基于微流控平台的载药纳米颗粒联合间充质干细胞双载体对脑胶质瘤靶向效应研究

Gliomas possess properties of diffuse growth and high resistant to chemoradiotherapy. Currently, it is still difficult to cure gliomas by using combination of surgery and chemoradiotherapy. New and efficient treatment systems are urgent to be developed for glioma therapy. Based on the tumor tropism in mesenchymal stem cells (MSCs), targeted glioma therapy using MSCs carrier shows unique advantages and prospects. However, there is no clear conclusion from related research so far, and the disadvantages of the existing models include deviation between the model system and the in vivo microenvironment, difficulties in dynamic detection of the cell behaviors in endpoint analysis. This project plans to utilize microfluidic chip as the core technology, taking full advantage of microfluidic technology in controllable 3D cell culture and precise fluid control, based on previous research work on functional chip preparation, 3D glioma microenvironment construction and drug carrier material synthesis, using the glioma cells and MSCs as objects of study, combining intensely investigating the cell-cell, cell-matrix interaction in the microenvironment, to establish and develop innovative integrated model system for stem cell based glioma targeting and explore the application of targeting effect of drug loaded nanoparticle-mesenchymal stem cell double carriers to glioma on the microfluidic platform. The project aims to establish a novel and effective technology and platform for glioma treatment and evaluation in vitro, to solve the bottleneck problems in current glioma therapy.

脑胶质瘤具有弥散性生长及放化疗高度抗性，运用目前手术切除-放化疗联合疗法难以对其根治，亟需发展新型高效治疗体系；基于间充质干细胞（MSCs）的肿瘤趋化特性，以MSCs为载体的靶向胶质瘤治疗显示了独具优势的前景。目前相关研究尚无明确结论，存在模型体系与体内真实微环境偏差大、终点分析难以实时动态监测细胞行为的重要缺陷。本项目拟以极具潜力的微流控芯片为核心技术，充分利用该技术细胞可控三维培养、流体精确操控等优势，在前期功能化芯片制备、胶质瘤三维微环境构建及药物载体材料合成的工作基础上，以胶质瘤细胞和MSCs为研究对象，兼顾细胞-细胞、细胞-细胞外基质等三维微环境作用机制，创新性地建立和发展基于微流控技术的MSCs胶质瘤靶向作用整合模型新体系，并探索载药纳米颗粒联合MSCs双载体对胶质瘤靶向效应芯片应用研究，从而建立一种体外胶质瘤治疗评价新技术、新平台，解决现阶段胶质瘤治疗面临的瓶颈问题。

自行设计一种可拆卸、组装型微流控装置以构建体外三维胶质母细胞瘤(Glioblastoma multiforme, GBM)模型，用于研究GBM侵袭性和抗GBM药物评价。胶质瘤微环境基本要素，包括3D组织构型、胞外基质、流体，以集成式芯片中连续操作的形式于体外得到重现，操作步骤包括胶质瘤细胞球自组装、基质包埋、连续灌流。同时实现了多细胞球平行操作、分区培养，方法灵活度高。运用此模型，对连续药物刺激下、3D微环境中GBM细胞存活状态、增殖、侵袭能力和表型转换进行了评价。结果发现，胶质瘤的侵袭伴随着增殖，证明了胶质瘤侵袭式增殖的特性；首次发现波形蛋白Vimentin主要表达于细胞球边缘及细胞球外部侵袭细胞中，证明波形蛋白表达与胶质瘤细胞侵袭具有特异性关联。天然多酚单体白藜芦醇抗侵袭作用，创新地于此3D胶质瘤模型中进行评价，同时考察替莫唑胺、白藜芦醇与替莫唑胺药对组合药效。白藜芦醇：替莫唑胺=1：2及2：1的药对组合，相对于同浓度的两种单药，具有更强的侵袭数量、间质形态转化方面的抑制效果。此外，将浓度梯度它莫西芬作用于GBM细胞，发现侵袭细胞的ER-α36(雌激素受体-α66，ER-α66新型变体)/Ki-67阳性表达率是非侵袭细胞的2.14倍，ER-α36表达与自噬蛋白P62一致。为研究神经干细胞(Neural stem cells, NSCs)对胶质瘤侵袭的影响，在芯片三维胶质瘤模型基础上构建了NSCs培养基、NSCs外泌体与胶质瘤相互作用模型。NSCs培养基对胶质瘤细胞有明显侵袭抑制效果，且对胶质瘤细胞存活率没有明显影响。构建了NSCs多样性培养微环境，实现了在一个芯片上同时平行比较多种独立培养条件对NSCs生长和分化的影响：NSCs在该微流控芯片体系中有良好活性，3D胞外基质培养有利于NSCs自我更新和增殖，2D静态培养和细胞球灌流培养有利于NSCs分化。上述研究内容紧密围绕胶质瘤与干细胞开展，相关结果丰富了胶质瘤治疗与评价体系，为肿瘤疾病靶向治疗提供新的技术平台。