基于同步辐射3D显微表征技术研究XBP1s对脑缺血后血管新生的作用及调控机制

Cerebral ischemia induces spontaneous angiogenesis.To promote endogenous angiogenesis is a key factor of stroke treatment and rehabilitation.Using synchrotron radiation technique in combination with histomorphologic methods,our previous studies have demonstrated that the process of angiogenesis initiates in an early stage and quickly reached the culmination. Newly generated microvessels showed an increase in tortuosity and branching, thus intertwining into a complicated network.According to recent studies,the transcription factor XBP1s in endoplasmic reticulum stress response may also positively regulate angiogenesis,thus performing crosstalk with classic pro-angiogenic signal pathway.However,up to now,it still remains unclear about its role and regulatory mechanism.In the present study, we plan to perform directional transfection of lentivirus vectors to oxygen-glucose deprivation model in vitro and middle cerebral artery occlusion model in vivo. Via up-regulation or down-regulation of XBP1s expression,the parameters of the angiogenesis will be examined, as well as the downstream target gene expression.Based on studies both in vivo and vitro, this research aims to figure out the positive effects and reveal the relationship between the XBP1s treatment and angiogenesis after cerebral ischemia, proceeding to elucidate the upstream XBP1s regulatory mechanism in the ischemic stroke process from the perspective of structure and function.All the above-mentioned investigations are expected to provide the theoretical basis and experimental evidences for the potential clinical application of the XBP1s as a novel gene-intervention therpeutic target in the mannagement of ischemic stroke.

脑缺血后可引发自体血管新生，促进内源性血管新生以改善缺血区血供是脑卒中治疗修复的关键。我们前期应用同步辐射显微血管3D成像方法联合2D组织形态学技术，发现血管新生在脑缺血后极早期即可启动，并迅速达到高峰，新生血管迂曲度增加。最新研究表明内质网应激反应的重要转录因子XBP1s亦可作为血管新生的正向调控因子，与经典促血管生成信号通路间存在crosstalk，但其在脑缺血后血管新生中的作用和调控机制尚不明确。本项目拟通过构建慢病毒载体，于体外脑微血管内皮细胞糖氧剥夺模型和体内大鼠大脑中动脉阻塞模型进行定向转染，通过上调或下调XBP1s的表达,观察其对脑缺血后血管新生及下游靶基因表达的影响。经由细胞、动物双重模型，力求从结构-功能角度揭示XBP1s 与脑缺血后血管新生的作用关系，为阐明XBP1s调控脑缺血后微血管网络重塑的生物学机制提供理论基础和实验依据，以期为脑卒中的药物治疗提供新的干预靶点。

脑缺血后可引发自体血管新生，促进内源性血管新生以改善缺血区血供是脑卒中治疗修复的关键。最新研究表明内质网应激反应的重要转录因子XBP1s亦可作为血管新生的正向调控因子，与经典促血管生成信号通路间存在crosstalk，但其在脑缺血后血管新生中的作用和调控机制尚不明确。本研究通过培养脑微血管内皮细胞(BMECs)，建立糖氧剥夺模型(OGD)，体外模拟缺血缺氧环境，进行慢病毒转染，通过MTS细胞增殖检测，流式细胞凋亡检测，迁移实验，Matrigel成管实验及下游通路检测，观测XBP1s不同干预对细胞生物学功能的影响，证实XBP1s促进OGD后cyclin D1, MMP-2, and MMP-9蛋白表达,抑制caspase-3 and caspase-9蛋白表达，通过激活PI3K/AKT, ERK, and HIF-1α/VEGF通路促进脑微血管内皮细胞的存活、细胞周期、迁移及成血管能力.于在体动物层面，通过建立大鼠大脑中动脉阻塞模型，构建慢病毒载体定向纹状体转染，使用TTC染色证实过表达XBP1s可减少梗死面积，修复缺血损伤，使用Hoechst/RECA-1，TUNEL，PCNA/CD31免疫荧光在2D形态学层面证实XBP1s能减少内皮细胞凋亡，促进其存活和增殖。同时，首次应用前沿的同步辐射显微血管成像技术在无需血管造影的条件下无损原位展示脑3D微血管网络，并证实XBP1s有效改善大鼠MCAO后损伤脑区3D血管构筑，促进血管新生。同时进一步拓展和优化同步辐射成像体系,实现脑神经网络3D可视化。本项目为神经系统微血管网络的3D形态表征精准研究提供了数字化虚拟解析的全新方式，经由细胞-动物双重模型，从结构-角度证实XBP1s对于增加缺血脑区血管新生改善局部缺血，增加血流灌注，以及促进组织细胞存活，减少梗死面积促进神经功能修复具有积极有效的作用，为外源性XBP1s干预应用于缺血性脑卒中治疗的临床前疗效评估提供了实验科学依据以及新的干预靶点。