经动脉输注骨髓干细胞条件培养基调控AQP4减轻犬缺血性脑水肿的分子机制及多模态MR研究

Cerebral edema is the key point for treatment of acute ischemic stroke. The water channel aquaporin 4 (AQP4) plays an important role in the regulation of brain edema. Bone marrow mesenchymal stem cells (BMSCs) could help to reduce brain edema by inhibiting AQP4 up-regulation. However, some problems of stem cell transplantation are still existed, such as limited survival rate of stem cells in the brain and limited number of stem cells in targeted place due to the systemic and pulmonary circulation. Current studies suggest that the therapeutic effects of BMSC are mainly mediated by stem cell secretome. Our preliminary experiment suggested that BMSC-conditioned medium (BMSC-CM) which contained many BMSC secreted proteins could down-regulate AQP4 expression in injured astrocytes. Therefore, we suppose that intra-arterial infusion of BMSC-CM at early stage after ischemic stroke could protect the blood-brain barrier (BBB) through regulating AQP4 and reduce brain edema. We aim to collect and concentrate the BMSC-CM in vitro and to investigate whether BMSC-CM could regulate the expression of AQP4 in astrocytes and the related signal pathways. Then the BMSC-CM will be intra-arterially infused into a canine stroke model in vivo to investigate whether BMSC-CM could protect BBB through down-regulation of AQP4, and hence reduce the ischemic edema. Multimodal MR imaging (mutil-b DWI and DCE-MRI) will be used to dynamically quantify the expression of AQP4 and BBB permeability. The MRI results will be compared with molecular biology findings. Finally, these results may provide a theoretical basis for BMSC-CM treatment in brain ischemic edema.

脑水肿是缺血性卒中的治疗关键，水通道蛋白AQP4是脑水肿的重要调控靶点。研究示骨髓间充质干细胞（BMSC）移植可下调AQP4表达，减轻脑水肿，然而干细胞移植存在一些问题：如细胞存活率低、体循环吞噬等。目前认为BMSC主要通过旁分泌途径发挥作用，我们体外预实验结果示富含旁分泌因子的BMSC条件培养基（BMSC-CM）可下调脑水肿效应细胞-星形胶质细胞AQP4表达，由此提出假说：单独利用BMSC-CM可下调AQP4表达，保护血脑屏障，减轻脑水肿。为验证假说，我们拟收集BMSC-CM，体外研究其调控星形胶质细胞AQP4表达的作用及信号通路，体内输注治疗犬缺血性脑水肿，采用多模态MRI（多b值DWI、DCE-MRI）活体监测AQP4及血脑屏障通透性变化，并与分子生物学结果对照，探讨BMSC-CM通过调控AQP4保护血脑屏障减轻脑水肿的疗效及分子机制，为临床应用BMSC-CM治疗脑水肿提供理论依据。

脑水肿是缺血性卒中的治疗关键，水通道蛋白AQP4是脑水肿的重要调控靶点。研究示骨髓间充质干细胞（BMSC）移植可下调AQP4表达，减轻脑水肿，然而干细胞移植存在一些问题：如细胞存活率低、体循环吞噬等。目前认为BMSC主要通过旁分泌途径发挥作用，本研究构建了犬自体血栓大脑中动脉脑梗死模型，收集犬骨髓间充质干细胞调节培养基（BMSC-CM）：①在体外，针对AQP4相关的脑水肿效应细胞（星形胶质细胞），通过糖氧剥夺方法模拟脑缺血损伤，研究BMSC-CM对星形胶质细胞AQP4表达的影响以及PKC信号通路在此过程中发挥的作用；②在体内，经动脉输注BMSC-CM，探索其抑制AQP4表达，保护血脑屏障，从而改善脑水肿的作用及机制；③采用多模态MRI作为研究工具，活体监测BMSC条件培养基治疗过程中AQP4表达水平、血脑屏障通透性及脑水肿的动态改变，并将多模态MRI结果与病理学、分子生物学结果及神经功能评分进行对照。此外，我们通过质谱分析和蛋白芯片分析了BMSC-CM，对其中可能发挥治疗作用的蛋白也进行了一些探讨。研究结果示：BMSC-CM能够下调脑水肿效应细胞（星形胶质细胞）AQP4表达水平，抑制细胞肿胀和凋亡；缺血性卒中后早期经动脉输注BMSC-CM可通过PKC信号通路下调AQP4表达，保护血脑屏障完整性，减少脑水肿体积，从而促进动物神经功能恢复。病理学结果与多模态MRI参数可相互印证。同时通过BMSC-CM蛋白组学分析，我们也发现了一些潜在的在脑水肿治疗中发挥积极作用的蛋白靶点。结论：BMSC-CM早期体内输注可下调AQP4表达，保护血脑屏障完整性，减少脑水肿体积，促进神经功能恢复。本研究可为BMSC-CM治疗缺血性脑水肿的“Cell-free therapy”提供实验性理论依据。