氯倍他索干预鼻黏膜来源外胚层间充质干细胞向少突胶质样细胞分化及机制
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摘要
背景:氯倍他索是临床工作中常用的糖皮质激素类药物,鼻黏膜来源外胚层间充质干细胞在体外具有向少突胶质样细胞分化的潜能,氯倍他索能否促进其向少突胶质样细胞分化及其潜在的机制尚不得知。目的:观察氯倍他索对鼻黏膜来源外胚层间充质干细胞向少突胶质样细胞分化的影响,探讨其可能的机制。方法:贴壁筛选法分离培养获取SD大鼠鼻黏膜来源外胚层间充质干细胞,免疫荧光法鉴定其特性和纯度。MTT法检测0-10μmol/L氯倍他索对鼻黏膜来源外胚层间充质干细胞增殖能力的影响。取生长良好的第3代鼻黏膜来源外胚层间充质干细胞,用含氯倍他索的无血清诱导分化培养液使其向少突胶质样细胞分化,氯倍他索浓度为0,2.5和5μmol/L。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少突胶质细胞相关蛋白MBP和OLIG2的表达,免疫印迹法检测SHH信号通路相关蛋白SHH表达水平。结果与结论:(1)体外培养的鼻黏膜来源外胚层间充质干细胞高表达vimentin和nestin;(2)低浓度的氯倍他索可促进细胞增殖;高浓度的氯倍他索则抑制细胞增殖,其中5μmol/L可能为最适增殖浓度;(3)鼻黏膜来源外胚层间充质干细胞成少突胶质样细胞诱导3 d后,氯倍他索组出现多极化形态,少突胶质细胞相关蛋白MBP、OLIG2以及SHH信号通路相关蛋白SHH的表达明显高于对照组;(4)结果表明,氯倍他索可能通过SHH信号通路促进鼻黏膜来源外胚层间充质干细胞向少突胶质样细胞分化。
BACKGROUND:Clobetasol propionate is a commonly used glucocorticoid in clinics. Nasal mucosa-derived ectomesenchymal stem cells(EMSCs) have the potential to differentiate into oligodendrocyte-like cells in vitro. However, the underlying mechanisms by which clobetasol propionate promote the differentiation into oligodendrocyte-like cells are unknown as yet.OBJECTIVE:To observe the effect of clobetasol propionate on the differentiation of nasal mucosa-derived EMSCs into oligodendrocyte-like cells and to explore its possible mechanisms.METHODS:Nasal mucosa-derived EMSCs were isolated and cultured by adherent screening method, and cell properties and purity were identified using immunofluorescence staining. Cell proliferation of EMSCs was detected by MTT after intervention with 0-10 μmol/L clobetasol propionate. Passage 3 nasal mucosa-derived EMSCs grew well and were induced to differentiate into oligodendrocyte-like cells in the serum-free medium containing clobetasol propionate(0, 2.5 and 5 μmol/L). Cell morphology was then observed by inverted microscope.Expression of oligodendrocyte-associated proteins, myelin basic protein and oligodendrocyte transcription factor 2, was measured by immunofluorescence and western blot assay. Expression of SHH related to the SHH signaling pathway was detected by western blot assay.RESULTS AND CONCLUSION:EMSCs cultured in vitro highly expressed vimentin and nestin. Clobetasol propionate at a low concentration promoted the proliferation of EMSCs; and on the contrary, clobetasol propionate at a high concentration inhibited the cell proliferation. Therein,5 μmol/L was the best concentration of clobetasol propionate for cell proliferation. After 3 days of induction, a multi-polarization pattern appeared in the clobetasol propionate group, and the expressions of MBP, OLIG2 and SHH were significantly higher than those in the control group. To conclude, clobetasol propionate may promote the differentiation of EMSCs into oligodendrocyte-like cells via the SHH signaling pathway.
BACKGROUND:Clobetasol propionate is a commonly used glucocorticoid in clinics. Nasal mucosa-derived ectomesenchymal stem cells(EMSCs) have the potential to differentiate into oligodendrocyte-like cells in vitro. However, the underlying mechanisms by which clobetasol propionate promote the differentiation into oligodendrocyte-like cells are unknown as yet.OBJECTIVE:To observe the effect of clobetasol propionate on the differentiation of nasal mucosa-derived EMSCs into oligodendrocyte-like cells and to explore its possible mechanisms.METHODS:Nasal mucosa-derived EMSCs were isolated and cultured by adherent screening method, and cell properties and purity were identified using immunofluorescence staining. Cell proliferation of EMSCs was detected by MTT after intervention with 0-10 μmol/L clobetasol propionate. Passage 3 nasal mucosa-derived EMSCs grew well and were induced to differentiate into oligodendrocyte-like cells in the serum-free medium containing clobetasol propionate(0, 2.5 and 5 μmol/L). Cell morphology was then observed by inverted microscope.Expression of oligodendrocyte-associated proteins, myelin basic protein and oligodendrocyte transcription factor 2, was measured by immunofluorescence and western blot assay. Expression of SHH related to the SHH signaling pathway was detected by western blot assay.RESULTS AND CONCLUSION:EMSCs cultured in vitro highly expressed vimentin and nestin. Clobetasol propionate at a low concentration promoted the proliferation of EMSCs; and on the contrary, clobetasol propionate at a high concentration inhibited the cell proliferation. Therein,5 μmol/L was the best concentration of clobetasol propionate for cell proliferation. After 3 days of induction, a multi-polarization pattern appeared in the clobetasol propionate group, and the expressions of MBP, OLIG2 and SHH were significantly higher than those in the control group. To conclude, clobetasol propionate may promote the differentiation of EMSCs into oligodendrocyte-like cells via the SHH signaling pathway.
引文
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[18]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.Mol Cells.2015;38(3):221-228.
[19]Yao X,Su T,Verkman AS.Clobetasol promotes remyelination in a mouse model of neuromyelitis optica.Acta Neuropathol Commun.2016;4(1):42.
[20]Porcu G,Serone E,De Nardis V,et al.Clobetasol and Halcinonide Act as Smoothened Agonists to Promote Myelin Gene Expression and Rx RγReceptor Activation.PLoS One.2015;10(12):e0144550.
[2]刘洋,王文己.硬膜切开减压术实施时间对急性脊髓损伤的影响[J].西安交通大学学报:医学版,2018,39(2):237-240,244.
[3]Marques S,Zeisel A,Codeluppi S,et al.Oligodendrocyte heterogeneity in the mouse juvenile and adult central nervous system.Science.2016;352(6291):1326-1329.
[4]Hamilton NB,Clarke LE,Arancibia-Carcamo IL,et al.Endogenous GABA controls oligodendrocyte lineage cell number,myelination,and CNS internode length.Glia.2017;65(2):309-321.
[5]Zhou Q,Anderson DJ.The b HLH transcription factors OLIG2and OLIG1 couple neuronal and glial subtype specification.Cell.2002;109(1):61-73.
[6]Huang TN,Yen MI,Nelson DW,et al.Monoacylglycerol Acyltransferase(MGAT)1 and 2 Play Divergent Roles in Adipogenesis/Lipogenesis in Mice.FASEB J.2017;31(1_supplement):781.6.
[7]Zhang Z,He Q,Deng W,et al.Nasal ectomesenchymal stem cells:multi-lineage differentiation and transformation effects on fibrin gels.Biomaterials.2015;49:57-67.
[8]张新萍,彭立新,高影,等.复方丙酸氯倍他索软膏治疗神经性皮炎和慢性湿疹疗效观察[J].临床皮肤科杂志,2015,44(5):320-322.
[9]Najm FJ,Madhavan M,Zaremba A,et al.Drug-based modulation of endogenous stem cells promotes functional remyelination in vivo.Nature.2015;522(7555):216-220.
[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,2004,43(5):310-312.
[11]Wang J,Lu J,Bond MC,et al.Identification of select glucocorticoids as Smoothened agonists:potential utility for regenerative medicine.Proc Natl Acad Sci U S A.2010;107(20):9323-9328.
[12]王振超,黄壮雄,许建鹏,等.复方丙酸氯倍他索乳膏对小型猪皮肤长程涂布的毒性观察[J].广东医学院学报,2004,22(6):563-565.
[13]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(11):490-493,498.
[14]Kim YC,Zhang AH,Yoon J,et al.Engineered MBP-specific human Tregs ameliorate MOG-induced EAE through IL-2-triggered inhibition of effector T cells.J Autoimmun.2018;92:77-86.
[15]Takebayashi H,Yoshida S,Sugimori M,et al.Dynamic expression of basic helix-loop-helix Olig family members:implication of Olig2 in neuron and oligodendrocyte differentiation and identification of a new member,Olig3.Mech Dev.2000;99(1-2):143-148.
[16]Rowitch DH,Lu QR,Kessaris N,et al.An'oligarchy'rules neural development.Trends Neurosci.2002;25(8):417-422.
[17]Farreny MA,Agius E,Bel-Vialar S,et al.FGF signaling controls Shh-dependent oligodendroglial fate specification in the ventral spinal cord.Neural Dev.2018;13(1):3.
[18]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.Mol Cells.2015;38(3):221-228.
[19]Yao X,Su T,Verkman AS.Clobetasol promotes remyelination in a mouse model of neuromyelitis optica.Acta Neuropathol Commun.2016;4(1):42.
[20]Porcu G,Serone E,De Nardis V,et al.Clobetasol and Halcinonide Act as Smoothened Agonists to Promote Myelin Gene Expression and Rx RγReceptor Activation.PLoS One.2015;10(12):e0144550.