Endorepellin表达变化在TBI后BBB损伤修复及血管新生中作用和机制研究

Functional repair of blood-brain barrier (BBB) and angiogenesis play key roles on neural functional recovery following traumatic brain injury (TBI).Our preliminary studies found that stretch injury promoted Perlecan proteolysis, protidtemns of Perlecan-Endorepellin which was also known as perlecan domain Ⅴ expressed increasingly. Down regulate Endorepellin levels may alleviate pro-apoptotic proteins expression, its impact on BBB and angiogenesis post TBI and the underline mechanisms remains elusive yet. We hypothesized that TBI induced Endorepellin over-expression may activate apoptotic and autophagic pathways, which may promote apoptosis and autophagy of endothelial cells, aggravate BBB breakdown, inhibition of Endorepellin expression could decrease BBB disruption and promote angiogenesis. To verify this hypothesis, we will analyze Endorepellin expression levels according to endothelial cell stretch injury and mice controlled cortex injury. To further determine the potential mechanisms, the adeno-associated virus (AAV) areconstructed to over express Endorepellin and silence Endorepellin expression in both endothelial cells and murine hippocampus, the transfection effects and influences on apoptosis-related factors levels within or without α2β1integrin will be testified. The impact of these changes on functions of endothelial cells, BBB damage repair and ability of angiogenesis post TBI will be discussed, and provide experimental gist for new treatment targets on microvascular protection after TBI.

创伤性脑损伤（TBI）后血脑屏障（BBB）功能修复及血管新生对神经功能修复具有重要作用。课题组前期发现，TBI后Perlecan水解增强，其产物Endorepellin（Ep）表达升高；抑制Perlecan水解可减轻细胞损伤，但Ep表达变化对TBI后BBB功能及脑微血管新生作用和机理尚不清楚。我们假设：TBI后Ep水平升高，激活相关凋亡及自噬通路，促进血管内皮细胞凋亡、自噬，加重BBB损伤；抑制Ep表达可改善BBB对抗损伤能力并促进微血管新生。为验证此假说，本项目拟通过建立细胞牵张损伤和小鼠控制性皮层损伤模型；进一步构建针对Ep过表达及干扰腺相关病毒，转染血管内皮细胞及小鼠海马立体定向注射，观察Ep表达变化及在整合素α2β1参与下对凋亡相关因子影响，探讨上述变化对TBI后脑血管内皮细胞功能、BBB损伤修复及血管新生中作用。为探寻TBI后微血管保护新的治疗靶点提供实验依据。

脑微血管功能与新生与创伤性颅脑损伤（TBI）后神经功能修复密切相关。课题组前期发现，TBI后Perlecan水解增强，其产物Endorepellin（Ep）表达升高；抑制Perlecan水解可减轻细胞损伤。在该课题中，课题组构建了稳定小鼠控制性皮层损伤模型和bEnd.3细胞牵张损伤模型，验证了Ep在小鼠创伤性颅脑损伤以及脑血管内皮细胞牵张损伤中的重要作用。随着颅脑损伤损伤程度加重，Ep的表达水平随之高。在bEnd.3与HUVEC细胞牵张损伤模型中，发现过抑制Ep可促进细胞成管，减轻细胞损伤。在小鼠皮层控制性损伤模型中，敲减Ep能显著减轻由损伤导致的脑水肿程度、神经炎症反应水平、促进血管新生、改善小鼠神经功能恢复等，验证了在Ep敲减后的bEnd.3细胞自噬、氧化应激相关蛋白表达降低，提示在颅脑创伤中Ep发挥作用很有可能是通过减轻细胞自噬与促进血管新生相关机制进行调节。以上实验结果可能为颅脑创伤治疗提供新策略。