骨髓干细胞调节培养基对犬脑梗死后神经血管重塑作用的多模态MRI研究

Stem cell therapy could improve the functional outcome after stroke. However, some problems have been reported, such as its limited survival rate in the brain, limited number of stem cells in targeted area due to the systemic and pulmonary circulation. Furthermore, decisive evidence indicate that tissue regeneration may not be the main source of functional recovery since only a small proportion of transplanted bone marrow mesenchymal stem cells (BMSC) appear to differentiate into neurons and astrocytes. Many studies suggest that the therapeutic effects of BMSC transplantation are mainly mediated by stem cell secretome. Our preliminary experiment suggested many secreted proteins which closely related to the neurovascular plasticity (VEGF, BDNF, HGF, IGF-1, et al) were existed in the BMSC-conditioned medium (BMSC-CM). Therefore, we suppose that the intravenous infusion of BMSC-CM at early stage could enhance the effect of neurovascular plasticity after cerebral ischemia. We aim to collect and concentrate the BMSC-CM in vitro, quantitatively analyze the levels of VEGF, BDNF, HGF, IGF-1. Then the extracted BMSC-CM will be intravenously infused into the canine stroke model after the middle cerebral artery occlusion. The effects of BMSC-CM on neurovascular plasticity after stroke will be investigated. Multimodal MR (DSC-PWI, DCE, DTI, 1H-MRS) will be used to dynamically evaluate the effect on neurovascular plasticity in vivo. The MRI results will be compared with the pathology and immunohistochemistry staining findings. Whether the hypoxic preconditioning can increase the levels of VEGF, BDNF, HGF, IGF-1 inside of BMSC-CM in vitro and therefore promote the therapeutic efficacy in vivo in the canine stroke model will be further evaluated. Finally, this study may provide a theoretical basis for the “cell-free therapy” hypothesis in treatment of stroke.

干细胞移植可改善脑梗死预后，但仍存在一些问题，如细胞存活率低、体循环吞噬等。目前研究认为骨髓间充质干细胞（BMSC）的分化替代作用并非主要治疗机制，而其丰富的旁分泌蛋白在脑梗死修复过程中发挥重要作用。我们的预实验结果提示BMSC调节培养基内含多种脑神经血管重塑相关分泌蛋白，由此提出假说：单独利用BMSC调节培养基可促进神经血管重塑，改善脑梗死预后。为验证这一假说，我们收集浓缩BMSC调节培养基，体外定量神经血管重塑相关分泌蛋白含量；体内早期输注治疗犬脑梗死；应用多模态MR（PWI、DCE、DTI、1H-MRS）活体监测血管新生及神经轴突修复，与病理学结果对照，探讨BMSC调节培养基在促进脑梗后神经血管重塑中的作用和机制；并进一步探索体外缺氧预处理能否提高BMSC调节培养基内相关分泌蛋白含量，增强疗效，为临床应用BMSC调节培养基治疗脑梗死提供理论依据。

干细胞移植可改善脑梗死预后，但仍存在一些问题，如细胞存活率低、体循环吞噬等。研究认为骨髓间充质干细胞（BMSC）的分化替代作用并非主要治疗机制，而其丰富的旁分泌蛋白在脑梗死修复过程中发挥重要作用。本研究采用与人脑结构更为类似的大动物犬，①构建犬自体血栓大脑中动脉脑梗死模型并进行MRI评估；②收集浓缩犬BMSC调节培养基，体外分析BMSC调节培养基内分泌蛋白谱，寻找神经血管重塑相关分泌蛋白；③体内分别输注常氧及缺氧预处理BMSC调节培养基用于治疗犬缺血性脑梗死，并与对照组进行对比；④应用多模态MRI（DSC-PWI、DCE-MRI、ASL、DTI等）在4周内活体监测新生血管生成、神经轴突重塑及脑梗死体积变化，与病理学结果及神经功能评分对照。研究结果示：犬BMSC调节培养基内富含大量分泌蛋白，其中包括与神经血管重塑相关分泌蛋白VEGF、HGF、IGF、TIMP1等；经动脉输注BMSC调节培养基治疗犬急性期脑梗死，4周时治疗组梗死侧脑血流量CBF值、Ktrans值及FA值较对照组高，犬脑梗死体积更小，动物神经功能评分恢复更好，提示BMSC调节培养基的早期输注有利于梗死区新生血管生成及神经轴突重塑，促进脑梗死后神经功能恢复。Ktrans值变化可较好的反映梗死早期的血脑屏障破坏及后期新生血管形成，可作为观察新生血管形成的MRI指标。病理学结果与多模态MRI参数可相互印证。常氧BMSC调节培养基治疗组与缺氧预处理BMSC调节培养基治疗组之间脑梗死体积、CBF值及FA值、动物神经功能评分均未见明显差异。结论：BMSC调节培养基早期体内输注可促进神经血管重塑，改善脑梗死预后；3.0T多模态MRI成像可以动态活体监测脑梗死治疗后新生血管生成及神经轴突重塑，评估脑梗死治疗疗效。本研究可为BMSC调节培养基治疗急性期脑梗死的“Cell-free therapy”提供实验性理论依据。