近交系长爪沙鼠Willis环变异缺失中Cystatin C 的作用及相关调控机制

Our previous study demonstrated that the expression level of Cystatin C (CST3) was significantly higher in absent anterior communicating artery (ACoA) than in complete ACoA in the circle of Willis in inbred Mongolian gerbil brain. However, what role does the CST3 play in anatomical variation of the circle of Willis is unclear. Many reports showed that CST3 could inhibit angiogenesis in tumor and has relationship with VEGFA. CST3 also could indirectly inhibit Notch procession in ischemia-induced neovascularization and Notch deficiency would results in under-development of the ACoA. So we speculate that CST3, Notch, and VEGFA would play important role and closely association each other in variation and malformation in the circle of Willis. To confirm this hypothesis, in this study, we will investigate CST3 expression in different patterns of Willis circle through temporal analyses in inbred Mongolian gerbil brain. We will also analyze the regulation of CST3, Notch, and VEGFA in proliferation and migration of vascular endothelial cell and vascular smooth muscle cell by electric cell-substrate impedance sensing (ECIS) and other methods; and investigate the function of CST3 in vascular development of chicken chorioallantoic membrane (CAM). Finally, we will establish CST3 knockout gerbil using the CRISPR-Cas9 genome editing, and reveal the function of CST3 and the relationship among CST3, Notch, and VEGFA. Our study would be useful in explaining the molecular mechanism of deformation of the circle of Willis and providing a new idea for angiogenesis and a potential therapeutic target for cerebrovascular disease.

我们研究发现Cystatin C(CST3) 在Willis环前交通支缺失的长爪沙鼠脑中表达显著升高，但它在Willis环的变异缺失中如何发挥作用尚不清楚。文献显示它能抑制肿瘤血管发生并和VEGFA有关联，血管发生时它能抑制Notch的表达，而Notch 缺失则会导致前交通支缺损，据此我们推测CST3与Notch和VEGFA在 Willis环变异缺失的发生中有重要关联。为验证这一假说，我们拟用长爪沙鼠近交系研究CST3在不同Willis环类型中的时相表达，利用ECIS等方法分析CST3、Notch和VEGFA在血管内皮细胞和平滑肌细胞增殖、迁移中的调控及对鸡胚血管生成的作用，制备Cas9-CST3基因敲除长爪沙鼠并分析其在Willis环变异缺失中的作用及与Notch和VEGFA的关系。本课题将有助于阐释Willis环变异缺失的发生机制，为血管发生机制研究提供新思路和脑血管病治疗提供新靶点。

脑血管病是全世界公认的严重威胁人类生命和健康的三大疾病之一，脑底动脉Willis环（circle of Willis, COW）的变异缺失是脑缺血和中风的一种重要危险性因素。长爪沙鼠由于先天存在Willis 环变异缺失的多种类型而备受重视。我们在前期应用抑制消减杂交技术（suppression subtractive hybridization, SSH），筛选获得4个基因，在长爪沙鼠Willis环缺失与完整不同类型中表达有显著差异，其中包括半胱氨酸蛋白酶抑制肽C (Cystatin C, CST3）。.在本课题中，我们选取长爪沙鼠胚胎和出生后仔鼠的脑组织，对不同胚龄和出生后不同日龄该基因的表达水平进行分析，发现CST3的表达高峰在胚胎第10天，既在胚胎血管形成关键期高表达，证明其可能与血管生成相关。我们分别构建CST3过表达和shRNA的内皮细胞后，用MTT实验、RTCAxCELLigence系统和鸡胚尿囊膜形成实验证明，CST3可以抑制血管形成。用CST3蛋白中和抗体和激动剂分别通过MTT实验、ELISA和免疫荧光实验研究发现，抑制VEGFA能显著提高CST3的表达，并且有剂量依赖关系。同时发现CST3和p53以及CAPN10有调控关系。我们应用7种行为测试实验检测了长爪沙鼠的基本行为学特征，结果发现，与大鼠相比，沙鼠在旷场实验、高架十字迷宫、抓力、社会交互和条件恐惧实验中表现出明显不同的行为特征。这些结果提示，沙鼠在行为脑研究探索中可能是一个敏感的动物模型，尤其在焦虑和条件恐惧方面。针对CST3基因的不同片段分别设计多个gRNA（guide RNA），用CRISPR-Cas9方法获得CST3基因敲除纯合子动物。经测试发现，CST3敲除动物其神经行为学发生了变化，脑缺血后脑梗死面积显著增加，并且Willis环前交通支缺失的比例增加。这些结果提示，CST3与血管生成相关，可能参与了Willis环前交通支的形成，并对脑神经有一定的保护作用。