多基因共修饰的骨髓间充质干细胞动脉内移植治疗犬脑梗死的研究

Stroke is seriously harmful to human health throuthout the world due to its high incidence, high morbidity and mortality. The last decade has witnessed the progress made in extensive studies suggesting the therapeutic potential of stem cell-based therapy for stroke, thus, may represent the direction of stroke therapy. However, some controversies still exist. ① Many studies were performed in rodent whose brain is not gyrencephalic like the human brain, making the differences or gap between the animal experiments and clinical studies. ② The mechanism of stem cell-based therapy is still on debating, to result in the unclear method for transplantation in dose, time window, and delivery route. In addition, both the the survival and differential rate of stem cells are still low due to the inflammation reaction after cell transplantation. ③ Short of an objective and accurate method to evaluate the treatment efficiency after in vivo stem cell transplantation. Recently, some studies pointed that the BMSC may work through their secretome. Gene modified stem cell is a potential way to improve the effect. Therefore, based on the successfully developed canine stroke model in our previous studies, the current study aims to combine the use of intra-arterially transplantation of muti-gene modified bone marrow mesenchymal stem cells and minocycline which helps to control the inflammation reaction, in order to improve the treatment efficency of cerebral infarction. Meanwhile, multimodal imaging including PET and MRI will be used to in vivo track the the survival, immigration, differentiation and transformation of stem cells after transplantation. This study will investigate the value and mechanism of gene-modified stem cells for treatment of cerebral infarction from molecular, cellular, tissue and whole body levels and provide the therotic and experimental evidence for clinical studies.

脑梗死严重危害人类健康，干细胞移植能促进神经细胞的再生和修复，可能代表脑梗死治疗的方向。但仍然存在一些问题:①常用的实验动物鼠与人脑的差别较大，造成动物实验与临床研究的差异；②干细胞移植治疗脑梗死的机制存在多种学说，致移植细胞的数量、时间点、途径都存在争议，移植后的炎性环境致疗效降低；③对于活体移植后疗效的评价尚缺乏客观准确的手段等。最新的观点认为BMSC可能主要是通过其分泌各种因子的作用改善脑梗死预后，基因修饰干细胞移植是研究的热点。为验证这一机制，本项目拟在已成功建立大动物-犬脑梗死模型的基础上，经动脉移植有互补和协同作用的多基因共修饰的BMSC以增加目的性的因子表达，联合使用米诺环素控制炎症反应可能会提高疗效；同时用PET和MR等分子影像学技术监测移植细胞的生存、迁移和分化。有望从分子、细胞、组织及动物整体水平多方面探讨基因修饰干细胞治疗脑梗死的价值和机制，具有重要的理论和现实意义。

脑梗死的发病率、致残率和死亡率均较高，临床治疗较发病的时间窗相对滞后，且无法挽救已发生缺血坏死的神经细胞，而促进脑梗死区神经细胞再生和修复成为临床治疗和改善预后的关键之一。近年来利用干细胞治疗脑梗死已在实验中取得了一些进展。本实验以比格犬为研究对象，研究缺氧处理骨髓间充质干细胞（BMSCs）对其生物学特性影响，通过细胞因子芯片技术明确缺氧处理后相关细胞因子及蛋白表达变化，选择明显上调的蛋白基因，通过慢病毒转染技术实现并证实该基因过表达，再以SPIO标记的基因修饰后的骨髓间充质干细胞通过移植注射，利用分子影像学技术监测，观察其治疗的疗效，为临床治疗脑梗塞的进一步研究提供参考。研究结果表明：利用自体血栓栓塞一侧大脑中动脉及联合同侧颈内动脉限时性堵塞可以建立稳定的犬脑梗塞模型；缺氧预处理可明显提供BMSC的增殖、迁移以及神经分化能力，蛋白组学分析缺氧预处理后的条件培养基，可发现多种上调及下调蛋白；经动脉途径移植SPIO标记的经基因修饰后骨髓间充质干细胞治疗犬脑梗塞，可以实时在体通过MR示踪，并且显著改善犬神经行为学，这一过程可能与基因修饰后BMSC旁分泌的外泌体的作用有关。