中国组织工程研究 ›› 2017, Vol. 21 ›› Issue (33): 5360-5368.doi: 10.3969/j.issn.2095-4344.2017.33.018

• 胚胎干细胞 embryonic stem cells • 上一篇    下一篇

人胚胎干细胞源多巴胺能神经元的功能性分化

彭雅南1,胡 兰2,王 埮1,李 科3,杨 柳1,陈 丽1,陈小武1,陈志斌1,赵振强1   

  1. 海南医学院第一附属医院,1神经内科,2检验科,3眼科,海南省海口市 570102
  • 修回日期:2017-09-12 出版日期:2017-11-28 发布日期:2017-12-01
  • 通讯作者: 赵振强,博士,主任医师,海南医学院第一附属医院神经内科,海南省海口市 570102
  • 作者简介:彭雅南,女,1990年生,江西省上饶市人,汉族,2017年海南医学院神经病学专业毕业,硕士,医师,主要从事干细胞源多巴胺能神经元的分化研究。
  • 基金资助:

    国家自然科学基金(3126023/C100308);海南省国际合作项目(KJHZ2015-09);海南省国际合作项目(2012-GH002);海南省研究生创新科研课题立项(Hys2016-84)

Functional differentiation of dopaminergic neurons derived from human embryonic stem cells

Peng Ya-nan1, Hu Lan2, Wang Tan1, Li Ke3, Yang Liu1, Chen Li1, Chen Xiao-wu1, Chen Zhi-bin1,Zhao Zhen-qiang1   

  1. 1Department of Neurology, 2Clinical Laboratory, 3Department of Ophtalmology, First Affiliated Hospital, Hainan Medical University, Haikou 570102, Hainan Province, China
  • Revised:2017-09-12 Online:2017-11-28 Published:2017-12-01
  • Contact: Zhao Zhen-qiang, M.D., Chief physician, Department of Neurology, First Affiliated Hospital, Hainan Medical University, Haikou 570102, Hainan Province, China
  • About author:Peng Ya-nan, Master, Physician, Department of Neurology, First Affiliated Hospital, Hainan Medical University, Haikou 570102, Hainan Province, China
  • Supported by:

    the National Natural Science Foundation of China, No. 3126023/C100308; the International Cooperation Projects of Hainan Province, No. KJHZ2015-09, 2012-GH002; Hainan Province Graduate Student Innovation Research Project, No. Hys2016-84

摘要:

文章快速阅读:

文题释义:
A9神经元:
中脑多巴胺能神经元根据投射区域和功能调控的不同,可分为A8,A9和A10这3个亚群。A9神经元位于黑质致密部,其发出的神经纤维投射到背侧纹状体,调控躯体的随意运动,其变性缺失可以引起帕金森病。定向诱导人胚胎干细胞分化为成熟的、有功能的多巴胺能神经元,用于移植治疗帕金森病,而这些多巴胺能神经元中A9神经元的纯度,与后期帕金森病动物模型的功能恢复呈正相关性。
膜片钳:实验采用膜片钳技术检测神经元的全细胞电压门控电流、配体门控电流、兴奋性突触后电流和动作电位,判定产物多巴胺能神经元具备成熟的电压、配体门控电流,具备发射动作电位的能力;同时参照体内多巴胺能神经元的电生理标准,对体外分化的多巴胺能神经元进行功能评价,最终证实在本实验方案下,诱导人胚胎干细胞分化而来的多巴胺能神经元具备成熟的电生理功能,且接近体内多巴胺能神经元。

 

摘要
背景:
干细胞源多巴胺能神经元作为帕金森病替代疗法的细胞来源,其体外分化方案被不断的优化和改良,后续的鉴定手段和检测指标也随之不断完善。
目的:通过观察人胚胎干细胞分化为多巴胺能神经元的形态发育过程,检测其电生理特性,以了解在目前分化方案下人胚胎干细胞能否发育为形态成熟、功能成熟的多巴胺能神经元。
方法:通过单层贴壁法,采用SMAD通道双抑制剂分化方案,定向诱导人胚胎干细胞分化为多巴胺能神经元,通过光镜、电镜和细胞免疫荧光等检测手段进行形态学和免疫化学鉴定,并应用膜片钳技术检测多巴胺能神经元的电生理特性。参照体内多巴胺能神经元的电生理标准,对体外分化的多巴胺能神经元进行功能评价。
结果与结论:①采用SMAD通道双抑制剂分化方案,成功诱导人胚胎干细胞定向分化为形态学上发育成熟的多巴胺能神经元;②膜片钳检测结果显示多巴胺能神经元具有成熟的电生理功能,且其电生理特性符合体内多巴胺能神经元的评价标准;③上述分化方案可以将人胚胎干细胞定向分化为成熟的、有功能的多巴胺能神经元。

 

关键词: 干细胞, 人胚胎干细胞, 细胞分化, 多巴胺能神经元, 帕金森病, 膜片钳, 细胞电生理, 国家自然科学基金

Abstract:

BACKGROUND: The in vitro differentiation methods of stem cell-derived dopaminergic neurons that serve as a cell source for the replacement therapy of Parkinson’s disease are continuously optimized and improved, as well as the subsequent identification methods and testing indicators.
OBJECTIVE: To observe the morphological development and electrophysiological characteristics of dopaminergic neurons differentiated from human embryonic stem cells so as to identify whether these differentiated cells have mature morphology and function under the current differentiation program. 
METHODS: Monolayer adherent method combined with dual-SMAD signaling inhibition was used to induce the directional differentiation of human embryonic stem cells into dopaminergic neurons. Then the cells were identified by light microscopy, electron microscopy and immunofluorescence, and the electrophysiological properties of dopaminergic neurons were detected by patch clamp electrophysiological technique. Herein, we evaluated the electrophysiological functions of dopaminergic neurons differentiated in vitro, with reference to the evaluation standard of dopaminergic neuron in vivo.
RESULTS AND CONCLUSION: In this study, we successfully obtained dopaminergic neurons with mature morphology and functions differentiated from human embryonic stem cells in vitro. Findings from the subsequent electrophysiological test confirmed that dopaminergic neurons we acquired had electrophysiological properties in accordance with the evaluation standards of dopaminergic neurons in vivo. To conclude, the monolayer adherent method combined with dual-SMAD signaling inhibition can successfully induce the directional differentiation of human embryonic stem cells into dopaminergic neurons with mature morphology and functions. 

 

Key words: Embryonic Stem Cells, Cell Differentiation, Neurons, Patch-Clamp Techniques, Parkinson Disease, Tissue Engineering

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