基于脂质体纳米金靶向递送ERG稳定表达及基因编辑系统维持脑血管内皮细胞稳态的实验研究

Disruption of blood brain barrier leads to brain edema and hemorrhage. The fundamental mechanism is the injury of inter-cellular junctions of endothelial cells. Previous studies from our groups and others indicated that transcriptional factor ERG is the key molecule to maintain the expression of multiple junctional proteins. Downregulation of ERG results in loss of tight and adherens junctions, and subsequent hyperpermeability. Based on these results, we hypothesize that increase of ERG expression in brain endothelial cells by gene transfer through targeted carrier will help maintain haemeostasis of brain vasculature under pathological conditions. We will develop gold nanocluster/lipid core–shell Nanocarrier with Angiopoitin1 ligand to carry ERG overexpression plasmid or Chrispr/Cas9 sgRNA Plasmid modifying the -387 NFIC binding motif of ERG promoter. The carriers will enter brain endothelial cells by binding with the Tie2 receptor. The carriers will be examined by in vitro culture of brain endothelial cells and HPRT targeted transgenic mice of ERG, Claudin-5, VE-cadherin and ICAM-2 promoter with brain edema. The most efficient carrier/plasmid combination will be determined. The project will provide new venue for prevention and treatment of cerebral vascular diseases.

脑血管屏障稳态失衡导致脑水肿和脑出血，其核心机制是脑血管内皮细胞连接的破坏。前期研究中发现，转录因子ERG是维持多种连接蛋白表达的关键分子，降低ERG表达导致脑血管内皮细胞连接减少和通透性增加。我们在前期基础上提出假设，通过靶向载体向脑血管内皮细胞进行基因导入,升高ERG表达从而实现在病理条件下维持脑血管内皮细胞稳态。我们将构建带有Angiopoitin1配体的脂质体纳米金载体,包裹ERG过表达质粒或修改ERG启动子NFIC抑制区的Chrispr/Cas9基因编辑质粒，通过识别Tie2受体进入脑血管内皮细胞。在体外细胞模型和前期构建的人ERG、Claudin-5、VE-cadherin和ICAM-1基因启动子HPRT定点转基因鼠进行基因表达检测，确定最有效的载入和调控方法。本项目的实施将为脑血管病的防治提供新的治疗思路。

脑血管稳态失衡导致脑血管内皮细胞连接的破坏，进一步导致脑水肿和脑出血。转录因子ERG是维持多种连接蛋白表达的关键分子，降低ERG表达导致血管内皮细胞连接减少和通透性增加。本研究的目的是通过靶向载体向脑血管内皮细胞进行基因导入,升高ERG表达从而实现在病理条件下维持脑血管内皮细胞稳态。在本研究中，我们构建了带有Angiopoitin1配体的脂质体纳米金载体,包裹ERG过表达质粒或修改ERG启动子NFIC抑制区的Chrispr/Cas9基因编辑质粒，通过识别Tie2受体进入脑血管内皮细胞。带有Angiopoitin1配体的脂质体纳米金载体包裹ERG过表达质粒复合物经过表征确定构建成功，通过体外实验表明：该复合物成功递送ERG过表达质粒进入人脑微血管内皮细胞，在TNFα刺激的细胞中升高ERG表达，并调节Claudin-5、VE-cadherin和ICAM-1表达。在小鼠脑水肿模型中，该复合物减少组织含水量。此外，我们还确定了ERG在Notch通路激活中的介导作用。本研究为脑血管病的防治提供新的治疗思路。