氯倍他索促进鼻粘膜间充质干细胞成神经样细胞分化
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摘要
目的:研究氯倍他索(Clo)对鼻粘膜外胚层间充质干细胞(EMSCs)向神经样细胞分化的影响。方法:贴壁筛选法分离培养EMSCs,免疫荧光法鉴定体外扩增的EMSCs;MTT法检测Clo对EMSCs增殖能力的影响;成神经样细胞诱导实验分为Clo组、音猬因子(SHH)组和空白对照组,采用全反式维甲酸(ATRA)诱导各组细胞向神经样细胞分化;倒置显微镜观察细胞形态的变化;免疫荧光和免疫印迹方法检测神经元细胞相关蛋白。结果:体外分离培养的EMSCs高表达间充质干细胞和神经嵴源干细胞标志物Vimentin和Nestin。各组EMSCs成神经样细胞诱导7 d后,Clo组和SHH组EMSCs呈现双极和多极的典型神经样细胞形态并且高表达神经元细胞相关蛋白。Clo组EMSCs表达SHH和SMO的水平明显高于空白对照组,与SHH组比较差异无统计学意义(P>0.05);Clo可促进EMSCs分泌内源性脑神经营养因子(BDNF)和神经生长因子(NGF)。结论:Clo可通过激活SHH信号通路和上调内源性神经营养因子水平促进EMSCs向神经样细胞分化。
Objective: To study the effect of Clobetasol propionate(Clo) on the differentiation of nasal mesenchymal stem cells(EMSCs) into neuron-like cells. Methods: EMSCs were isolated and cultured by the adherent screening method, and the EMSCs amplified in vitro were identified by immunofluorescence. The effect of Clo on the proliferation of EMSCs was examined by MTT assay. Induced neuroblast-like cells were divided into the Clo group, Sonic hedgehog(SHH) group, and control group. All-trans retinoic acid(ATRA) was used to induce differentiation into neuron-like cells. Changes in cell morphology were observed under inverted microscope, and detection of neuronal cell-associated proteins was accomplished by immunohistochemistry and immunoblot methods. Results: EMSCs isolated and cultured in vitro displayed high expression levels of mesenchymal and neural stem cell markers vimentin and nestin. After induction of EMSCs into neuroblast-like cells for 7 days, the EMSCs in the Clo and SHH groups showed bipolar and multipolar typical neuron-like cell morphology and high expression of neuronal cell-associated proteins. At the same time, the expression levels of SHH and SMO in the EMSCs of the Clo group were significantly higher than those of the control group, and there was no significant difference compared with that of the SHH group(P>0.05). Data showed that Clo could promote the secretion of endogenous brain derived neurotrophic factor(BDNF) and nerve growth factor(NGF)by EMSCs. Conclusion: Clo can promote the differentiation of EMSCs into neuron-like cells by activating the SHH signaling pathway and up-regulating the levels of endogenous neurotrophic factors.
Objective: To study the effect of Clobetasol propionate(Clo) on the differentiation of nasal mesenchymal stem cells(EMSCs) into neuron-like cells. Methods: EMSCs were isolated and cultured by the adherent screening method, and the EMSCs amplified in vitro were identified by immunofluorescence. The effect of Clo on the proliferation of EMSCs was examined by MTT assay. Induced neuroblast-like cells were divided into the Clo group, Sonic hedgehog(SHH) group, and control group. All-trans retinoic acid(ATRA) was used to induce differentiation into neuron-like cells. Changes in cell morphology were observed under inverted microscope, and detection of neuronal cell-associated proteins was accomplished by immunohistochemistry and immunoblot methods. Results: EMSCs isolated and cultured in vitro displayed high expression levels of mesenchymal and neural stem cell markers vimentin and nestin. After induction of EMSCs into neuroblast-like cells for 7 days, the EMSCs in the Clo and SHH groups showed bipolar and multipolar typical neuron-like cell morphology and high expression of neuronal cell-associated proteins. At the same time, the expression levels of SHH and SMO in the EMSCs of the Clo group were significantly higher than those of the control group, and there was no significant difference compared with that of the SHH group(P>0.05). Data showed that Clo could promote the secretion of endogenous brain derived neurotrophic factor(BDNF) and nerve growth factor(NGF)by EMSCs. Conclusion: Clo can promote the differentiation of EMSCs into neuron-like cells by activating the SHH signaling pathway and up-regulating the levels of endogenous neurotrophic factors.
引文
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[4]Luo Y,Zou Y,Yang L,et al.Transplantation of NSCs with OECs alleviates neuropathic pain associated with NGF downregulation in rats following spinal cord injury[J].Neurosci Lett,2013,549:103-108.
[5]Cai X,Lin Y,Hauschka PV,et al.Adipose stem cells originate from perivascular cells[J].Biol Cell,2011,103:435-447.
[6]Song K,Yang Y,Li S,et al.In vitro culture and oxygen consumption of NSCs in size-controlled neurospheres of Ca-alginate/gelatin microbead[J].Mater Sci Eng C Mater Biol Appl,2014,40:197-203.
[7]Chen Q,Zhang Z,Liu J,et al.A fibrin matrix promotes the differentiation of EMSCs isolated from nasal respiratory mucosa to myelinating phenotypical Schwann-like cells[J].Mol cells,2015,38:221-228.
[8]Zhang XM,Ramalho-Santos M,McMahon AP.Smoothened mutants reveal redundant roles for Shh and Ihh signaling including regulation of L/R asymmetry by the mouse node[J].Cell,2001,105:781-792.
[9]Zhang Z,Li Z,Deng W,et al.Ectoderm mesenchymal stem cells promote differentiation and maturation of oligodendrocyte precursor cells[J].Biochem Biophys Res Commun,2016,480:727-733.
[10]毕志刚,倪容之,秦万章,等.复方丙酸氯倍他索软膏治疗寻常性银屑病临床疗效观察[J].临床皮肤科杂志,2004,33:310-312.
[11]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.
[12]Yao X,Su T,Verkman AS.Clobetasol promotes remyelination in a mouse model of neuromyelitis optica[J].Acta Neuropathol Commun,2016,4:42-42.
[13]Harlow DE,Honce JM,Miravalle AA.Remyelination therapy in multiple sclerosis[J].Front Neurol,2015,6:257.
[14]Mullard A.Remyelination researchers regroup after proof-of-concept setback in multiple sclerosis[J].Nat Rev Drug Discov,2016,15:519-521.
[15]Wang J,Lu J,Bond MC,et al.Identification of select glucocorticoids as Smoothened agonists:potential utility for regenerative medicine[J].Proc Natl Acad Sci U S A,2010,107:9323-9328.
[16]Zhang Z,He Q,Deng W,et al.Nasal ectomesenchymal stem cells:multi-lineage differentiation and transformation effects on fibrin gels[J].Biomaterials,2015,49:57-67.
[17]黄秋生,陆汉强,周月鹏,等.成人鼻黏膜间充质干细胞的体外培养和多向诱导分化[J].临床耳鼻咽喉头颈外科杂志,2012,26:490-493,498.
[18]Benowitz LI,Routtenberg A.GAP-43:an intrinsic determinant of neuronal development and plasticity[J].Trends Neurosci,1997,20:84-91.
[19]Tischfield MA,Baris HN,Wu C,et al.Human TUBB3 mutations perturb microtubule dynamics,kinesin interactions,and axon guidance[J].Cell,2010,140:74-87.
[20]Zhao L,Yu Y,Deng C.Protein and mRNA expression of Shh,Smo and Gli1 and inhibition by cyclopamine in hepatocytes of rats with chronic fluorosis[J].Toxicol Lett,2014,225:318-324.
[21]Hyman C,Hofer M,Barde Y A,et al.BDNF is a neurotrophic factor for dopaminergic neurons of the substantia nigra[J].Nature,1991,350:230.
[22]Rizzi C,Tiberi A,Giustizieri M,et al.NGF steers microglia toward a neuroprotective phenotype[J].Glia,2018,13:1395-1416.