Wdpcp基因在心脏及冠脉发育中的实验研究

发布时间：2018-06-21 02:01

本文选题：Wdpcp基因 + 房间隔缺损　；参考：《上海交通大学》2013年博士论文

【摘要】：先天性心脏发育畸形是儿童主要的致死性疾病之一,其遗传学机制目前仍旧不甚清楚。本研究运用基因遗传工具对Wdpcp基因导致的一系列心脏异常表型,包括房、室间隔缺损,肺动脉闭锁,流出道障碍,冠脉发生障碍等,进行实验研究。力图揭示该基因与心脏发育之间的关系。第一部分,我们将正常的"#$%细胞进行培养,然后进行'()*)蛋白及微管蛋白的免疫荧光染色。结果表明,'()*)蛋白与微管共同构成了纤毛,是纤毛的组成部分。因此证实!"#$#基因是一个与纤毛生长有关的基因。!第二部分,用!"#$%'()*小鼠示踪!"信号通路的标志物!"#$在+,-'-基因突变鼠中的表达。结果发现野生型鼠胚#$$%'的心脏的()*结构有表达,但在+,-'-基因突变鼠中表达明显降低,()*结构也发育不良。由于()*结构是发育成原发房间隔的重要组织,其形成受!"信号通路的调控,因此能够推论+,-'-小鼠的心房间隔发育畸形与!"信号通路相关。另外我们发现#$$%'的突变小鼠胚胎第六咽弓动脉完全缺如,而以+,-'-'./012)*,!"#$%'()*小鼠来示踪流出道的细胞,发现野生型小鼠流出道有大量!"#$表达阳性细胞,而突变鼠在同一位置没有!"#$基因的表达。我们推论因咽弓动脉缺如,导致!"信号通路受阻,心脏神经棘细胞无法正常的迁移至流出道,进行生长。+第三部分,我们观察到Wdpcp基因突变鼠E18.5d的冠脉前降支短小、分支稀疏甚至消失、发育明显障碍。我们用内皮细胞特异性抗体CD31进行PECAM染色,发现在E12.5d、E13.5d、E14.5d均没有明显的血管生成障碍。我们又针对该阶段的胚胎心脏进行了一系列和冠脉发育相关的细胞因子检测,将E13.5d小鼠胚胎心脏的RNA提取出来,对Gli1、Gli2、Gli3、Shh、Patch1等相关因子进行定量检测,没有发现统计学差异。但是在进行的EMT的标志物检测方面,发现了差异。Wdpcp基因突变鼠Snail1、Snail3、Twist1表达均有显著性降低。Snail1是最主要的EMT标志物,其受到上游Wt1的调控。为了确认这一发现,我们利用'+,%'()*+,--./0123.*./作为工具,在雌鼠孕,456天时注射雌激素,通过绿色荧光示踪表达'+,的心外膜细胞和心外膜来源的细胞在孕,656天时的情况。结果发现野生型小鼠胚心心外膜,心外膜下间质的绿色荧光明显多于Wdpcp基因突变鼠胚心。该结果能够直观的说明Wdpcp基因突变鼠胚心心外膜来源细胞减少,心外膜EMT受到影响,这会导致平滑肌细胞生成减少,冠脉在重构阶段遇到障碍。在本章的最后部分,我们选取了25例膜部巨大室缺合并肺动脉高压的患者,进行了手术治疗,并抽取患者血样,进行了Wdpcp基因的检测,结果为阴性。本研究首次就Wdpcp基因在小鼠心脏及冠脉发育方面的作用机制进行了探讨和研究,对于揭示该基因与心脏发育的关系有重要意义。
[Abstract]:Congenital cardiac malformation is one of the leading fatal diseases in children, and its genetic mechanism is still unclear. A series of abnormal cardiac phenotypes caused by Wdpcp gene including atrium ventricular septal defect pulmonary atresia outflow tract dysfunction coronary artery dysfunction and so on were studied by genetic tools. Try to reveal the relationship between the gene and heart development. In the first part, we cultured the normal #N% cells, and then immunofluorescence staining for the protein and tubulin. The results showed that the protein and the microtubules formed the cilium and were part of the cilia. So confirmed! "#China # gene is a gene related to cilia growth.!" The second part, use! A marker of the signaling pathway! "the expression of #$ in mutated mice." The results showed that there was expression in the heart of the wild mouse embryo #zhiya', but the expression decreased significantly in the mutant mice. Because the structure is an important tissue which develops into the primary atrial septum, it is formed by! " The regulation of the signaling pathway, therefore, can infer the development of atrial septal malformation and the development of the atrial septum in mice. " Signal pathway correlation. In addition, we found that the sixth arcuate artery of the mouse embryo with the mutant #China 'was completely absent, and that the mouse had a large number of positive cells in the outflow tract of the wild-type mouse. But mutant mice are not in the same position! "#$ gene expression. We infer that the absence of the pharyngeal arch leads to it! " The signal pathway was blocked and the cardiac nerve spine cells could not normally migrate to the outflow tract for growth. In the third part, we observed that the anterior descending coronary artery of Wdpcp mutant mice was short, the branches were sparse or even disappeared, and the development was obviously impaired. We stained PECAM with endothelial cell specific antibody CD31 and found that there was no significant angiogenesis disorder at E12.5 d E13.5d E14.5d. We also carried out a series of cytokines related to coronary artery development in the embryonic heart at this stage. We extracted RNA from the embryonic heart of E13.5d mouse, and quantitatively detected the related factors, such as Gli1, Gli2, Gli2Gli2, Gli3Sh, Patch1, and so on. No statistical difference was found. However, in the detection of EMT markers, it was found that the expression of Snail1pp gene mutation mouse Snail1Snail3twist1 was significantly decreased. Snail1 was the most important marker of EMT, which was regulated by upstream Wt1. In order to confirm this finding, we used the method of estradiol injection on day 456 of gestation to express' the epicardial cells and epicardial cells at day 656 of gestation. The results showed that the green fluorescence in the epicardium and mesenchyme of wild mouse embryo was significantly higher than that in the mouse embryo heart with Wdpcp gene mutation. This result can directly explain the decrease of epicardial cells and the influence of epicardial EMT in mouse embryos with Wdpcp gene mutation, which may lead to the decrease of smooth muscle cell formation and the obstruction of coronary artery remodeling. In the last part of this chapter, we selected 25 patients with massive membranous ventricular defect associated with pulmonary hypertension, performed surgical treatment, and took blood samples from the patients, and detected the Wdpcp gene, the results were negative. In this study, the mechanism of Wdpcp gene in the development of heart and coronary artery in mice was studied for the first time, which is of great significance to reveal the relationship between Wdpcp gene and heart development.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R725.4

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