中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (25): 3974-3979.doi: 10.12307/2022.401

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

持续释放神经生长因子的壳聚糖/聚乳酸羟基乙酸/聚乳酸支架促进骨髓间充质干细胞向神经元分化

季航宇1，2，顾  军2，谢凌寒1，鲍军平1，彭  鑫1，吴小涛1，3   

  1. 1东南大学医学院，江苏省南京市   210009；2无锡市锡山人民医院骨科，江苏省无锡市   214105；3东南大学附属中大医院骨科，江苏省南京市   210009
  • 收稿日期:2020-12-29 接受日期:2021-02-07 出版日期:2022-09-08 发布日期:2022-01-25
  • 通讯作者: 吴小涛，博士，教授，主任医师，博士生导师，东南大学医学院，江苏省南京市 210009；东南大学附属中大医院骨科，江苏省南京市 210009
  • 作者简介:季航宇，男，1979 年生，辽宁省喀左县人，满族，东南大学毕业，博士，副主任医师，主要从事脊柱外科相关研究。 顾军，男，1972年生，江苏省无锡市人，汉族，博士，副主任医师，主要从事脊柱外科相关研究。
  • 基金资助:
    江苏大学2018年度临床医学科技发展基金(JLY20180027)，项目负责人：季航宇；2018年江苏卫健委面上项目卫生厅项目(H2018023)，项目负责人：顾军；无锡市社会发展科技示范性项目(N20192001)，项目负责人：顾军

Chitosan/poly(lactic-co-glycolic acid)/polylactic acid scaffold with sustained release of nerve growth factor promotes the differentiation of bone marrow mesenchymal stem cells into neurons

Ji Hangyu1, 2, Gu Jun2, Xie Linghan1, Bao Junping1, Peng Xin1, Wu Xiaotao1, 3   

  1. 1School of Medicine, Southeast University, Nanjing 210009, Jiangsu Province, China; 2Department of Orthopedics, Wuxi Xishan People’s Hospital, Wuxi 214105, Jiangsu Province, China; 3Department of Orthopedics, Zhongda Hospital, Southeast University, Nanjing 210009, Jiangsu Province, China
  • Received:2020-12-29 Accepted:2021-02-07 Online:2022-09-08 Published:2022-01-25
  • Contact: Wu Xiaotao, MD, Professor, Chief physician, Doctoral supervisor, School of Medicine, Southeast University, Nanjing 210009, Jiangsu Province, China; Department of Orthopedics, Zhongda Hospital, Southeast University, Nanjing 210009, Jiangsu Province, China
  • About author:Ji Hangyu, MD, Associate chief physician, School of Medicine, Southeast University, Nanjing 210009, Jiangsu Province, China; Department of Orthopedics, Wuxi Xishan People’s Hospital, Wuxi 214105, Jiangsu Province, China Gu Jun, MD, Associate chief physician, Department of Orthopedics, Wuxi Xishan People’s Hospital, Wuxi 214105, Jiangsu Province, China Ji Hangyu and Gu Jun contributed equally to this article.
  • Supported by:
    Clinical Medicine Technology Development Fund of Jiangsu University in 2018, No. JLY20180027 (to JHY); the General Project of Jiangsu Health Commission and Health Department Project in 2018, No. H2018023 (to GJ); the Science and Technology Demonstration Project of Social Development in Wuxi, No. N20192001 (to GJ)

PDF

43

摘要：

文题释义：
骨髓间充质干细胞：是一种来源于骨髓的间充质干细胞，它具有增殖能力强、低免疫原性等优点，可以在体外被诱导为骨细胞、软骨细胞、脂肪细胞等多种细胞。该实验将其种植在壳聚糖/神经生长因子-聚乳酸羟基乙酸/聚乳酸支架表面，在神经生长因子持续存在的情况下成功将其诱导分化为神经元细胞。
组织工程支架：支架是组织工程技术的核心要素之一，为改善脊髓损伤的修复效果，该研究设计了一种薄膜状支架，由壳聚糖/聚乳酸羟基乙酸/聚乳酸组成，其中壳聚糖一侧作为骨髓间充质干细胞的黏附部位，聚乳酸羟基乙酸可以负载神经生长因子，能够在脊髓损伤部位持续释放神经生长因子，为细胞的神经分化创造了条件。

背景：越来越多的证据显示，神经生长因子对神经元的存活、分化等有着重要的促进作用，但是神经生长因子半衰期短，稳定性较差，限制了它在临床上的广泛应用。
目的：探讨大鼠骨髓间充质干细胞在可持续释放神经生长因子的壳聚糖/聚乳酸羟基乙酸/聚乳酸支架上向神经元细胞分化的可行性。
方法：构建可持续释放神经生长因子的壳聚糖/聚乳酸羟基乙酸/聚乳酸支架和不含有神经生长因子的壳聚糖/聚乳酸羟基乙酸/聚乳酸支架，将大鼠骨髓间充质干细胞分别接种在两种支架表面，采用低糖DMEM完全培养基中加入碱性成纤维生长因子、脑源性神经营养因子以及N2和B27神经细胞生长添加剂的诱导方案进行成神经诱导分化。诱导18 d后，免疫荧光染色检测神经元标志物(微管相关蛋白2和巢蛋白)的表达，Western blot检测p-TrkA和p-ERK蛋白表达。
结果与结论：①经过连续18 d的诱导，免疫荧光检测显示微管相关蛋白2阳性细胞在空白支架组约为30%，可持续释放神经生长因子组约为70%，而巢蛋白阳性细胞在空白支架组为40%，可持续释放神经生长因子组约为60%，两组比较差异有显著性意义；②Western blot检测结果显示可持续释放神经生长因子组的p-TrkA 和p-ERK蛋白表达量明显高于空白支架组，差异有显著性意义；③结果表明，在持续性释放神经生长因子的壳聚糖/聚乳酸羟基乙酸/聚乳酸支架表面，骨髓间充质干细胞可以更好地向神经元细胞分化，其机制可能是通过TrkA/ERK通路来实现的。
缩略语：壳聚糖/聚乳酸羟基乙酸/聚乳酸：chitosan/polyglycolic acid/polylactic acid，CS/PLGA/PLA；含神经生长因子的CS/PLGA/PLA：nerve growth factor CS/PLGA/PLA，CS/NGF-PLGA/PLA

https://orcid.org/0000-0001-8505-0231 (季航宇) 

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: 干细胞, 骨髓间充质干细胞, 神经生长因子, 壳聚糖, 聚乳酸羟基乙酸, 聚乳酸, 支架, 神经元

Abstract: BACKGROUND: More and more evidences demonstrate that nerve growth factor plays important roles in promoting neuronal survival and differentiation. However, nerve growth factor has a short half-life and poor stability, which limits its wide clinical application.  
OBJECTIVE: To investigate the feasibility of rat bone marrow mesenchymal stem cells to differentiate into neuronal cells on chitosan/poly(lactic-co-glycolic acid)/polylactic acid scaffolds that can continuously release nerve growth factors.
METHODS:  Chitosan/poly(lactic-co-glycolic acid)/polylactic acid scaffolds that sustainably released nerve growth factor and chitosan/poly(lactic-co-glycolic acid)/polylactic acid scaffolds that did not contain nerve growth factor were constructed, and rat bone marrow mesenchymal stem cells were inoculated on the surface of two kinds of scaffolds. The induction program of basic fibroblast growth factor, brain-derived neurotrophic factor and N2 and B27 nerve cell growth additives added to the low-glucose DMEM complete medium was used to induce neurogenesis and differentiation. After 18 days of induction, immunofluorescence staining was used to detect the expression of neuronal markers (microtubule-associated protein 2 and nestin), and western blot assay was utilized to determine the expression of p-TrkA and p-ERK proteins.  
RESULTS AND CONCLUSION: (1) After 18 consecutive days of induction, immunofluorescence detection displayed that the microtubule-associated protein 2-positive cells were approximately 30% in the blank scaffold group, and approximately 70% in the sustained release nerve growth factor group. Nestin-positive cells were 40% in the blank scaffold group, and approximately 60% in the sustained release nerve growth factor group. The difference between the two groups was significant. (2) Western blot assay exhibited that the expression levels of p-TrkA and p-ERK in the sustained release nerve growth factor group were significantly higher than those of the blank scaffold group. (3) The results have confirmed that on the surface of the chitosan/poly(lactic-co-glycolic acid)/polylactic acid scaffold that continuously releases nerve growth factor, bone marrow mesenchymal stem cells can achieve better differentiation of neuronal cells through the TrkA/ERK pathway.

Key words: stem cells, bone marrow mesenchymal stem cells, nerve growth factor, chitosan poly(lactic-co-glycolic acid), polylactic acid, scaffold, neuron

中图分类号: