中国组织工程研究 ›› 2020, Vol. 24 ›› Issue (13): 2055-2060.doi: 10.3969/j.issn.2095-4344.2059

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

羊水干细胞定向及稳定分化为神经元样细胞体系的建立

宗  凌1,2,陈观贵1,张兰珍3,翟锦明1,龙艳波1,刘晓岚3   

  1. 广州医科大学附属第二医院,1耳鼻咽喉科,3产科,广东省广州市  510260;2中山大学附属第一医院耳鼻咽喉科;中山大学耳鼻咽喉科学研究所,广东省广州市  510080
  • 收稿日期:2019-10-22 修回日期:2019-10-24 接受日期:2019-11-25 出版日期:2020-05-08 发布日期:2020-03-09
  • 通讯作者: 陈观贵,博士,副主任医师,副教授,广州医科大学附属第二医院耳鼻咽喉科,广东省广州市 510260
  • 作者简介:宗凌,女,1987年生,2014年中山大学附属第一医院毕业,博士,主治医师,主要从事耳神经外科临床和基础研究。
  • 基金资助:
    国家自然科学基金项目(81700912);广东省自然科学基金项目(2016A030310286)

The system for directional and stable differentiation of amniotic fluid stem cells into neuron-like cells

Zong Ling1, 2, Chen Guangui1, Zhang Lanzhen3, Zhai Jinming1, Long Yanbo1, Liu Xiaolan3   

  1. 1Department of Otorhinolaryngology, 3Department of Obstetrics, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, Guangdong Province, China; 2Department of Otorhinolaryngology, the First Affiliated Hospital of Sun Yat-sen University and Institute of Otorhinolaryngology, Sun Yat-sen University, Guangzhou 510080, Guangdong Province, China
  • Received:2019-10-22 Revised:2019-10-24 Accepted:2019-11-25 Online:2020-05-08 Published:2020-03-09
  • Contact: Chen Guangui, MD, Associate chief physician, Associate professor, Department of Otorhinolaryngology, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, Guangdong Province, China
  • About author:Zong Ling, MD, Attending physician, Department of Otorhinolaryngology, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, Guangdong Proince, China; Department of Otorhinolaryngology, the First Affiliated Hospital, Sun Yat-sen University and Institute of Otorhinolaryngology, Sun Yat-sen University, Guangzhou 510080, Guangdong Province, China
  • Supported by:
    the National Natural Science Foundation of China, No. 81700912; the Natural Science Foundation of Guangdong Province, No. 2016A030310286

摘要:

文题释义:

羊水干细胞:羊水是由多种细胞组成的异质细胞群,其中包括胎儿脱落的表皮细胞、呼吸道上皮细胞、消化道和泌尿道等体腔管道的脱落细胞、羊膜脱落细胞等。羊水中包含有向内、中、外3个胚层分化潜能的干细胞,约占1%,近年来研究发现羊水干细胞在体外通过特殊的诱导微环境能够分化成多种不同类型的细胞,使羊水来源干细胞成为干细胞研究的一个新热点。

悬滴培养:与传统的二维培养模式不同,悬滴培养提供的三维环境有利于促进各种干细胞的增殖与分化,可以使细胞聚集,相互紧密接触,从而促进细胞之间的相互作用,进而提高或增强干细胞的生物学功能。羊水干细胞悬滴培养可形成拟胚体结构,具有较强的分化潜能,有助于提高定向诱导分化效率。

背景:利用干细胞分化代替受损神经元的设想,已经逐渐受到科研工作者的关注。胚胎干细胞和诱导性多能干细胞虽然具有良好的神经元分化潜能,但由于接种活体动物具有致瘤性,限制了其进一步的深入研究。
目的:建立稳定的羊水干细胞分选、培养、成神经诱导分化体系,探讨其作为神经元再生种子细胞的可行性。
方法:经B超引导下,穿刺获取孕19-22周期间的羊水样本10 mL,磁珠分选其中c-Kit阳性的羊水干细胞。免疫荧光染色鉴定羊水干细胞标记物Oct-4、Sox2;RT-PCR检测羊水干细胞多次传代后干性标记物c-Kit、Oct-4、Sox2、Nestin的表达;羊水干细胞悬滴培养4 d观察拟胚体的形成情况;采用“两阶段”分步诱导羊水干细胞向神经元方向分化,免疫荧光染色观察Neuro D、Tuj1的表达。
结果与结论:①羊水中可分选出大约1%的c-Kit阳性羊水干细胞;②(75.0±4.6)%的羊水干细胞表达Oct-4,(86.0±2.8)%的羊水干细胞表达Sox2;③RT-PCR方法检测干性标记物c-Kit、Oct-4、Sox2、Nestin的表达量并不随细胞传代数的增加而改变;④经悬滴培养可形成拟胚体并表达Oct-4;⑤免疫荧光证实未经诱导的羊水干细胞表达神经元标记物Tuj1,但缺乏典型的神经元形态特征;RT-PCR证实不同羊水干细胞标本都能检测到Tuj1的表达,同一样本多次传代也能稳定检测到Tuj1的表达;⑥羊水干细胞经碱性成纤维细胞生长因子、脑源性神经营养因子、神经营养因子3的两阶段分步诱导后表达神经干细胞标记物Neuro D和神经元标记物Tuj1,具有神经元样细胞的形态特征。

ORCID: 0000-0001-6488-2055(宗凌)

中国组织工程研究杂志出版内容重点:干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程

关键词: 羊水干细胞, 拟胚体, 悬滴培养, 神经元分化, 神经营养因子

Abstract:

BACKGROUND: Neuronal regeneration using stem cell differentiation has gained a lot of attentions from researchers. Although embryonic stem cells and induced pluripotent stem cells have good potential for neuronal differentiation, a high risk of tumor development in vivo limits the further study.

OBJECTIVE: To establish a stable system for sorting, culture and neuronal differentiation of amniotic fluid stem cells, and to explore the feasibility as seed cells for neuronal regeneration.

METHODS: Amniotic fluid sample (10 mL) was obtained at 19-22 weeks of pregnancy under B-ultrasound guidance, and amniotic fluid stem cells were isolated by c-Kit magnetic beads. The markers Oct-4 and Sox2 of amniotic fluid stem cells were identified by immunofluorescence. The expression levels of c-Kit, Oct-4, Sox2 and Nestin in amniotic fluid stem cells after multiple passages were detected by RT-PCR. Then, the cells were cultured by hanging drop for 4 days to observe the embryoid bodies-like structures. Amniotic fluid stem cells were induced to differentiate into neurons using two-stage method. The expression levels of Neuro D and Tuj1 were observed by immunofluorescence.

RESULTS AND CONCLUSION: (1) About 1% of amniotic fluid stem cells were positive for c-Kit. (2) (75.0±4.6)% of amniotic fluid stem cells expressed Oct-4 and (86.0±2.8)% of the cells expressed Sox2. (3) The expression levels of c-Kit, Oct-4, Sox2 and Nestin detected by RT-PCR did not change with passage times. (4) Embryoid bodies-like structures formed after hanging drop culture. (5) Immunofluorescence results showed that amniotic fluid stem cells expressed neuronal marker Tuj1, but without the typical morphological features. RT-PCR detected the expression of Tuj1 in different amniotic fluid stem cell specimens as well as in the same sample after several passages. (6) Amniotic fluid stem cells could have the characteristics of neuron-like cells after induction with basic fibroblast growth factor, brain-derived neurotrophic factor, and neurotrophin factor 3 in two stages, and could express neural stem cell marker Neuro D and neuronal marker Tuj1.

Key words: amniotic fluid stem cells, embryonic body, hanging drop, neuronal differentiation, neurotrophic factor

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