中国组织工程研究

中国组织工程研究 ›› 2026, Vol. 30 ›› Issue (19): 4942-4948.doi: 10.12307/2026.246

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

miR-9调控小鼠大脑皮质神经干细胞的分化

刘颖赵1，马艳霞2，林耀发1，张国桥1，苗炜亮1，贾燕丽1，池宸申1，宋旺胜1，李  迪3，刘成龙4，张浩楠1   

  1. 1上海市第一人民医院嘉定医院(上海市嘉定区江桥医院)，上海市   201803；2苏州大学骨科研究所，江苏省苏州市   215006；3苏州大学附属第一医院神经外科，江苏省苏州市   215000；4高邮市人民医院麻醉科，江苏省高邮市   225600
  • 收稿日期:2025-09-02 接受日期:2025-11-28 出版日期:2026-07-08 发布日期:2026-02-14
  • 通讯作者: 张浩楠，硕士，医师，上海市第一人民医院嘉定医院(上海市嘉定区江桥医院)，上海市 201803； 共同通讯作者：刘成龙，博士，主任医师，高邮市人民医院麻醉科，江苏省高邮市 225600
  • 作者简介:刘颖赵，男，1979年生，硕士，副主任医师，主要从事骨科(关节、创伤、修复重建、运动医学及基础)研究。 共同第一作者：马艳霞，女，1986年生，硕士，实验师，主要从事神经干细胞与神经修复方向研究。
  • 基金资助:
    上海市嘉定区自然科学研究课题(JDKW-2024-0037)，项目负责人：刘颖赵；江苏大学临床医学科技发展基金项目(JLY20180167)，项目负责人：刘成龙；国家自然科学基金青年项目(81801238)，项目负责人：李迪

miR-9 regulates the differentiation of neural stem cells in mouse cerebral cortex

Liu Yingzhao1, Ma Yanxia2, Lin Yaofa1, Zhang Guoqiao1, Miao Weiliang1, Jia Yanli1, Chi Chenshen1, Song Wangsheng1, Li Di3, Liu Chenglong4, Zhang Haonan1   

  1. 1Jiading Hospital (Jiangqiao Hospital), Shanghai General Hospital, Shanghai 201803, China; 2Institute of Orthopedics, Soochow University, Suzhou 215006, Jiangsu Province, China; 3Department of Neurosurgery, The First Affiliated Hospital of Soochow University, Suzhou 215000, Jiangsu Province, China; 4Department of Anesthesiology, Gaoyou People’s Hospital, Gaoyou 225600, Jiangsu Province, China
  • Received:2025-09-02 Accepted:2025-11-28 Online:2026-07-08 Published:2026-02-14
  • Contact: Zhang Haonan, MS, Physician, Jiading Hospital (Jiangqiao Hospital), Shanghai General Hospital, Shanghai 201803, China; Co-corresponding author: Liu Chenglong, MD, Chief physician, Department of Anesthesiology, Gaoyou People’s Hospital, Gaoyou 225600, Jiangsu Province, China
  • About author:Liu Yingzhao, MS, Associate chief physician, Jiading Hospital (Jiangqiao Hospital), Shanghai General Hospital, Shanghai 201803, China; Ma Yanxia, MS, Experimentalist, Institute of Orthopedics, Soochow University, Suzhou 215006, Jiangsu Province, China. Liu Yingzhao and Ma Yanxia contributed equally to this article.
  • Supported by:
    Shanghai Jiading District Natural Science Research Project, No. JDKW-2024-0037 (to LYZ); Clinical Medicine Science and Technology Development Fund Project of Jiangsu University, No. JLY20180167 (to LCL); National Natural Science Foundation of China (Youth Project), No. 81801238 (to LD)

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摘要：

文题释义：

miR-9的成熟过程：miR-9的成熟始于初级miR-9的转录，其茎环结构被Drosha/DGCR8复合物识别并切割生成前体miR-9，茎区的GU摇摆配对会影响加工效率。前体miR-9随后通过输出蛋白5转运至胞质，再由Dicer酶切割5’端种子序列(5’-UCU UUG GU-3’)及互补3’端，形成22个核苷酸的双链RNA。引导链miR-9-5p整合至Argonaute 2蛋白形成RNA诱导沉默复合体，靶向mRNA的3’非翻译区进行调控；而过客链miR-9-3p同样具有调控功能。成熟miR-9的稳定性受3’端尿苷酸化/腺苷酸化修饰调控，并在神经元活动中发挥重要作用。
脑室区和脑室下区：是胚胎和成体大脑中神经发生的重要区域。脑室区位于脑室附近，包含放射状胶质细胞，是胚胎期神经干细胞增殖和分化的主要场所，产生神经元和胶质细胞。脑室下区位于脑室区外侧，在胚胎发育后期及成体脑中持续存在，含有神经干细胞，可生成中间神经元并参与脑修复。两者共同维持神经发生能力，脑室下区在成体中尤为活跃，与神经再生相关，是神经退行性疾病研究的潜在靶点。

摘要
背景：位于脑室区和脑室下区的神经干细胞对大脑皮质的神经发育及神经退行性疾病的治疗至关重要，但具体调控机制尚未完全阐明。miRNA-9(miR-9)是脊椎动物早期及成年大脑中表达最丰富的miRNAs之一，在发育过程中具有多种功能，然而miR-9在神经干细胞分化中的作用尚不清楚。
目的：探讨miR-9在调控脑室区和脑室下区神经干细胞分化中的作用。
方法：分离胚胎14.5 d ICR小鼠脑室区和脑室下区神经干细胞，采用原代培养技术在增殖培养基中培养三四天形成神经球后，通过Pax6/Nestin免疫荧光双染鉴定细胞干性。采用qRT-PCR检测胚胎12.5，14.5，16.5，18.5 d和出生后0，7 d不同发育阶段端脑组织以及胚胎14.5 d体外培养神经干细胞中miR-9的表达谱。使用转染试剂miR-9抑制剂和模拟物对神经干细胞进行转染，转染24 h后继续分化三四天(神经元)和6-8 d(胶质细胞)，通过Tuj1(神经元标志物)、髓鞘碱性蛋白(少突胶质细胞标志物)和胶质纤维酸性蛋白(星形胶质细胞标志物)免疫荧光染色定量各谱系分化情况。
结果与结论：qRT-PCR结果显示，miR-9在胚胎早期(胚胎12.5 d-14.5 d)端脑组织中呈现高表达，随发育进程(胚胎16.5 d至出生后7 d)表达量逐渐降低。胚胎14.5 d神经干细胞中miR-9表达水平接近内参RNU6B表达水平的90%。功能实验表明，与对照组相比，miR-9抑制组Tuj1阳性神经元和髓鞘碱性蛋白阳性少突胶质细胞比例降低，而胶质纤维酸性蛋白阳性星形胶质细胞比例升高。相反，miR-9过表达组Tuj1阳性神经元和髓鞘碱性蛋白阳性少突胶质细胞比例升高，胶质纤维酸性蛋白阳性星形胶质细胞比例降低，差异均有显著性意义(P < 0.001)。结果表明，miR-9在神经干细胞分化中呈现双向调控作用：①通过发育阶段特异性表达模式(早期高表达，后期下调)参与神经发生时序调控；②通过促进神经干细胞向神经元和少突胶质细胞分化、同时抑制星形胶质细胞生成来维持三系分化平衡。

关键词: miR-9, 神经干细胞, 分化, 神经元, 少突胶质细胞, 星形胶质细胞

Abstract: BACKGROUND: Neural stem cells located in the ventricular zone and subventricular zone are crucial for cortical neurodevelopment and the treatment of neurodegenerative diseases. However, their precise regulatory mechanisms remain incompletely understood. miRNA-9 is one of the most abundantly expressed miRNAs in the vertebrate embryonic and adult brain, playing diverse roles during development. Yet, the function of miRNA-9 in neural stem cell differentiation remains unclear.
OBJECTIVE: To investigate the role of miRNA-9 in regulating the differentiation of neural stem cells in the ventricular zone and subventricular zone. 
METHODS: Neural stem cells were isolated from the ventricular zone and subventricular zone of embryonic day 14.5 ICR mice and cultured in proliferation medium for 3-4 days to form neurospheres. The stemness of these cells was confirmed by Pax6/Nestin immunofluorescence staining. The expression profile of miRNA-9 was assessed using qRT-PCR in forebrain tissues at different developmental stages (embryonic days 12.5, 14.5, 16.5, 18.5, postnatal days 0, and 7) as well as in in vitro cultured neural stem cells at embryonic day 14.5. Neural stem cells were transfected with either a miRNA-9 inhibitor or mimic using a transfection reagent. After 24 hours of transfection, cells were induced to differentiate for 3-4 days (for neurons) or 6-8 days (for glial cells). The differentiation ratios of neuronal and glial lineages were quantified via immunofluorescence staining for Tuj1 (a neuronal marker), myelin basic protein (an oligodendrocyte marker), and glial fibrillary acidic protein (an astrocyte marker). 
RESULTS AND CONCLUSION: qRT-PCR results revealed that miRNA-9 was highly expressed during early embryonic stages (embryonic days 12.5-14.5), with its expression gradually decreasing as development progressed (embryonic day 16.5 to postnatal day 7). The expression level of miRNA-9 in neural stem cells at embryonic day 14.5 was approximately 90% of the expression level of the internal reference RNU6B. Functional experiments demonstrated that compared with the control group, the proportion of Tuj1-positive neurons and myelin basic protein-positive oligodendrocytes decreased in the miRNA-9 inhibition group, while the proportion of glial fibrillary acidic protein-positive astrocytes increased. Conversely, the proportion of Tuj1-positive neurons and myelin basic protein-positive oligodendrocytes increased in the miRNA-9 overexpression group, while the proportion of glial fibrillary acidic protein-positive astrocytes decreased. All differences were significant (P < 0.001). Results indicate that miRNA-9 plays a bidirectional regulatory role in neural stem cell differentiation: (1) It participates in the temporal regulation of neurogenesis through a developmental-stage-specific expression pattern (high expression early, downregulation later); (2) It maintains a balance in tri-lineage differentiation by promoting neural stem cell differentiation into neurons and oligodendrocytes while inhibiting astrocyte formation. 

Key words: miRNA-9, neural stem cells, differentiation, neurons, oligodendrocytes, astrocytes

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