中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2021, Vol. 16 ›› Issue (9): 1856-1864.doi: 10.4103/1673-5374.306097

• 原著:周围神经损伤修复保护与再生 • 上一篇    下一篇

三维胶原水凝胶培养常规培养和冷冻保存背根神经节的神经生长特征

  

  • 出版日期:2021-09-15 发布日期:2021-02-05
  • 基金资助:

    国家自然科学基金项目（82000871，81871495）；湖南省自然科学基金项目（2020JJ5001）；爱尔眼科医院集团科研资助项目（AF1913D2）；中南大学博士后基金项目。

Characteristics of neural growth and cryopreservation of the dorsal root ganglion using three-dimensional collagen hydrogel culture versus conventional culture

Ze-Kai Cui1, 2, 3, Shen-Yang Li3, Kai Liao3, Zhi-Jie Wang3, Yong-Long Guo4, 5, 6, Luo-Sheng Tang1, Shi-Bo Tang2, 3, 7, Jacey Hongjie Ma1, 2, 8, *, Jian-Su Chen2, 3, 4, 5, 6, *#br#   

  1. 1Department of Ophthalmology, the Second Xiangya Hospital, Central South University, Changsha, Hunan Province, China; 2Aier Eye Institute, Changsha, Hunan Province, China; 3Aier School of Ophthalmology, Central South University, Changsha, Hunan Province, China; 4Institute of Ophthalmology, Medical College, Jinan University, Guangzhou, Guangdong Province, China; 5Department of Ophthalmology, First Affiliated Hospital of Jinan University, Guangzhou, Guangdong Province, China; 6Key Laboratory for Regenerative Medicine of Ministry of Education, Jinan University, Guangzhou, Guangdong Province, China; 7CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, China; 8Imaging and Functional Center, Guangzhou Aier Eye Hospital, Guangzhou, Guangdong Province, China
  • Online:2021-09-15 Published:2021-02-05
  • Contact: Jian-Su Chen, MD, PhD, chenjiansu2000@163.com; Jacey Hongjie Ma, MD, PhD, ma.hongjie12@gmail.com.
  • Supported by:
    This study was supported by the National Natural Science Foundation of China, Nos. 82000871 (to ZKC), 81871495 (to JSC); the Natural Science Foundation of Hunan Province, China, No. 2020JJ5001 (to ZKC); the Science Research Grant of Aier Eye Hospital Group, China, No. AF1913D2 (to ZKC), and Central South University Postdoctoral Funds, China.

PDF

41

摘要：

在脊椎动物中，大多数体感感觉始于背根神经节神经元的激活。适当的背根神经节的体外培养方法是周围神经疾病模型构建和药物筛选的基础，此次实验比较了三维胶原水凝胶培养和传统二维培养的鸡胚背根神经节的形态、分子生物学和转录组学的变化，并进一步比较两者在冷冻保存后的变化。（1）三维或二维培养7d后，鸡胚背根神经节转录组学差异很大，其中三维胶原水凝胶培养的背根神经节中上调基因主要与神经发生、轴突导向和突触可塑性有关，下调基因主要与细胞增殖和细胞分裂有关，同时突触小泡周期、环磷酸腺苷信号通路、钙信号通路等相关的基因被激活，而与细胞周期通路有关的基因被下调。在三维胶原水凝胶培养的背根神经节中，神经发生和髓鞘形成相关基因均为高表达，而上皮-间充质转化、细胞凋亡和细胞分裂相关基因被抑制；（2）形态学观察可见，三维胶原水凝胶培养的背根神经节的分支、结点、终点体素的数量均大于二维培养的背根神经节。二维培养的背根神经节中许旺细胞分布零散，而三维胶原水凝胶培养的背根神经节中许旺细胞分布集中。与二维培养的背根神经节相比，三维胶原水凝胶培养的背根神经节中EdU阳性细胞的比例较低，S100B阳性反应较高，α-SMA阳性反应较低；（3）在48h血清饥饿培养后，两种培养的背根神经节中TUNEL阳性细胞减少；（4）冷冻保存后，与二维培养的背根神经节相比，三维胶原水凝胶培养的背根神经节保持了更好的形态特征，存活的许旺细胞较多，TUNEL阳性细胞较少，新形成的神经束可以沿着现有的许旺细胞生长；（5）三维胶原水凝胶培养的背根神经节可能是一种有希望的体外培养模型，具有更好的神经生长和抗凋亡能力。实验构建的三维胶原水凝胶培养的背根神经节用于组织工程神经的构建、储存和运输提供了实验的依据。研究于2020年3月15日经中南大学爱尔眼科学院伦理委员会批准（批准号2020-IRB16）。

https://orcid.org/0000-0003-3670-6219 (Ze-kai Cui)

关键词:

背根神经节, 胶原水凝胶, 许旺细胞, 冷冻保存, 组织工程, 神经发生, RNA序列, 抗凋亡

Abstract: In vertebrates, most somatosensory pathways begin with the activation of dorsal root ganglion (DRG) neurons. The development of an appropriate DRG culture method is a prerequisite for establishing in vitro peripheral nerve disease models and for screening therapeutic drugs. In this study, we compared the changes in morphology, molecular biology, and transcriptomics of chicken embryo DRG cultured on tissue culture plates (T-DRG) versus three-dimensional collagen hydrogels (C-DRG). Our results showed that after 7 days of culture, the transcriptomics of T-DRG and C-DRG were quite different. The upregulated genes in C-DRG were mainly related to neurogenesis, axon guidance, and synaptic plasticity, whereas the downregulated genes in C-DRG were mainly related to cell proliferation and cell division. In addition, the genes related to cycles/pathways such as the synaptic vesicle cycle, cyclic adenosine monophosphate signaling pathway, and calcium signaling pathway were activated, while those related to cell-cycle pathways were downregulated. Furthermore, neurogenesis- and myelination-related genes were highly expressed in C-DRG, while epithelial–mesenchymal transition-, apoptosis-, and cell division-related genes were suppressed. Morphological results indicated that the numbers of branches, junctions, and end-point voxels per C-DRG were significantly greater than those per T-DRG. Furthermore, cells were scattered in T-DRG and more concentrated in C-DRG, with a higher ratio of 5-ethynyl-2′-deoxyuridine (EdU)-positive cells in T-DRG compared with C-DRG. C-DRG also had higher S100 calcium-binding protein B (S100B) and lower α-smooth muscle actin (α-SMA) expression than T-DRG, and contained fewer terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells after 48 hours of serum starvation. After cryopreservation, C-DRG maintained more intact morphological characteristics, and had higher viability and less TUNEL-positive cells than T-DRG. Furthermore, newly formed nerve bundles were able to grow along the existing Schwann cells in C-DRG. These results suggest that C-DRG may be a promising in vitro culture model, with better nerve growth and anti-apoptotic ability, quiescent Schwann cells, and higher viability. Results from this study provide a reference for the construction, storage, and transportation of tissue-engineered nerves. The study was approved by the Ethics Committee of Aier School of Ophthalmology, Central South University, China (approval No. 2020-IRB16), on March 15, 2020.

Key words: anti-apoptosis, collagen hydrogel, cryopreservation, dorsal root ganglion, neurogenesis, RNA-seq, Schwann cell, tissue engineering