莫德(FTY720)对神经干细胞(NSCs)和星形胶质细胞迁移及增殖的影响,阐明FTY720对小鼠脊髓损伤(SCI)的保护作用及其机制。方法:体外培养NSCs和星形胶质细胞,每种细胞随机分为对照组(采用PBS进行处理)和实验组(采用0.1μmol·L~(-1) FTY720进行处理)。SCI小鼠随机分为对照组(口服PBS)和实验组(口服1mg·kg~(-1)·d~(-1) FTY720),每组10只。采用Transwell实验检测2组NSCs和星形胶质细胞的迁移细胞数,免疫荧光染色检测NSCs的迁移细胞数和增殖神经球数,HE染色检测2组小鼠SCI空洞面积,Basso Mouse Scale (BMS)评分评估小鼠运动能力恢复情况。结果:Transwell检测,与对照组比较,实验组NSCs迁移细胞数增多(P<0.05),星形胶质细胞迁移细胞数减少(P<0.05)。免疫荧光检测,与对照组比较,实验组NSCs增殖神经球数增多(P<0.05),实验组小鼠SCI震中星形胶质细胞数减少(P<0.05)。HE染色检测,与对照组比较,SCI空洞面积缩小(P<0.05)。BMS评分,与对照组比较,实验组小鼠BMS评分升高(P<0.05)。结论:FTY720可以促进NSCs增殖和迁移,抑制星形胶质细胞迁移,减少SCI后胶质疤痕形成,促进小鼠运动功能恢复。
Objective:To investigate the effects of fingolimod(FTY720)on the migration and proliferation of neural stem cells(NSCs)and astrocytes of the mice,and to elucidate the protective effect of FTY720 on spinal cord injury(SCI)of the mice and its mechanism.Methods:The NSCs and astrocytes were cultured in vitro and divided into control group(treated with PBS)and experiment group(treated with 0.1μmol·L~(-1) FTY720).The SCI mice were divided into control group(given PBS by oral,n=10)and expriment group(given 1 mg·kg~(-1)·d~(-1) FTY720 by oral,n=10).The number of migration cells of NSCs and astrocytes in two groups was detected by Transwell experiment,and the number of migration cells and the number of proliferative neurospheres of NSCs were detected by immunofluorescence staining.The SCI void areas of the mice in two groups were detected by HE staining,and the dynamic recovery of motor function of the mice was assessed by Basso Mouse Scale(BMS)behavioral score.Results:The Transwell detection results showed that compared with control group,the number of migration cells of NSCs in expriment group was increased(P<0.05),and the number of migration cells of astrocytes in expriment group was decreased(P<0.05).The immunofluorescence detection results showed that compared with control group,the number of proliferative neurospheres of NSCs in expriment group was increased(P<0.05),and the number of astrocytes in damaged SCI in expriment group was decreased(P<0.05).The HE staining results showed that compared with control group,the SCI void area was decreased(P<0.05).The BMS score results showed that the BMS score of the mice in expriment group was higher than that in control group(P<0.05).Conclusion:FTY720 can promote the proliferation and migration of NSCs,inhibit the migration of astrocytes,decrease the formation of glial scar,and promote the motor function recovery of the mice after SCI.
Objective:To investigate the effects of fingolimod(FTY720)on the migration and proliferation of neural stem cells(NSCs)and astrocytes of the mice,and to elucidate the protective effect of FTY720 on spinal cord injury(SCI)of the mice and its mechanism.Methods:The NSCs and astrocytes were cultured in vitro and divided into control group(treated with PBS)and experiment group(treated with 0.1μmol·L~(-1) FTY720).The SCI mice were divided into control group(given PBS by oral,n=10)and expriment group(given 1 mg·kg~(-1)·d~(-1) FTY720 by oral,n=10).The number of migration cells of NSCs and astrocytes in two groups was detected by Transwell experiment,and the number of migration cells and the number of proliferative neurospheres of NSCs were detected by immunofluorescence staining.The SCI void areas of the mice in two groups were detected by HE staining,and the dynamic recovery of motor function of the mice was assessed by Basso Mouse Scale(BMS)behavioral score.Results:The Transwell detection results showed that compared with control group,the number of migration cells of NSCs in expriment group was increased(P<0.05),and the number of migration cells of astrocytes in expriment group was decreased(P<0.05).The immunofluorescence detection results showed that compared with control group,the number of proliferative neurospheres of NSCs in expriment group was increased(P<0.05),and the number of astrocytes in damaged SCI in expriment group was decreased(P<0.05).The HE staining results showed that compared with control group,the SCI void area was decreased(P<0.05).The BMS score results showed that the BMS score of the mice in expriment group was higher than that in control group(P<0.05).Conclusion:FTY720 can promote the proliferation and migration of NSCs,inhibit the migration of astrocytes,decrease the formation of glial scar,and promote the motor function recovery of the mice after SCI.
引文
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[8] Ambrosius B,Pitarokoili K,Schrewe L,et al.Fingolimod attenuates experimental autoimmune neuritis and contributes toSchwanncell-mediatedaxonalprotection[J].J Neuroinflammat,2017,14(1):92.
[9] Kretzschmar B,Pellkofer H,Weber MS.The use of oral disease-modifying therapies in multiple sclerosis[J].Curr Neurol Neurosci,2016,16(4):38.
[10]Chun J,Hartung HP.Mechanism of action of oral fingolimod(FTY720)in multiple sclerosis[J].Clin Neuropharmacol,2010,33(2):91-101.
[11]Dusaban SS,Chun J, Rosen H,et al.Sphingosine 1-phosphatereceptor3 andRhoAsignalingmediate inflammatorygeneexpressioninastrocytes[J].J Neuroinflammat,2017,14(1):111.
[12]Zhu Z,Fu Y,Tian D,et al.Combination of an immune modulator fingolimod with alteplase in acute ischemic stroke:a pilot trial[J].Circulation,2015,132(12):1104-1112.
[13]Zhang J,Zhang A, Sun Y, et al. Treatment with immunosuppressants FTY720 andtacrolimuspromotes functional recovery after spinal cord injury in rats[J].Tohoku J Exp Med,2009,219(4):295-302.
[14]Norimatsu Y,Ohmori T,Kimura A,et al.FTY720improves functional recovery after spinal cord injury by primarily nonimmunomodulatory mechanisms[J].Am J Pathol,2012,180(4):1625-1635.
[15]Chen L,Li J,Wu L,et al.Synergistic actions of olomoucine and bone morphogenetic protein-4in axonal repair after acute spinal cord contusion[J].Neural Regen Res,2014,9(20):1830-1838.
[16]Ren H,Han M,Zhou J,et al.Repair of spinal cord injury by inhibition of astrocyte growth and inflammatory factor synthesis through local delivery of flavopiridol in PLGA nanoparticles[J].Biomaterials,2014,35(24):6585-6594.
[17]Choi JW,Gardell SE,Herr DR,et al.FTY720(fingolimod)efficacy in an animal model of multiple sclerosis requires astrocyte sphingosine 1-phosphate receptor 1(S1P1)modulation[J].Proc Natl Acad Sci USA,2011,108(2):751-756.
[18]Snyder EY,Teng YD.Stem cells and spinal cord repair[J].N Engl J Med,2012,366(20):1940-1942.
[19] Hsuan YC,Lin C,Chang C,et al.Mesenchymal stem cell‐based treatments for stroke, neural trauma, and heat stroke[J].Brain Behav,2016,6(10):1-11.
[2] Singh A,Tetreault L,Kalsi-Ryan S,et al.Global prevalence and incidence of traumatic spinal cord injury[J]. Clin Epidemiol,2014,6:309-331.
[3] Iyer NR,Wilems TS,Sakiyama-Elbert SE.Stem cells for spinal cord injury:Strategies to inform differentiation and transplantation[J].Biotechnol Bioeng,2017,114(2):245-259.
[4] Dumont RJ,Okonkwo DO,Verma S.et al.Acute spinal cord injury,part I:pathophysiologic mechanisms[J]. Clin Neuropharmacol,2001,24(5):254-264.
[5] Tator CH.Strategies for recovery and regeneration after brain and spinal cord injury[J].Inj Prev,2002,8(Suppl 4):V33-V36.
[6] Chen L,Li J,Wu L,et al.Synergistic actions of olomoucine and bone morphogenetic protein-4in axonal repair after acute spinal cord contusion[J].Neural Regen Res,2014,9(20):1830-1838.
[7]任浩.局部释放夫拉平度调控局部星形胶质细胞和炎症反应促进脊髓损伤修复[D].杭州:浙江大学,2017.
[8] Ambrosius B,Pitarokoili K,Schrewe L,et al.Fingolimod attenuates experimental autoimmune neuritis and contributes toSchwanncell-mediatedaxonalprotection[J].J Neuroinflammat,2017,14(1):92.
[9] Kretzschmar B,Pellkofer H,Weber MS.The use of oral disease-modifying therapies in multiple sclerosis[J].Curr Neurol Neurosci,2016,16(4):38.
[10]Chun J,Hartung HP.Mechanism of action of oral fingolimod(FTY720)in multiple sclerosis[J].Clin Neuropharmacol,2010,33(2):91-101.
[11]Dusaban SS,Chun J, Rosen H,et al.Sphingosine 1-phosphatereceptor3 andRhoAsignalingmediate inflammatorygeneexpressioninastrocytes[J].J Neuroinflammat,2017,14(1):111.
[12]Zhu Z,Fu Y,Tian D,et al.Combination of an immune modulator fingolimod with alteplase in acute ischemic stroke:a pilot trial[J].Circulation,2015,132(12):1104-1112.
[13]Zhang J,Zhang A, Sun Y, et al. Treatment with immunosuppressants FTY720 andtacrolimuspromotes functional recovery after spinal cord injury in rats[J].Tohoku J Exp Med,2009,219(4):295-302.
[14]Norimatsu Y,Ohmori T,Kimura A,et al.FTY720improves functional recovery after spinal cord injury by primarily nonimmunomodulatory mechanisms[J].Am J Pathol,2012,180(4):1625-1635.
[15]Chen L,Li J,Wu L,et al.Synergistic actions of olomoucine and bone morphogenetic protein-4in axonal repair after acute spinal cord contusion[J].Neural Regen Res,2014,9(20):1830-1838.
[16]Ren H,Han M,Zhou J,et al.Repair of spinal cord injury by inhibition of astrocyte growth and inflammatory factor synthesis through local delivery of flavopiridol in PLGA nanoparticles[J].Biomaterials,2014,35(24):6585-6594.
[17]Choi JW,Gardell SE,Herr DR,et al.FTY720(fingolimod)efficacy in an animal model of multiple sclerosis requires astrocyte sphingosine 1-phosphate receptor 1(S1P1)modulation[J].Proc Natl Acad Sci USA,2011,108(2):751-756.
[18]Snyder EY,Teng YD.Stem cells and spinal cord repair[J].N Engl J Med,2012,366(20):1940-1942.
[19] Hsuan YC,Lin C,Chang C,et al.Mesenchymal stem cell‐based treatments for stroke, neural trauma, and heat stroke[J].Brain Behav,2016,6(10):1-11.