基于微流控的神经干细胞与内皮细胞三维微环境构建及相互作用机制研究

Two-dimension cell culturing has been the most widely used research method for the in-vitro study of neural stem cells proliferation and differentiation. However,two-dimension method fails to consider the important cell-cell and cell-matrix interactions in niche. This study proposed a novel protocol based on microfluidics that better simulates the three-dimension niche environment. This study focused on the interaction mechanism between neural stem cells and brain vascular endothelial cells in three-dimension symbiotic conditions, which includes the study of contacting alternative co-growth of neural stem cells and vascular endothelial cells on microfluidic chips,the screening and optimization of the non-serum co-culturing conditions of neural stem cells and vascular endothelial cells, and the study of molecular mechanisms by which pigment epithelium derived factor (PEDF) promotes the proliferation and differentiation of neural stem cells under the condition of the brain vascular endothelial cells exist. This work would provide great value in theory and practical application for promoting the studies of mammalian nervous system development, clinical transplantation of nervous tissues, and drug screening.

目前用于体外研究神经干细胞增殖与分化机制的细胞模型主要采用细胞二维培养方式，忽略了神经干细胞微环境（niche）中细胞与细胞间、细胞与细胞外基质间的相互作用影响。为此本项目首次提出一种基于微流控构建神经干细胞三维微环境的新方法，并创新性地开展了神经干细胞与血管内皮细胞在三维微环境中相互作用机制的研究。重点开展微流控芯片上神经干细胞与血管内皮细胞间隔式接触共生长模型的构建，神经干细胞与血管内皮细胞的无血清共生长条件的筛选与优化，和上皮衍生因子（PEDF）在血管内皮细胞存在条件下促进神经干细胞增殖与分化的机制研究。项目成果对于研究哺乳动物神经系统的发育、神经组织临床移植以及体外药物筛选等具有重要的理论意义和实际应用价值。

微流控芯片因其精准的操控性、多功能集成性以及可构建与细胞体内生存环境相似的封闭系统等优势，近年来被广泛用于细胞的体外培养研究。本课题设计并制作了模拟神经干细胞(Neural stem cells, NSCs)微环境的三维微流控芯片，利用主腔室和侧通道之间的微柱阵列实现NSCs与血管内皮细胞(Vascular endothelial cells, VECs)在空间上的有序分隔又相互接触的共生模式。采用正交试验筛选了NSCs与VECs共培养的无血清培养基，四个关键组分(VEGF、bFGF、EGF、EPO)的最佳水平的组合为（100ng/mL、10 ng/mL、20 ng/mL、0.5 U/mL）。为了进一步研究和分析NSCs在模拟的三维微环境中的增殖与分化机制，课题组采用微加工技术将一种三电极系统集成于NSCs的三维培养主腔室中，通过电化学检测方法实时监控处于微环境中的NSCs向多巴胺能神经元诱导分化过程中多巴胺(Dopamine, DA)的分泌量。实验结果表明，采用未修饰的金电极，获得的多巴胺检出限为3.92μM，线性范围为10μM-500μM，片间检测的重复精度为4%。当采用聚吡咯/聚对苯乙烯磺酸钠对金电极进行修饰后，获得的多巴胺检出限可达0.69µM。目前，对NSCs三维的诱导分化研究工作仍在进行中。综上所述，本课题的研究成果将为研究和开发以DA缺失为主要特征的神经退行性疾病，如帕金森综合症的研究及相关药物的研发提供理论依据和实验平台。