CADASIL相关突变型Notch3受体导致人脑血管周细胞病变的机制

The relationship between the NOTCH3 mutation and microvessels damage has become the research hotspot of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Prominent damage of the vessel walls are the basic pathological characteristics of CADASIL. The pericyte is an important component of microvessels and is highly consistent with pathological changes of CADASIL patients. Thus, the pericyte may play the leading role in microvascular damage of CADASIL, but the mechanism needs to be studied. The Notch3 gene knockout models and CADASIL cases related Notch3 mutations (c. 128C > G, c. 430 T > A and c. 465 C > G) models are build on the pricytes by using the CRISPR-Cas9 system. The effects of the CADASIL related mutant Notch3 protein on the growth and development of the pericyte, the Notch3 pathway and PDGFR-β pathway will be studied. Based on this study, the mechanism for the pericyte degeneration caused by mutant Notch3 will be elucidated. We speculate the new insight might provide protective and therapeutic benefits in CADASIL.

NOTCH3突变与小血管病变的关系已成为常染色体显性遗传性脑动脉病伴皮层下梗死和白质脑病(CADASIL)的研究热点。小血管壁损伤是CADASIL最基本的病理特征。血管周细胞是小血管壁的重要组份，且与CADASIL病变血管的分布高度一致，提示其在血管损伤中起着核心作用，但具体机制亟待研究。本项目在前期研究基础上拟利用CRISPR-Cas9系统，在人脑血管周细胞上构建NOTCH3基因敲除模型和CADASIL病例相关NOTCH3突变(c.128C>G, c.430T>A, c.465C>G)模型，深入研究人脑血管周细胞生长发育情况，Notch3蛋白表达的时空变化与沉积情况，及相关的信号通路（Notch3通路，PDGFR-β通路），从新的角度揭示“CADASIL相关突变型Notch3蛋白导致人脑血管周细胞损害的分子机制”，以期为CADASIL的防治提供新的思路。

常染色体显性遗传性脑动脉病伴皮层下梗死和白质脑病(CADASIL)是最常见的单基因遗传性脑小血管病，其临床、影像学特征与散发性脑小血管病(SVD)患者高度一致，且在治疗上同样缺乏有效的防治手段以及存在抗凝抗栓等防治的类似困扰，目前已成为研究SVD发病机制与防治手段的良好疾病模型。因此，阐明CADASIL的发病机制，将有利于SVD的防治。 本项目运用CRISPR-Cas9系统靶向构建与CADASIL相关的NOTCH3突变型(c.128C>G)和NOTCH3EXON4 Knock-out的人血管内皮细胞模型，着重研究Notch3敲除或突变对血管内皮细胞生长发育的影响，Notch3蛋白的代谢变化、GOM沉积与血管内皮细胞病变的关系，以及对Notch3信号通路和PDGFR-β信号通路的影响 。我们发现与野生型人血管内皮细胞组（HUVEC）相比， HUVEC-NOTCH3-Exon4 KO 和 HUVEC-NOTCH3-C128G组的细胞存活变差，细胞凋亡增多，电镜下可见线粒体肿胀明显，但并没有观察到典型的GOM形成；Notch3的表达和合成明显下降，但没有观察到Notch3在细胞内定位分布的异常；当NOTCH3基因Exon4被敲除或NOTCH3突变时，Notch3信号通路的下游转录因子HES1表达明显下降，但没有检测到PDGFR-β信号通路的异常。当NOTCH3敲除或突变细胞与表达人源性Notch3配体Jagged1的细胞共培养时，可以明显改善细胞存活等情况，提高Notch3信号通路中HES1的表达。以上结果表明，Notch3对维持人静脉血管内皮细胞的正常存活起着至关重要的作用，可能与其Notch3信号通路的损害有关，初步揭示了Notch3突变损害血管内皮细胞的可能机制，为CADASIL的防治提供新的思路。