本文選題：Wdpcp基因 + 房間隔缺損　； 參考：《上海交通大學》2013年博士論文

【摘要】：先天性心臟發(fā)育畸形是兒童主要的致死性疾病之一,其遺傳學機制目前仍舊不甚清楚。本研究運用基因遺傳工具對Wdpcp基因?qū)е碌囊幌盗行呐K異常表型,包括房、室間隔缺損,肺動脈閉鎖,流出道障礙,冠脈發(fā)生障礙等,進行實驗研究。力圖揭示該基因與心臟發(fā)育之間的關(guān)系。第一部分,我們將正常的"#$%細胞進行培養(yǎng),然后進行'()*)蛋白及微管蛋白的免疫熒光染色。結(jié)果表明,'()*)蛋白與微管共同構(gòu)成了纖毛,是纖毛的組成部分。因此證實!"#$#基因是一個與纖毛生長有關(guān)的基因。!第二部分,用!"#$%'()*小鼠示蹤!"信號通路的標志物!"#$在+,-'-基因突變鼠中的表達。結(jié)果發(fā)現(xiàn)野生型鼠胚#$$%'的心臟的()*結(jié)構(gòu)有表達,但在+,-'-基因突變鼠中表達明顯降低,()*結(jié)構(gòu)也發(fā)育不良。由于()*結(jié)構(gòu)是發(fā)育成原發(fā)房間隔的重要組織,其形成受!"信號通路的調(diào)控,因此能夠推論+,-'-小鼠的心房間隔發(fā)育畸形與!"信號通路相關(guān)。另外我們發(fā)現(xiàn)#$$%'的突變小鼠胚胎第六咽弓動脈完全缺如,而以+,-'-'./012)*,!"#$%'()*小鼠來示蹤流出道的細胞,發(fā)現(xiàn)野生型小鼠流出道有大量!"#$表達陽性細胞,而突變鼠在同一位置沒有!"#$基因的表達。我們推論因咽弓動脈缺如,導致!"信號通路受阻,心臟神經(jīng)棘細胞無法正常的遷移至流出道,進行生長。+第三部分,我們觀察到Wdpcp基因突變鼠E18.5d的冠脈前降支短小、分支稀疏甚至消失、發(fā)育明顯障礙。我們用內(nèi)皮細胞特異性抗體CD31進行PECAM染色,發(fā)現(xiàn)在E12.5d、E13.5d、E14.5d均沒有明顯的血管生成障礙。我們又針對該階段的胚胎心臟進行了一系列和冠脈發(fā)育相關(guān)的細胞因子檢測,將E13.5d小鼠胚胎心臟的RNA提取出來,對Gli1、Gli2、Gli3、Shh、Patch1等相關(guān)因子進行定量檢測,沒有發(fā)現(xiàn)統(tǒng)計學差異。但是在進行的EMT的標志物檢測方面,發(fā)現(xiàn)了差異。Wdpcp基因突變鼠Snail1、Snail3、Twist1表達均有顯著性降低。Snail1是最主要的EMT標志物,其受到上游Wt1的調(diào)控。為了確認這一發(fā)現(xiàn),我們利用'+,%'()*+,--./0123.*./作為工具,在雌鼠孕,456天時注射雌激素,通過綠色熒光示蹤表達'+,的心外膜細胞和心外膜來源的細胞在孕,656天時的情況。結(jié)果發(fā)現(xiàn)野生型小鼠胚心心外膜,心外膜下間質(zhì)的綠色熒光明顯多于Wdpcp基因突變鼠胚心。該結(jié)果能夠直觀的說明Wdpcp基因突變鼠胚心心外膜來源細胞減少,心外膜EMT受到影響,這會導致平滑肌細胞生成減少,冠脈在重構(gòu)階段遇到障礙。在本章的最后部分,我們選取了25例膜部巨大室缺合并肺動脈高壓的患者,進行了手術(shù)治療,并抽取患者血樣,進行了Wdpcp基因的檢測,結(jié)果為陰性。本研究首次就Wdpcp基因在小鼠心臟及冠脈發(fā)育方面的作用機制進行了探討和研究,對于揭示該基因與心臟發(fā)育的關(guān)系有重要意義。
[Abstract]:Congenital cardiac malformation is one of the leading fatal diseases in children, and its genetic mechanism is still unclear. A series of abnormal cardiac phenotypes caused by Wdpcp gene including atrium ventricular septal defect pulmonary atresia outflow tract dysfunction coronary artery dysfunction and so on were studied by genetic tools. Try to reveal the relationship between the gene and heart development. In the first part, we cultured the normal #N% cells, and then immunofluorescence staining for the protein and tubulin. The results showed that the protein and the microtubules formed the cilium and were part of the cilia. So confirmed! "#China # gene is a gene related to cilia growth.!" The second part, use! A marker of the signaling pathway! "the expression of #$ 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.
【學位授予單位】：上海交通大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R725.4

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1 胡振雷;Wdpcp基因在心臟及冠脈發(fā)育中的實驗研究[D];上海交通大學;2013年

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