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浙江大学学报（医学版）
浙江大学学报（医学版）  2012, Vol. 41 Issue (4): 373-380    DOI: 10.3785/j.issn.1008-9292.2012.04.004
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基于光镜表型的促神经元亚型分化药物初筛法
高益宁1，王丹音1，2，潘宗富1，梅宇钦1，王志强1，朱丹雁1，楼宜嘉1
1.浙江大学药学院 药理毒理与生化药学研究所，浙江 杭州 310058； 2.浙江大学医学院附属第二医院药剂科，浙江 杭州 310009
Phenotype-based primary screening for drugs promoting neuronal subtype differentiation in embryonic stem cells with light microscope
GAO Yi-ning1, WANG Dan-ying 1,2, PAN Zong-fu1, MEI Yu-qin1, WANG Zhi-qiang1, ZHU Dan-yan1, LOU Yi-jia1
1.Institute of Pharmacology, Toxicology and Biochemical Pharmaceutics, Zhejiang University, Hangzhou 310058, China; 2．Department of Pharmacy, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, China
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摘要： 目的：建立基于光镜表型的促胚胎干（embryonic stem，ES）细胞神经元亚型分化药物筛选法，供早期发现促神经再生药初筛用。
方法：采用悬滴培养4-/4+法，形成拟胚体，继而贴壁培养向神经细胞分化，光镜确认表型呈神经元样的基础上进一步评价各类亚型。以10-7 mol/L异补骨脂甲素（Isobavachin，IBA）和10-6 mol/L淫羊藿苷（Icariin，ICA）为例，分化终点以光镜下轴突为胞体三倍长以上者为神经元样细胞，继续以免疫荧光成像法确认神经元（β-tubulinⅢ阳染），并进一步以特异性抗体（ChAT、GABA）阳染共定位鉴别相应神经元亚型。
结果：基于光镜表型评价，在分化终点即可剔除轴突分化不全的细胞，取代了以往免疫荧光成像所需的人力、物力与时间；并避免进一步ELISA或流式细胞术评价的假阳性。光镜显示IBA处理者大部分显示神经元样表型，而ICA处理的细胞却不然，免疫荧光成像结果与此相一致。因此，仅对IBA处理的神经元做亚型鉴定，显示胆碱能神经元以及γ-氨基丁酸能神经元亚型。
结论：基于光镜表型的促神经元分化初筛法在化合物促神经元亚型分化上具有简便易行、节约物力与时间，避免假阳性的独到之处；经该法初筛后供后续研究，可见IBA具有促胆碱能和γ-氨基丁酸能神经元分化活性。
关键词： 胚胎干细胞； 神经元/细胞学； 神经元表型； 异补骨脂甲素； 淫羊藿苷； 乙酰胆碱能神经元； γ  -氨基丁酸能神经元    
Abstract: Objective： To set up a platform for phenotype-based primary screening of drug candidates promoting neuronal subtype differentiation in embryonic stem cells (ES) with light microscope.
Methods： Hanging drop culture 4-/4+ method was employed to harvest the cells around embryoid body (EB) at differentiation endpoint. Morphological evaluation for neuron-like cells was performed with light microscope. Axons for more than three times of the length of the cell body were considered as neuron-like cells. The compound(s) that promote neuron-like cells was further evaluated. Icariin (ICA, 10-6mol/L) and Isobavachin (IBA, 10-7mol/L) were selected to screen the differentiation-promoting activity on ES cells. Immunofluorescence staining with specific antibodies (ChAT, GABA) was used to evaluate the neuron subtypes.
Results： The cells treated with IBA showed neuron-like phenotype, but the cells treated with ICA did not exhibit the morphological changes. ES cells treated with IBA was further confirmed to be cholinergic and GABAergic neurons.
Conclusions： Phenotypic screening with light microscope for molecules promoting neuronal differentiation is an effective method with advantages of less labor and material consuming and time saving, and false-positive results derived from immunofluorescence can be avoided. The method confirms that IBA is able to facilitate ES cells differentiating into neuronal cells, including cholinergic neurons and GABAergic neurons.
Key words: Embryonic stem cells    Neurons/cytol    Neuron phenotype    Isobavachin    Icariin    cholinergic neurons    GABAergic neurons
出版日期: 2012-07-25
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高益宁;王丹音;潘宗富;梅宇钦;王志强;朱丹雁;楼宜嘉. 基于光镜表型的促神经元亚型分化药物初筛法[J]. 浙江大学学报（医学版）, 2012, 41(4): 373-380.

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https://www.zjujournals.com/med/CN/Y2012/V41/I4/373

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