[1]王淑辉 王薇 张海廷等. Wnt3a调控大鼠骨髓间充质干细胞向胆碱能神经元分化的实验研究[J].卒中与神经疾病杂志,2016,23(05):309-313.[doi:10.3969/j.issn.1007-0478.2016.05.001]
　Wang Shuhui,Wang Wei,Zhang Haiting,et al. Experimental study of Wnt3a regulating the cholinergic neuron differentiation of rat bone marrow mesenchymal stem cells[J].Stroke and Nervous Diseases,2016,23(05):309-313.[doi:10.3969/j.issn.1007-0478.2016.05.001]
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 Wnt3a调控大鼠骨髓间充质干细胞向胆碱能神经元分化的实验研究(/HTML)
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《卒中与神经疾病》杂志[ISSN:1007-0478/CN:42-1402/R]

卷:
第23卷
期数:
2016年05期
页码:
309-313
栏目:
论 著
出版日期:
2016-10-26

文章信息/Info

Title:
 Experimental study of Wnt3a regulating the cholinergic neuron differentiation of rat bone marrow mesenchymal stem cells
作者:
 王淑辉 王薇 张海廷等
100050 北京,首都医科大学附属北京友谊医院神经科[王淑辉 王薇(共同第一作者)张海廷 张拥波 李继梅]; 首都医科大学基础医学院生物化学与分子生物学系(程杉); 解放军总医院老年神经科(尚延昌)
Author(s):
 Wang Shuhui; Wang Wei; Zhang Haiting;  et al.
 Department of Neurology, Beijing Friendship Hospital, Capital Medical University, Beijing 100050
关键词:
 ; 骨髓间充质干细胞 Wnt3a 胆碱能神经元 阿尔茨海默病
Keywords:
MSCs Wnt3a Cholinergic neuron Alzheimer’s disease
分类号:
R741
DOI:
10.3969/j.issn.1007-0478.2016.05.001
文献标志码:
A
摘要:
 目的 探讨经典Wnt/β-catenin通路对大鼠MSCs在体外分化为神经元和胆碱能神经元的调节作用。方法 取SD大鼠股骨和胫骨的骨髓,利用差速贴壁法分离、扩增及纯化MSCs,绘制生长曲线; 应用免疫荧光和Western-Blot的方法检测Wnt3a处理后β-catenin蛋白的分布变化; 取第4代MSCs分为3组; A组为空白对照组,用空白DMEM培养基培养细胞; B组为诱导分化对照组,用含100 ng/mL碱性成纤维细胞生长因子(basic fibroblast grown factor, bFGF)、5 umol/L维甲酸(retinoic acid, RA)的DMEM诱导培养基培养细胞; C组为Wnt3a诱导分化组,在上述诱导培养基中加入50 ng/mL Wnt3a培养细胞; 用形态学观察和Western-Blot的方法比较各组对MSCs向神经元及胆碱能神经元分化的影响; 应用形态学观察和Western-Blot的方法比较各组对MSCs向神经元及胆碱能神经元分化的影响。结果 利用差速贴壁法细胞传至P3代时形态趋于一致,呈均匀分布生长。P3代细胞的生长曲线显示,接种后的第1、2 d细胞处于潜伏期; 第3、4 d细胞进入对数生长期; 第5 d进入平台期。P4代细胞高度表达CD29和CD44(阳性率分别为99.9%和73.2%)。Wnt3a处理组细胞的β-catenin在细胞核的分布较对照组细胞显著增多(P<0.01)。诱导分化细胞组中A组细胞可检测到少量神经元的标记物,但未检测到胆碱能神经元的标志物,B组和C组细胞均可检测到神经元和胆碱能神经元的标志物,B组和C组分化为神经元的比例较A组显著增高(P<0.01); C组分化为胆碱能神经元的比例较B组显著增高(P<0.01)。结论 Wnt3a能够促进MSCs内的β-catenin的核转移激活经典Wnt信号通路,促进体外培养的MSCs向胆碱能神经元分化。
Abstract:
ObjectiveTo investigate the effects of canonical Wnt/β-catenin signaling pathway on differentiation of MSCs into neuron and cholinergic neuron.Methods Rat MSCs were isolated and purified through differential anchoring method. Observe the morphological changes of cells under optical microscope, drew the cell growth curves, and detect the surface antigens by FACScan. The distribution of β-catenin was detected by Western blot and immunofluorescence stain after Wnt3a treatment. Then the fourth generation of MSCs was divided into three groups. Group A: cells were cultured in DMEM; Group B: cells were cultured in DMEM supplemented with 100 ng/ml basic fibroblast growth factor(bFGF)and 5 umol/L retinoic acid(RA); Group C: cells were cultured in DMEM supplemented with 100 ng/ml bFGF, 5 umol/L RA and 50ng/ul Wnt3a. The morphological changes of the cells were observed and the markers of neurons and cholinergic neurons were detected by Western blot after inducing neuronal differentiation.Results MSCs were purified after passage 3 through differential anchoring method. The growth kinetics of MSCs showed cells grew slowly in the first two days. After inoculation, entered a logarithmic phase from the third day and became slowly in the fifth day. Positive rate of CD29 and CD44 was 99.9% and 73.2%. Compared with control group, Wnt3a treated MSCs showed a higher level of β-catenin in the nucleus(P<0.01). The marker of neurons was detected in Group A, but the marker of cholinergic neurons couldn’ t be detected. The marker of both neuron and cholinergic could be detected in Group B and Group C. Compared to Group A, the level of the neurons’ marker was higher in both Group B and Group C(P<0.01). Compared with Group B, the level of the cholinergic neurons’ marker was significantly higher in the Group C(P<0.01).Conclusion Wnt3a could induce beta-catenin nuclear translocation and activate the canonical Wnt pathway in MSCs. The canonical Wnt signaling pathway enhances the differentiation of MSCs into cholinergic neurons.

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备注/Memo

备注/Memo:
 (2016-03-05收稿)
更新日期/Last Update: 2016-10-30