脂联素联合骨髓间充质干细胞治疗脑缺血的保护作用及其分子机制

Adiponectin(APN), an adipcyte-specific adipocytokine characterized by its anti-inflamatory and anti-therosclerosis, attenuates ischemic brain injury via angiogenesis, induced mobilization of endothelial progenitor cells(EPCs), and anti-inflamatory. However the mechanism of APN effect remains unclear. At present, transplantation of bone marrow-derived mesenchymal stem cells (MSCs) is one of the most promising therapies for cerebral ischemia. MSCs-induced angiogenesis enhances cerebral tissue perfusion and creates a suitable microenvironment within the ischemic brain, which in turn accelerates endogenous neurogenesis and leads to improved functional recovery. Nevertheless, the disadvantages of low survival rate and poor integration significantly reduce the effectiveness of transplantation MSCs in stimulating the migration and proliferation of endogenous stem cells for brain plasticity and stroke therapy. This study targets to determine whether injury induced by cerebral ischemia is effectively attenuated by transplantation MSCs modified by adiponectin (APN) in the rat brain following middle cerebral artery occlusion. The study is performed in the following aspects. 1) To verify whether transplantation MSC modified by APN stimulates the migration, homing action, and survival of endogenous nerve stem cells and EPCs, benefiting the functional recovery in neurological outcomes. 2) To investigate the therapeutic effects of transplantation MSCs transfected with APN on the neurological recovery after rats' cerebral ischemia. To explore, both in vivo and in vitro, any undiscovered APN-mediating signaling pathway of the gene, which significantly alters under the treatment of MSCs modified by APN in the ischemia brain. In conclusion, this study aims to provide a theoretical basis for the treatment of neurological functional recovery after cerebral ischemia.

脂联素（APN）具有抗炎、抗动脉粥样硬化等特性，可通过抗炎、促进血管新生及内皮祖细胞迁移存活促进缺血脑组织神经功能的修复。采用干细胞移植促进神经再生和血管新生以修复和重建脑功能是目前脑缺血治疗的研究热点。但单纯干细胞移植，细胞存活率低、与原有系统整合性差，对促进内源干细胞迁移、存活及脑功能重建的治疗作用有限。本项目拟采用腺相关病毒转染APN基因修饰的骨髓间充质干细胞（MSC）治疗脑缺血大鼠，探讨1）APN基因修饰的MSC（MSC-APN）是否能提高MSC自身存活和分化，并同时促进内源性神经干细胞和内皮祖细胞的迁移、归巢和存活并提高其修复缺血脑组织的疗效；2）MSC-ANP修复和重建缺血脑组织功能的保护作用及机制。本研究结果将为临床开拓脑缺血治疗的新路提供理论基础。

脂联素（APN）对缺血性心脑血管疾病具有保护作用。脂联素通过保护内皮细胞功能、促进血管新生、减轻炎症反应等促进脑缺血后神经功能恢复。内皮祖细胞（EPC）具有干细胞增殖分化的能力，可以参与内皮细胞成管，促进血管新生和缺血后的血管新生。目前尚未有利用内皮祖细胞联合脂联素转基因治疗脑缺血的研究，我们设想将脂联素与内皮祖细胞联合治疗，观察对脑缺血神经功能恢复的影响。本研究通过（1）分离培养内皮祖细胞；构建携带脂联素基因的慢病毒（lentivirus-GFP-APN）以及携带绿色荧光的对照病毒（lentivirus-GFP），使用慢病毒转染内皮祖细胞，并鉴定转染后脂联素的表达。（2）比较APN-EPC和GFP-EPC的增殖能力，迁移能力及体外成管能力。（3）评估APN-EPC和GFP-EPC细胞移植治疗对大鼠大脑中动脉缺血模型神经功能的改善及血管新生促进作用。结果1）采用单核细胞分离方式从脐带血中分离的内皮祖细胞经鉴定符合国际上对内皮祖细胞的定义，培养的内皮祖细胞具有增殖、分化等干细胞的功能。2）携带脂联素基因的慢病毒转染内皮祖细胞后可以稳定表达脂联素和绿色荧光蛋白，并且可以随着增殖传代稳定传给后代。3）脂联素对EPC增殖：GFP-EPC组OD值小于APN-EPC组；迁移率：GFP-EPC组5h细胞迁移面积小于APN-EPC组；体外成管实验：5h APN-EPC组成管长度是GFP-EPC组的1.36倍；24h APN-EPC组成管长度是GFP-EPC组的1.34倍。4）大鼠短暂性大脑中动脉阻塞缺血模型颈静脉注射APN-EPC、GFP- EPC后，APN-EPC组移植3天后APN表达水平最高；术后3天时APN-EPC治疗组脑梗死面积明显缩小。术后7天、14天及28天时，APN-EPC组的神经行为学功能显著优于GFP-EPC组、PBS组（p<0.05）；脑缺血术后14天时，APN-EPC组梗死周围区CD31阳性，DCX阳性细胞数量明显高于GFP-EPC组、PBS组（p<0.05）。结论：（1）成功实现内皮祖细胞体外培养和传代。（2）脂联素可以促进内皮祖细胞的增殖、迁移和体外成管能力。（3）动物实验我们发现用脂联素联合内皮祖细胞可以促进血管再生，减少细胞凋亡，改善行为学功能，进而促进缺血性脑卒中的康复。本研究为缺血性脑卒中提供了新的治疗靶点。