Notch3基因变异介导Notch信号通路在非综合征性胎儿颈部淋巴水囊瘤中的作用及机制研究

Non syndromic cystic hygroma (CH) is a common congenital disease which characterised by accumulating cervical lymphatics and surrounding structure. Unknown etiology, difficult to evaluate the prognosis and poor treatment effect are the major existing issues of non syndromic CH. Our previous research on non syndromic CH fetuses have showed that ①double immunofluorescence method have confirmed the pathological nature of the disease is the primary lymphatic and capillary dysplasia;②whole exome sequencing have identified NOTCH3 gene mutation in fetuses with non-syndromic CH;③protein expression have confirmed that the NOTCH3 expression was significantly reduced in those fetal cases who had NOTCH3 gene mutation;④we have successfully constructed NOTCH3 mutation C57BL/6 mouse model; ⑤ the abnormal phenotype of cervical lymphangoncus, cardiovascular and skeletal abnormalities were found in F2 mutation mice. The objective of this study is to investigate the variant effects and mechanism of NOTCH3 gene defect and key factors(LYVE-1，prox-1，VEGF-3，VEGFA，JAG1 and D114.) in Notch3 pathway through lymphatic and capillary development& differentiation via molecular,celluar and animal researchlevel and research view, and to explore vascular endothelial cells damage caused by application of NOTCH3-mRNA dynamic NOTCH3 repair defects, provide clues for the pathogenesis and treatment of fetal non syndromic CH.

非综合征性淋巴水囊瘤（CH）是一种常见的主要累积颈部淋巴管及周边结构的先天性疾病，病因未明、预后评估困难及治疗效果差。本项目前期研究：①应用双重免疫荧光方法在非综合征性CH胎儿病例中证实该类疾病的病理本质是原发性淋巴管和毛细血管发育不良；②应用全外显子测序在非综合征性CH胎儿病例中发现NOTCH3基因突变；③蛋白质表达证实该类CH病例中NOTCH3表达显著下调；④成功构建了NOTCH3突变C57BL/6小鼠模型；⑤发现F2代NOTCH3突变小鼠发生明显的颈部淋巴管肿大、心血管及骨骼异常等表型。本项目拟从细胞、分子、动物水平及正反面论证NOTCH3缺陷对非综合征性CH形成的影响；探讨Notch信号通路对淋巴管和毛细血管发育及分化的调节机制；并摸索应用活力型NOTCH3-mRNA修复NOTCH3缺陷引起的脉管内皮细胞损伤，为阐明胎儿非综合征性CH的发生机制及诊治提供新的科学依据。

胎儿非综合征性颈部淋巴水囊瘤（CH）是一种严重的先天性发育异常，目前仍有多数胎儿病例的病因及分子机制不明。本研究利CH胎儿病例颈部组织，通过细胞实验和动物模型探讨了非综合征性颈部淋巴水囊瘤的发病机制。既往研究认为淋巴管发育过程中出现的紊乱或延迟是非综合征性颈部淋巴水囊瘤的原因。我们通过单细胞测序技术研究发现：①NOTCH3基因突变所致的非综合征性颈部淋巴水囊瘤胎儿病例中，淋巴管内皮细胞和淋巴细胞祖细胞基因表达异常（分别发现21和23个差异表达基因）；②病例组淋巴管内皮细胞NOTCH受体非标准结合蛋白DLK1较健康组表达下调明显；③病例组内皮细胞祖细胞分化调节基因MGP较健康组上调明显，通过基因调控网路分析发现MGP表达与NOTCH通路活性相关，同时细胞评分分析发现内皮细胞祖细胞分化异常；因此我们推断颈部淋巴水囊瘤病例组淋巴内皮细胞DLK1基因表达降低，抑制内皮细胞祖细胞NOTCH通路及其细胞分化调节基因MGP高表达，致内皮细胞祖细胞分化异常，最终导致发病；④在非NOTCH3基因突变CH胎儿病例中，同样发现淋巴管内皮细胞出现明显差异，同时VEGFR2基因在淋巴管内皮细胞内出现高表达现象，差异表达基因EDN1、TAGLN、CLDN5存在与KDR基因协同调控VEGF通路的可能性。CREB1、TP63、FOXM1、STAT1这四个潜在转录因子在调控VEGFR2基因转录调节中发挥重要作用。总之，上述结果证实了无论在NOTCH3基因突变所致非综合征性颈部淋巴水囊瘤或非NOTCH3基因突变所致非综合征性颈部淋巴水囊瘤病例中，均出现淋巴管内皮细胞显著差异。同时证明了上述两种非综合征性颈部淋巴水囊瘤类型由不同信号通路参与，NOTCH-DLK1-MGP通路在内皮细胞在NOTCH3基因突变所致CH发挥关键作用，但VEGFR2基因在非NOTCH3基因突变所致CH的起重要作用。这为了解淋巴管内皮细胞在非综合征性颈部淋巴水囊瘤中的发生机制中的作用和诊治提供科学依据。