【作者】 王薇；

【导师】 桂永浩；

【作者基本信息】 复旦大学 ， 儿科学， 2011， 博士

【摘要】 DiGeorge综合征(DGS)是人类最常见的染色体缺失综合征之一,活产婴儿发病率约1/4000-5000。由于DGS累及组织器官较多,对患者的危害较大,特别是复杂心血管畸形,严重影响患者的生活质量和生命安全。因此,研究DGS的发病机理,探索预防策略十分重要。近年来研究表明,DGS的发生与tbxl基因单倍剂量不足(haploinsufficiency)、神经嵴细胞(Neural crest cells, NCCs)发育异常等多种因素有关。鉴于此,我们研究了两者之间的关系。NCCs中tbxl基因不表达,可能存在介导tbxl基因作用的因子。视黄酸(Retinoic acid, RA)是胚胎发育过程中必需的微量营养素,也是一种作用广泛的信号分子,与心脏发育的多个阶段有关。动物实验证实,RA缺乏会造成心血管系统发育异常,与DGS特征相似。为此,我们考虑RA是否为tbxl基因表达与NCCs发育之间的“桥梁”?我们以斑马鱼为模式生物,研究了tbxl基因表达对NCCs发育的影响,并探讨RA在其中所起的作用。为了模拟DGS患者tbxl基因单倍剂量不足,在第一部分,我们首先构建了tbxl基因表达下调的斑马鱼模型。通过显微注射特异性的tbxl基因反义寡核苷酸tbxl-MO,下调tbxl基因表达,导致斑马鱼表型异常,表现为心脏环化障碍、心包水肿、下颌骨减小、耳囊减小、眼睛减小。通过tbxl-MO效率验证,证实tbxl基因表达下调成功。在tbxl基因表达下调的模型中,我们研究NCCs的发育过程。选取NCCs不同发育阶段的标记基因,通过胚胎整体原位杂交的方法,发现NCCs的起始、剥离、迁移、分化等多个发育环节都受到了tbxl基因表达下调的影响,发育过程受到抑制,心脏神经嵴细胞(Cardiac neural crest cells, CNCCs)向心脏细胞的分化障碍。在第二部分,我们构建了RA缺乏的斑马鱼模型。通过显微注射视黄醛脱氢酶基因(raldh2)的反义寡核苷酸,下调raldh2基因表达,并通过效率验证,证实raldh2基因表达下调成功。Raldh2基因表达下调的斑马鱼,表型异常,表现为心脏环化障碍,心包水肿、体长短。而且我们发现,NCCs的起始、剥离、迁移与分化过程也受到了抑制,CNCCs向心脏细胞分化障碍。鉴于tbxl基因表达下调与raldh2基因表达下调,NCCs发育抑制的表现非常相似,在第三部分,我们研究了tbxl基因表达与RA信号分子之间的关系。通过原位杂交和real-time PCR的检测,我们发现,tbxl基因表达下调的斑马鱼中,RA合成酶基因-raldh2表达降低,而RA降解酶基因-cyp26al,cyp26b1, cyp26c1表达增强,说明tbxl基因表达下调可以从合成和降解两方面降低斑马鱼体内RA的水平。我们在体外人工合成斑马鱼raldh2mRNA,对tbxl基因表达下调的斑马鱼进行拯救。实验证明,外源raldh2基因可以拯救tbxl基因表达下调的结果,异常的斑马鱼表型以及NCCs标记基因的表达都大部分得到拯救。因此,我们得出结论,RA是介导tbxl基因对NCCs发育调节的“中间桥梁”,tbxl基因表达通过RA来调节NCCs的发育。更多还原

【Abstract】 DiGeorge syndrome (DGS) is the most frequent known chromosomal microdeletion syndrome, with an incidence of1in4000-5000livebirths. Unveiling the genetic basis of DGS is important for understanding its pathogenesis.Tbxl was the most likely candidate gene responsible for the syndrome. Many of the affected tissues have contributions of neural crest origin. While tbxl is expressed in the non-neural crest mesoderm and endoderm of the pharyngeal arches. The special expression pattern suggests that there may be a cell non-autonomous function in NCCs development regulated by tbx1gene.It is also known that deficiency of vitamin A and its biologically active derivative, retinoic acid (RA), is detrimental to pharyngeal development and causes a DGS phenotype in humans and rodents. Therefore, genes involved in RA metabolism or signalling may be candidate modifier of tbxl gene to regulate NCCs.We try to understand whether NCCs development was altered in tbxl-knockdown zebrafish, whether RA acts on these phenomena?We established a zebrafish model which the expression of tbxl gene was knockdown by microinjection of tbxl-MO. The results showed tbxl-kd zebrafish present non-looping heart, pericardial edema, hypoplastic jaw, otic vesicles and eyes. We examined several NCC markers expression in different stages, and found that the initiation, delamination, migration and differentiation of NCCs were all impaired as the expression of tbx1gene was knockdown.Raldh2gene encodes the enzyme for RA synthesis. We established another zebrafish model which the expression of raldh2gene was knockdown by microinjection of raldh2-MO. The mutants showed non-looping heart and pericardial edema. And the NCC markers expression was altered as the mutants of tbxl knockdown.Regarding raldh2gene related to RA synthesis and cyp26genes related to the degrading of RA, we analyzed the expression of these genes in tbx1-knockdown zebrafish by in situ hybridization and real-time PCR. The results showed that raldh2was down regulated, cyp26al and two families, cyp26b1and cyp26c1encoding enzymes for degrading RA, were up regulated in tbxl-knockdown zebrafish.By co-injecting raldh2mRNA, we rescued the tbxl-knockdown zebrafish effectively. In the rescued zebrafish, the expression of several NCC markers was similar with that of wild type zebrafish. Therefore, we conclude that tbxl gene regulates NCCs development via RA signlling in zebrafish.更多还原