Akt对IL-1β诱导神经干细胞分化为多巴胺能神经元的作用
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摘要
目的探讨蛋白激酶B/Akt信号途径对IL-1β诱导神经干细胞(neutral stem cells,NSCs)分化多为巴胺能神经元(dopaminergic neurons,DNs)的作用。方法原代取材培养中脑来源大鼠NSCs,用IL-1β诱导NSCs分化;免疫荧光染色法检测βIII-tubulin和酪氨酸羟化酶(TH)表达; Motic Digital Class 1. 1/Motic Images Advanced3. 1图像分析软件测量多巴胺能神经元突起长度; Western blot分析法检测分化细胞Akt磷酸化程度。结果培养细胞表达nestin蛋白,分化为神经元和神经胶质细胞;βIII-tubulin阳性细胞分化率分别为13. 4%±0. 8%(A组)、16. 8%±1. 5%(B组)、28. 8%±2. 4%(C组)、29. 2%±1. 5%(D组)、12. 5%±0. 6%(对照组),βⅢ-tubulin阳性细胞百分率随着IL-1β剂量增加而增加(P <0. 05); TH细胞阳性率分别为1. 6%±0. 5%(A组)、9. 5%±0. 6%(B组)、16. 8%±1. 5%(C组)、16. 6%±1. 2%(D组)、1. 3%±0. 5%(对照组),TH阳性分化细胞随着IL-1β剂量增加而增加(P <0. 01);多巴胺能神经元突起长度分别为(48. 6±8. 3)μm(A组)、(56. 4±5. 2)μm(B组)、(80. 0±12. 8)μm(C组)、(88. 2±10. 5)μm(D组)、(45. 5±6. 5)μm(对照组),多巴胺能神经元突起长度随着IL-1β剂量增加而增加(P <0. 05);经IL-1β诱导后分化细胞Akt的磷酸化水平升高(P <0. 01)。结论 IL-1β作为一种重要的信号分子参与诱导NSCs向DNs分化,分化过程中Akt磷酸化水平升高,蛋白激酶B/Akt信号途径通过磷酸化修饰激活后可能参与NSCs向多巴胺能神经元分化的调节过程。
Objective To investigate the effect of Protein kinase B/Akt signal pathway in the process of neural stem cells on the differentiation of dopaminergic neurons induced by IL-1β. Methods The rat neural stem cells form the middle brain were obtained and cultured primitively and the differentiation was induced by IL-1β. Immunofluorescene staining method was used to detect the expression of beta III-tubulin and tyrosine hydroxylase( TH); Motic Digital Class 1. 1/Motic Images Advanced 3. 1 image analysis software was used to measure neurite's length of the dopaminergic neurons; Western blot was used to analyze the degree of Akt phosphorylation of differentiated cells induced by IL-1β. Results The cultured cells expressed nestin protein and differentiated into neurons and glial cells; The differentiation rate of beta III-tubulin positive cells was 13. 4% ± 0. 8%( group A),16. 8% ± 1. 5%( group B),28. 8% ± 2. 4%( group C),29. 2% ± 1. 5%( group D),12. 5% ± 0. 6%( control group),the percentage of beta III-tubulin positive cells increased with the increase of IL-1βdose( P < 0. 05); The positive rate of TH positive cells was 1. 6% ± 0. 5%( group A),6. 0% ± 1. 6%( group B),24. 8% ±1. 5%( group C) 、24. 6% ± 1. 4%( group D) 、1. 5% ± 0. 5%( control group),TH positive differentiated cells increased with the increase of IL-1β dose( P < 0. 01); The length of dopaminergic neurons' Bneurites was( 48. 6 ± 8. 3) μm( group A),( 56. 4 ± 5. 2) μm( group B),( 80. 0 ± 12. 5) μm( group C),( 80. 2 ± 10. 5) μm( group D),( 45. 5 ± 6. 5) μm( control group),the neurites' length of dopaminergic neurons increased with the increase of IL-1β dose( P < 0. 05),and the level of phosphorylation of Akt in differentiated cells increased after IL-1 beta( P < 0. 01). Conclusion IL-1β induced neural stem cells differentiate into dopaminergic neurons as a type of crucial signal molecules,the phosphorylation level of Akt increases during the differentiation,and Protein kinase B/Akt signal pathway may be activated by phosphorylation and involved in the regulation of neural stem cells to dopaminergic neurons.
Objective To investigate the effect of Protein kinase B/Akt signal pathway in the process of neural stem cells on the differentiation of dopaminergic neurons induced by IL-1β. Methods The rat neural stem cells form the middle brain were obtained and cultured primitively and the differentiation was induced by IL-1β. Immunofluorescene staining method was used to detect the expression of beta III-tubulin and tyrosine hydroxylase( TH); Motic Digital Class 1. 1/Motic Images Advanced 3. 1 image analysis software was used to measure neurite's length of the dopaminergic neurons; Western blot was used to analyze the degree of Akt phosphorylation of differentiated cells induced by IL-1β. Results The cultured cells expressed nestin protein and differentiated into neurons and glial cells; The differentiation rate of beta III-tubulin positive cells was 13. 4% ± 0. 8%( group A),16. 8% ± 1. 5%( group B),28. 8% ± 2. 4%( group C),29. 2% ± 1. 5%( group D),12. 5% ± 0. 6%( control group),the percentage of beta III-tubulin positive cells increased with the increase of IL-1βdose( P < 0. 05); The positive rate of TH positive cells was 1. 6% ± 0. 5%( group A),6. 0% ± 1. 6%( group B),24. 8% ±1. 5%( group C) 、24. 6% ± 1. 4%( group D) 、1. 5% ± 0. 5%( control group),TH positive differentiated cells increased with the increase of IL-1β dose( P < 0. 01); The length of dopaminergic neurons' Bneurites was( 48. 6 ± 8. 3) μm( group A),( 56. 4 ± 5. 2) μm( group B),( 80. 0 ± 12. 5) μm( group C),( 80. 2 ± 10. 5) μm( group D),( 45. 5 ± 6. 5) μm( control group),the neurites' length of dopaminergic neurons increased with the increase of IL-1β dose( P < 0. 05),and the level of phosphorylation of Akt in differentiated cells increased after IL-1 beta( P < 0. 01). Conclusion IL-1β induced neural stem cells differentiate into dopaminergic neurons as a type of crucial signal molecules,the phosphorylation level of Akt increases during the differentiation,and Protein kinase B/Akt signal pathway may be activated by phosphorylation and involved in the regulation of neural stem cells to dopaminergic neurons.
引文
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[20]Shu T,Liu C,Pang M,et al. Salvianolic acid B promotes neural differentiation of induced pluripotent stem cells via PI3K/AKT/GSK32/2-catenin pathway[J]. Neuroscience Letters,2018,671:154-160.
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[3]Huang BX,Ning S,Zhang QJ,et al. Bisphenol a represses dopaminergic neuron differentiation from Human Embryonic Stem Cells through down regulating the expression of insulin-like growth factor[J]. Molecular Neurobiology,2017,54(5):3798–3812.
[4]Ying B,Li Y,Wang JP,et al. Neuroprotective effects of ginsenoside Rg1-induced neural stem cell transplantation on hypoxic-ischemic encephalopathy[J]. Neural Regeneration Research,2015,10(5):753-759.
[5]王光辉,钟鸣,张敏娜,等.二十二碳五烯酸对PC12细胞神经突起生长的诱导作用[J].中华行为医学与脑科学杂志,2017,(5):390-394.
[6]陈孝祥,宋晓斌,王向鹏,等. GFP-Nurr1基因修饰神经干细胞的建立及过表达Nurr1对神经干细胞向多巴胺神经元分化的影响[J].中风与神经疾病杂志,2017,34(5):388-392.
[7]Paweena W,Jasadee K. Osteogenic differentiation of mesenchymal stem cells is impaired by bone morphogenetic protein[J]. Advances in Medical Sciences,2017,62(2):2233-2238.
[8]Shabana K,Simon RW,Stott,et al. Survival of a novel subset of midbrain dopaminergic neurons projecting to the lateral septum is dependent on neuron proteins[J]. Journal of Neuroscience,2017,37(9):2305-2316.
[9]朱晓瓞,余资江,孙宝飞,等.小鼠骨髓间充质干细胞体外扩增和分化为神经元样细胞的实验研究[J].中风与神经疾病杂志,2016,33(10):868-872.
[10]Vukasin MJ,Ahmad S,Hadas T,et al. BMP/SMAD pathway promotes neurogenesis of midbrain dopaminergic neurons in vivo and in human induced pluripotent and neural stem cells[J]. Journal of Neuroscience,2018,38(7):1662-1676.
[11]Marie CT,Christophe B,Sabrina M,et al. Zic-Proteins are repressors of dopaminergic forebrain fate in mice and C. elegans[J]. Journal of Neuroscience,2017,37(44):10611-10623.
[12]Varshney MK,Inzunza J,Lupu D,et al. Role of estrogen receptor beta in neural differentiation of mouse embryonic stem cells[J]. Proc Natl Acad Sci USA,2017,114(48):10428.
[13]Khasnavis S,Pahan K. Cinnamon treatment upregulates neuroprotective proteins Parkin and DJ-1 and protects dopaminergic neurons in a mouse model of Parkinson’s disease[J]. J Neuroimmune Pharmacol,2014,9(4):569-581.
[14 Patterson SL. Immune dysregulation and cognitive vulnerability in the aging brain:Interactions of microglia,IL-1β,BDNF and synaptic plasticity[J]. Neuropharmacology,2015,96:11-18.
[15]Jaakko JK,Anne P,Sara B,et al. Dampened amphetamine-stimulated behavior and altered dopamine transporter function in the absence of brain GDNF[J]. Journal of Neuroscience,2017,37(6):1581-1590.
[16]Berdichevsky Y,Dryer AM,Saponjian Y,et al. PI3K-Akt signaling activates m TOR-mediated epileptogenesis in organotypic hippocampal culture model of post-traumatic epilepsy[J]. Journal of Neuroscience the Official Journal of the Society for Neuroscience,2013,33(21):9056.
[17]Zhang Y,Gong XG,Wang ZZ,et al. Overexpression of DJ-1/PARK7,the Parkinson’s disease-related protein,improves mitochondrial function via Akt phosphorylation on threonine 308 in dopaminergic neuron-like cells[J]. European Journal of Neuroscience,2016,43(10):1379-1388.
[18]Zhang C,Yuan X,Liu Q,et al. Valproic acid protects primary dopamine neurons from MPP+-Induced neurotoxicity:involvement of GSK3βphosphorylation by Akt and ERK through the mitochondrial intrinsic apoptotic pathway[J]. Biomed Research International,2017,128:1-12.
[19]Torra A,Parent A,Cuadros T,et al. Overexpression of TFEB Drives a Pleiotropic Neurotrophic Effect and Prevents Parkinson’s DiseaseRelated Neurodegeneration[J]. Molecular Therapy,2018,24:3193-3203.
[20]Shu T,Liu C,Pang M,et al. Salvianolic acid B promotes neural differentiation of induced pluripotent stem cells via PI3K/AKT/GSK32/2-catenin pathway[J]. Neuroscience Letters,2018,671:154-160.