中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2019, Vol. 14 ›› Issue (3): 501-505.doi: 10.4103/1673-5374.245476

• 原著:神经损伤修复保护与再生 • 上一篇    下一篇

非整合性质粒载体介导人源成纤维细胞直接诱导为神经元的方法

  

  • 出版日期:2019-03-15 发布日期:2019-03-15
  • 基金资助:

    中国国家自然科学基金项目（81471126，81771216），浙江省自然科学基金项目（LY17H090005），浙江省医学科技计划项目（2016KYB119）

Plasmid-based generation of neural cells from human fibroblasts using non-integrating episomal vectors

Shao-Bing Dai 1 , Ting Shen 2 , Ting-Ting Zheng 2 , Jia-Li Pu 2, Xin-Zhong Chen 1   

  1. 1 Department of Anesthesiology, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China
    2 Department of Neurology, Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China
  • Online:2019-03-15 Published:2019-03-15
  • Contact: Xin-Zhong Chen, MD, chenxinz@zju.edu.cn; Jia-Li Pu, PhD, jialipu@zju.edu.cn, carrie_1105@163.com.
  • Supported by:

    This study was supported by the National Natural Science Foundation of China, No. 81471126 (to XZC) and 81771216 (to XZC); the Natural Science Foundation of Zhejiang Province of China, No. LY17H090005 (to JLP); a grant from the Medical Science and Technology Plan Project of Zhejiang Province of China, No. 2016KYB119 (to JLP).

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摘要：

人成纤维细胞分化为功能性神经元依赖于病毒介导的转录因子的导入，这种方法存在病毒基因整合和致瘤等风险；近年来虽然有一些研究可通过小分子化合物持续表达成功直接诱导神经元，但仅证实在小鼠源性细胞上有效。为此，实验设计将含有Epstein-Barr病毒衍生的oriP/EBNA1编码神经元转录因子Ascl1、神经元特异性微小RNAmiR124和P53抑制剂shP53的episomal载体转染人成纤维细胞，以神经元诱导培养基诱导培养，以免疫荧光染色检测诱导后细胞中Tuj-1、微管相关蛋白2、神经元特异性核蛋白NeuN和神经细胞粘附分子的免疫阳性反应。诱导11d时，Tuj1阳性细胞比例达36.7%；诱导后21d时可见微管相关蛋白2、NeuN和神经细胞粘附分子表达。实验提出的这种简单的基于质粒的方法将人成纤维细胞直接重编程为神经元，为未来疾病建模和神经退行性疾病的研究提供了模型支持。

orcid: 0000-0002-6719-4060(Jia-Li Pu)

关键词: 诱导分化, 神经元, 质粒, 人源成纤维细胞, 核转染, Ascl1, miRNA124, P53, 重编程, 神经再生

Abstract:

Differentiation of human fibroblasts into functional neurons depends on the introduction of viral-mediated transcription factors, which present risks of viral gene integration and tumorigenicity. In recent years, although some studies have been successful in directly inducing neurons through sustained expression of small molecule compounds, they have only been shown to be effective on mouse-derived cells. Thus, herein we delivered vectors containing Epstein-Barr virus-derived oriP/Epstein-Barr nuclear antigen 1 encoding the neuronal tran¬scription factor, Ascl1, the neuron-specific microRNA, miR124, and a small hairpin directed against p53, into human fibroblasts. Cells were incubated in a neuron-inducing culture medium. Immunofluorescence staining was used to detect Tuj-1, microtubule-associated protein 2, neuron-specific nucleoprotein NeuN and nerve cell adhesion molecules in the induced cells. The proportion of Tuj1-positive cells was up to 36.7% after induction for 11 days. From day 21, these induced neurons showed neuron-specific expression patterns of mi-crotubule-associated protein 2, NeuN and neural cell adhesion molecule. Our approach is a simple, plasmid-based process that enables direct reprogramming of human fibroblasts into neurons, and provides alternative avenues for disease modeling and neurodegenerative medicine.

Key words: nerve regeneration, induced neurons, plasmid-based, human fibroblasts, nucleofection, Ascl1, miR124, p53, reprogramming, neural regeneration