两种人胚胎干细胞系向神经元方向诱导分化的比较研究  

A comparative study of the neural induction of two human embryonic stem cell lines

作　　者：陈安迪 李子杰 赵又谊 武胜昔 刘加强 王亚周 Chen Andi;Li Zijie;Zhao Youyi;Wu Shengxi;Liu Jiaqiang;Wang Yazhou(Department of Neurobiology,School of Basic Medicine,Air Force Medical University,Xi'an 710032;Department of Anesthesiology,School of Stomatology,Air Force Medical University,Xi'an 710032;Department of Health Care Division,Guard Bureau of the Joint Staff,Beijing 100017,China)

机构地区：[1]空军军医大学基础医学院神经生物学教研室,西安710032 [2]空军军医大学口腔医院口腔麻醉学教研室,西安710032 [3]联合参谋部警卫局卫生保健处,北京100017

出　　处：《神经解剖学杂志》2022年第1期21-27,共7页Chinese Journal of Neuroanatomy

基　　金：国家自然科学基金(81730035,31970907);陕西省重点研发计划项目(2020ZDLSF01-09)。

摘　　要：目的:比较研究两种人胚胎干细胞系H8和H9在维持培养、向神经前体细胞和神经元方向分化的特性。方法:采用mTeSR1做为基本培养基,Matrigel做为基质胶,进行无滋养层的维持培养。采用白细胞抑制因子1(Smad-1)双抑制剂的神经前体细胞诱导,和含B27的Neurobasal联合脑源性神经营养因子(BDNF)、胶质细胞源性神经营养因子(GDNF)的神经元诱导方案。采用免疫荧光化学染色检测胚胎干细胞标记蛋白(Nanog和OCT4)、神经前体细胞标记蛋白(Nestin、Sox2和Pax6)、神经元标记蛋白Tuj-1的表达。结果:在维持培养和神经诱导的早期:H8和H9细胞在胚胎干细胞标记蛋白表达及类胚体形成方面无显著差异。神经前体细胞阶段:诱导分化相同时间(诱导14 d),两种细胞系的玫瑰花环样结构形成,及巢蛋白(Nestin)、SRY转录因子2(Sox2)的表达均无显著差异;相对而言,更高比例的H9细胞表达神经前体细胞标记蛋白成对同源基因盒6(Pax6)。神经元阶段:诱导相同时间(诱导30 d),约89%的H9细胞表达神经元标记蛋白Tuj-1,而仅约34%的H8细胞表达Tuj-1。结论:H8、H9细胞的胚胎干细胞特性相似。与H8相比,H9细胞的神经元方向分化效率更高,更适合进行神经系统疾病机制及转化研究。Objective:To compare the properties of two human embryonic stem cell(ESC)lines(H8 and H9)under maintaining,neural induction and neuronal differentiation conditions.Methods:Feeder free culture was adopted by using mTeSR1 as basic medium and Matrigel as extracellular matrix.Double Smad-1 inhibitors were adopted for neural induction.Neurobasal containing B27,brain-derived neurotrophic factor(BDNF)and glia derived neurotrophic factor(GDNF)was used for neuronal differentiation.Immunocytochemistry was conducted to evaluate the expression of ESC markers(Nanog,OCT4),neural stem cell markers(Nestin,Sox2,and Pax6)and neuronal marker(Tuj-1).Results:In the maintaining phase,there is no difference of ESC markers and embryonic body formation between H8 and H9 cell lines.In the early phase of neural induction,H8 and H9 cells exhibit similar expression levels of Nestin and SRY-transcription factor 2(Sox2)and similar efficiency in rosette formation.Interestingly,H9 cells show significantly higher percentages of paired box protein 6(Pax6)expression in comparison with H8 cells.In the neuronal differentiation phase,significantly more neuron-specific tubulin III(Tuj-1)positive cells(approximately 89%)were found in H9 cells,while only 34%H8 cells were Tuj-1-positive.Conclusion:H8 and H9 cells are similar in terms ESC properties.H9 cells are more efficient in neuronal induction,as compared with H8.Our data suggested that H9 cells,are relatively suitable for neuroscience research and translation.

关 键 词：胚胎干细胞 神经干细胞 诱导分化 神经前体细胞 神经元 

分 类 号：R321[医药卫生—人体解剖和组织胚胎学]