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  

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

作　　者：季航宇 顾军 谢凌寒[1] 鲍军平 彭鑫 吴小涛[1,3] Ji Hangyu;Gu Jun;Xie Linghan;Bao Junping;Peng Xin;Wu Xiaotao(School of Medicine,Southeast University,Nanjing 210009,Jiangsu Province,China;Department of Orthopedics,Wuxi Xishan People’s Hospital,Wuxi 214105,Jiangsu Province,China;Department of Orthopedics,Zhongda Hospital,Southeast University,Nanjing 210009,Jiangsu Province,China)

机构地区：[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

出　　处：《中国组织工程研究》2022年第25期3974-3979,共6页Chinese Journal of Tissue Engineering Research

基　　金：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。

摘　　要：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。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.Nestinpositive 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/p

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 

分 类 号：R459.9[医药卫生—治疗学] R318.08[医药卫生—临床医学]