氯倍他索对大鼠神经干细胞自发分化的影响
|
摘要
目的:研究氯倍他索(Clo)对神经干细胞自发分化的影响,为Clo治疗中枢性神经损伤提供医学理论基础。方法:利用无血清悬浮法分离培养及鉴定大鼠NSCs;选取第三代NSCs,以含10%胎牛血清完全培养基贴壁诱导分化7 d,培养基中各加入同体积二甲基亚砜、2. 5、5μmol/L的Clo;免疫荧光染色和Western Blot方法检测NSCs分化后轴突膜蛋白(GAP-43)、酸性胶质蛋白(GFAP)、髓磷脂碱性蛋白(MBP)表达情况,进一步探讨Clo的作用机制。结果:分离培养的NSCs高表达Nestin; Clo作用组表达GAP-43和MBP较对照组明显提高(P <0. 05),GFAP表达水平低于对照组(P <0. 05),Clo作用组表达音猬因子(SHH)和锌指蛋白(Gli 1)均高于对照组(P <0.05)。结论:Clo可在体外通过SHH信号通路促进NSCs向神经元细胞和少突胶质细胞分化,与此同时Clo可抑制NSCs向星形胶质细胞分化。
Objective: To study the effects of clobetasol( Clo) on the spontaneous differentiation of rat neural stem cells,provide a theoretical basis for Clo to treat central nerve injury.Methods: The serum-free suspension method was used to isolate and culture the rat NSCs.The third-generation NSCs were selected and adhered to the 10% FBS serumcontaining medium to induce differentiation for 7 days.Clobetasol 0( Same volume of Dimethyl sulfoxide),2.5 and 5μmol/L were added to the medium.Immunofluorescent staining and Western Blot methods were used to detect the expression of growth associated protein-43( GAP-43),glial fibrillary acidic protein( GFAP) and myelin basic protein( MBP)after differentiation of NSCs,and Nestin was used to identify NSCs.Results: Nestin was highly expressed in NSCs.The expression of GAP-43 and MBP in Clo treated group was significantly higher than those in control group( P < 0.05); the expression level of GFAP was lower than that in control group( P < 0.05) and the expression of sonic hedeahog( SHH)and Gli 1 were higher than those of control group( P < 0.05).Conclusion: Clo could promote the differentiation of NSCs into neurons and oligodendrocytes through the SHH signal pathway in vitro,and meanwhile while inhibit the differentiation of NSCs into astrocytes.
Objective: To study the effects of clobetasol( Clo) on the spontaneous differentiation of rat neural stem cells,provide a theoretical basis for Clo to treat central nerve injury.Methods: The serum-free suspension method was used to isolate and culture the rat NSCs.The third-generation NSCs were selected and adhered to the 10% FBS serumcontaining medium to induce differentiation for 7 days.Clobetasol 0( Same volume of Dimethyl sulfoxide),2.5 and 5μmol/L were added to the medium.Immunofluorescent staining and Western Blot methods were used to detect the expression of growth associated protein-43( GAP-43),glial fibrillary acidic protein( GFAP) and myelin basic protein( MBP)after differentiation of NSCs,and Nestin was used to identify NSCs.Results: Nestin was highly expressed in NSCs.The expression of GAP-43 and MBP in Clo treated group was significantly higher than those in control group( P < 0.05); the expression level of GFAP was lower than that in control group( P < 0.05) and the expression of sonic hedeahog( SHH)and Gli 1 were higher than those of control group( P < 0.05).Conclusion: Clo could promote the differentiation of NSCs into neurons and oligodendrocytes through the SHH signal pathway in vitro,and meanwhile while inhibit the differentiation of NSCs into astrocytes.
引文
[1]张志强,黄永辉,沈铁城.嗅鞘细胞移植修复脊髓损伤的研究进展[J].中国矫形外科杂志,2007,15:275-277.
[2]任春兰,许燕,吴维秀.神经营养素高亲和力受体在脊髓损伤大鼠运动神经末梢的分布[J].神经解剖学杂志,2004,20:497-500.
[3]Lee HY,Lee M,Bae YS. Formyl peptide receptors in cellular differentiation and inflammatory diseases[J]. J Cell Biochem,2017,118:1300-1307.
[4]Green DW,Watson GS,Watson JA,et al. Diversification and enrichment of clinical biomaterials inspired by Darwinian evolution[J]. Acta Biomater,2016,42:33-45.
[5]Hidasi AO,Groh KJ,Suter MJ,et al. Clobetasol propionate causes immunosuppression in zebrafish(Danio rerio)at environmentally relevant concentrations[J]. Ecotoxicol Environ Saf,2017,138:16-24.
[6]Najm FJ,Madhavan M,Zaremba A,et al. Drug-based modulation of endogenous stem cells promotes functional remyelination in vivo[J]. Nature,2015,522:216-220.
[7]Kameda T,Imamura T,Nakashima K. Epigenetic regulation of neural stem cell differentiation towards spinal cord regeneration[J].Cell Tissue Res,2018,371:189-199.
[8]陈孝祥,宋晓斌,王向鹏,等.大鼠胚胎中脑腹侧来源神经干细胞的分离和培养[J].神经解剖学杂志,2017,33:75-79.
[9]Reynolds BA,Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system[J].Science,1992,255:1707-1710.
[10]吕颖,张念平,徐丽萍,等.环孢菌素A结合神经干细胞移植对6-OHDA大鼠PD模型的作用[J].神经解剖学杂志,2017,33:723-730.
[11] Carriel V,GarzónI,Campos A,et al. Differential expression of GAP-43 and neurofilament during peripheral nerve regeneration through bio-artificial conduits[J]. J Tissue Eng Regen Med,2017,11:553-563.
[12]Benowitz LI,Routtenberg A. GAP-43:an intrinsic determinant of neuronal development and plasticity[J]. Trends Neurosci,1997,20:84-91.
[13] Eng LF,Ghirnikar RS,Lee YL. Glial fibrillary acidic protein:GFAP-thirty-one years(1969-2000)[J]. Neurochem Res,2000,25:1439-1451.
[14]Hol EM,Pekny M. Glial fibrillary acidic protein(GFAP)and the astrocyte intermediate filament system in diseases of the central nervous system[J]. Curr Opin Cell Biol,2015,32:121-130.
[15]Zhan X,Stamova B,Sharp FR. Lipopolysaccharide associates with amyloid plaques,neurons and oligodendrocytes in Alzheimer’s disease brain:A review[J]. Front Aging Neurosci,2018,10:42-56.
[16] Lamers KJ,Vos P,Verbeek MM,et al. Protein S-100B,neuronspecific enolase(NSE),myelin basic protein(MBP)and glial fibrillary acidic protein(GFAP)in cerebrospinal fluid(CSF)and blood of neurological patients[J]. Brain Res Bull,2003,61:261-264.
[17] Stanton BZ,Peng LF. Small-molecule modulators of the Sonic Hedgehog signaling pathway[J]. Mol Biosyst,2010,6:44-54.
[18]Schaefer GI,Perez JR,Duvall JR,et al. Discovery of small-molecule modulators of the Sonic Hedgehog pathway[J]. J Am Chem Soc,2013,135:9675-9680.
[19] Frank-Kamenetsky M,Zhang XM,Bottega S,et al. Small-molecule modulators of Hedgehog signaling:identification and characterization of Smoothened agonists and antagonists[J]. J Biol,2002,1:10-21.
[20]Yao PJ,Petralia RS,Mattson MP. Sonic Hedgehog signaling and hippocampal neuroplasticity[J]. Trends Neurosci,2016,39:840-850.
[21]Dessaud E,Mc Mahon AP,Briscoe J. Pattern formation in the vertebrate neural tube:a sonic hedgehog morphogen-regulated transcriptional network[J]. Development,2008,135:2489-2503.
[22]Hatton BA,Knoepfler PS,Kenney AM,et al. N-myc is an essential downstream effector of Shh signaling during both normal and neoplastic cerebellar growth[J]. Cancer Res,2006,66:8655-8661.
[23]Fuccillo M,Joyner AL,Fishell G. Morphogen to mitogen:the multiple roles of hedgehog signalling in vertebrate neural development.Nat Rev Neurosci[J],2006,7:772-783.
[24]黄奇,徐祝军.脊髓损伤的药物治疗的研究进展[J].中国药物经济学,2014,9:287-288.
[2]任春兰,许燕,吴维秀.神经营养素高亲和力受体在脊髓损伤大鼠运动神经末梢的分布[J].神经解剖学杂志,2004,20:497-500.
[3]Lee HY,Lee M,Bae YS. Formyl peptide receptors in cellular differentiation and inflammatory diseases[J]. J Cell Biochem,2017,118:1300-1307.
[4]Green DW,Watson GS,Watson JA,et al. Diversification and enrichment of clinical biomaterials inspired by Darwinian evolution[J]. Acta Biomater,2016,42:33-45.
[5]Hidasi AO,Groh KJ,Suter MJ,et al. Clobetasol propionate causes immunosuppression in zebrafish(Danio rerio)at environmentally relevant concentrations[J]. Ecotoxicol Environ Saf,2017,138:16-24.
[6]Najm FJ,Madhavan M,Zaremba A,et al. Drug-based modulation of endogenous stem cells promotes functional remyelination in vivo[J]. Nature,2015,522:216-220.
[7]Kameda T,Imamura T,Nakashima K. Epigenetic regulation of neural stem cell differentiation towards spinal cord regeneration[J].Cell Tissue Res,2018,371:189-199.
[8]陈孝祥,宋晓斌,王向鹏,等.大鼠胚胎中脑腹侧来源神经干细胞的分离和培养[J].神经解剖学杂志,2017,33:75-79.
[9]Reynolds BA,Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system[J].Science,1992,255:1707-1710.
[10]吕颖,张念平,徐丽萍,等.环孢菌素A结合神经干细胞移植对6-OHDA大鼠PD模型的作用[J].神经解剖学杂志,2017,33:723-730.
[11] Carriel V,GarzónI,Campos A,et al. Differential expression of GAP-43 and neurofilament during peripheral nerve regeneration through bio-artificial conduits[J]. J Tissue Eng Regen Med,2017,11:553-563.
[12]Benowitz LI,Routtenberg A. GAP-43:an intrinsic determinant of neuronal development and plasticity[J]. Trends Neurosci,1997,20:84-91.
[13] Eng LF,Ghirnikar RS,Lee YL. Glial fibrillary acidic protein:GFAP-thirty-one years(1969-2000)[J]. Neurochem Res,2000,25:1439-1451.
[14]Hol EM,Pekny M. Glial fibrillary acidic protein(GFAP)and the astrocyte intermediate filament system in diseases of the central nervous system[J]. Curr Opin Cell Biol,2015,32:121-130.
[15]Zhan X,Stamova B,Sharp FR. Lipopolysaccharide associates with amyloid plaques,neurons and oligodendrocytes in Alzheimer’s disease brain:A review[J]. Front Aging Neurosci,2018,10:42-56.
[16] Lamers KJ,Vos P,Verbeek MM,et al. Protein S-100B,neuronspecific enolase(NSE),myelin basic protein(MBP)and glial fibrillary acidic protein(GFAP)in cerebrospinal fluid(CSF)and blood of neurological patients[J]. Brain Res Bull,2003,61:261-264.
[17] Stanton BZ,Peng LF. Small-molecule modulators of the Sonic Hedgehog signaling pathway[J]. Mol Biosyst,2010,6:44-54.
[18]Schaefer GI,Perez JR,Duvall JR,et al. Discovery of small-molecule modulators of the Sonic Hedgehog pathway[J]. J Am Chem Soc,2013,135:9675-9680.
[19] Frank-Kamenetsky M,Zhang XM,Bottega S,et al. Small-molecule modulators of Hedgehog signaling:identification and characterization of Smoothened agonists and antagonists[J]. J Biol,2002,1:10-21.
[20]Yao PJ,Petralia RS,Mattson MP. Sonic Hedgehog signaling and hippocampal neuroplasticity[J]. Trends Neurosci,2016,39:840-850.
[21]Dessaud E,Mc Mahon AP,Briscoe J. Pattern formation in the vertebrate neural tube:a sonic hedgehog morphogen-regulated transcriptional network[J]. Development,2008,135:2489-2503.
[22]Hatton BA,Knoepfler PS,Kenney AM,et al. N-myc is an essential downstream effector of Shh signaling during both normal and neoplastic cerebellar growth[J]. Cancer Res,2006,66:8655-8661.
[23]Fuccillo M,Joyner AL,Fishell G. Morphogen to mitogen:the multiple roles of hedgehog signalling in vertebrate neural development.Nat Rev Neurosci[J],2006,7:772-783.
[24]黄奇,徐祝军.脊髓损伤的药物治疗的研究进展[J].中国药物经济学,2014,9:287-288.