人胚胎干细胞源多巴胺能神经元的功能性分化

发布时间：2019-03-20 10:59

【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。
[Abstract]:Background: stem cell-derived dopaminergic neurons as a cell source of Parkinson's disease replacement therapy, its in vitro differentiation scheme has been continuously optimized and improved, and the subsequent identification methods and detection indexes have been continuously improved. Objective: to observe the morphological development process of human embryonic stem cells differentiated into dopaminergic neurons and detect their electrophysiological characteristics in order to understand whether human embryonic stem cells can develop into morphologically mature cells under the current differentiation scheme. Functional dopaminergic neurons. Methods: human embryonic stem cells were induced to differentiate into dopaminergic neurons by means of monolayer attachment and SMAD channel double inhibitor differentiation. The morphology and immunochemistry of dopaminergic neurons were identified by electron microscopy and cellular immunofluorescence, and the electrophysiological characteristics of dopaminergic neurons were detected by patch clamp technique. According to the electrophysiological standard of dopaminergic neurons in vivo, the function of dopaminergic neurons differentiated in vitro was evaluated. Results & conclusion: (1) Human embryonic stem cells were successfully induced to differentiate into morphologically mature dopaminergic neurons by using SMAD channel double inhibitor differentiation scheme. (2) patch clamp test showed that dopaminergic neurons had mature electrophysiological function, and their electrophysiological characteristics accorded with the evaluation criteria of dopaminergic neurons in vivo. (3) the above differentiation scheme can differentiate human embryonic stem cells into mature, functional dopaminergic neurons.
【作者单位】：海南医学院第一附属医院神经内科;海南医学院第一附属医院检验科;海南医学院第一附属医院眼科;
【基金】：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)~~
【分类号】：R742.5

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