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.
作者
史文涛
戴瑶
毕士奇
陆浩
吕德民
张志坚
SHI Wen-tao;DAI Yao;BI Shi-qi;LU Hao;LV De-min;ZHANG Zhi-jian(Department of Orthopaedics,Nanjing Gaochun People’s Hospital,Nanjing 211300,China;Department of Histology and Embryology,School of Medicine,Jiangsu University,Jiangsu 212001,China)
出处
《神经损伤与功能重建》
2019年第1期1-5,共5页
Neural Injury and Functional Reconstruction
基金
国家自然科学基金地区科学基金(No.81571830)
镇江市科技局发展项目(No.SH2008044)
