【作者】 孔博；

【导师】 刘迎龙； 吕小东；

【作者基本信息】 中国协和医科大学 ， 心血管外科学， 2008， 博士

【摘要】 第一部分:法乐四联症相关基因的筛选与鉴定【目的】:通过比较法乐四联症患儿心肌与发育正常心肌组织间的全基因组表达谱差异,进一步揭示法乐四联症发生、发展的内在分子机制。【方法】:采用含21 329条Oligo DNA的人类全基因组寡核苷酸表达谱芯片,对5例法乐四联症患儿心肌组织与5例发育正常的心肌组织配对检测差异表达基因,通过生物信息学分析,我们将上述差异表达基因进行分类并挑选出了与法乐四联症发生相关的差异表达基因,并采用实时定量PCR及免疫组化技术在更多样本中验证芯片的结果。鉴于与本组法乐四联症患儿年龄相匹配的正常心肌组织难以获取,在基因芯片的实验过程中我们采用了发育正常引产胎儿心肌组织作为对照,为弥补芯片实验中由于年龄不匹配及手术因素对实验结果的影响,在接下来的实时定量PCR及免疫组化验证实验中我们采用了法乐四联症患儿心肌、发育正常胎儿心肌、与TOF患儿年龄匹配的单纯室间隔缺损患儿心肌及发育正常的成人心肌组织各10例来进一步验证芯片结果。【结果】:共有242个基因在全部5张基因芯片中存在共同的差异表达,依据上述基因所参与的主要的生物学过程,我们将上述242个基因分为13类,在“发育相关基因”中我们发现圆锥动脉干畸形候选基因CSPG2与NTRK3的表达在TOF组患儿中存在明显的表达减低,在接下来的扩大样本量的实时定量PCR和免疫组织化学实验中我们发现NTRK3基因的表达在mRNA水平和蛋白水平TOF组患儿中均特异性表达减低,而CSPG2基因的表达则随年龄的增加而增加。【结论】:我们的研究结果提示,NTRK3基因的表达减低可能与人类法乐四联症右心室流出道畸形的发生,发展相关。第二部分法乐四联症候选致病基因HEY2突变的初步分析【目的】:探讨中国法乐四联症患儿HEY2基因突变的情况。【方法】:法乐四联症患者52例,其中男性30例,女性22例,年龄5.74±2.50(3个月～9岁),均为我院小儿外科中心住院患儿,其中心脏畸形为单发法乐四联症患者38例,伴发其他心脏畸形14例,其中5例合并动脉导管未闭+房间隔缺损,2例合并动脉导管未闭,2例合并卵圆孔未闭,1例合并房间隔缺损,合并单发右位心,单冠畸形、左肺动脉缺如、永存左上腔静脉患者各1例。经心脏超声检查和(或)心导管、心外科手术明确诊断。所有研究对象无血缘关系,均行全面完整的体格检查,合并其他心外畸形患儿在本研究中予以排除,以除外综合征型TOF患儿。50例健康人作为正常研究对照。征得患儿家长同意后,抽取患儿外周血3mL,以EDTA抗凝,—70℃保存,常规胍盐酸法抽提基因组DNA,应用递减聚合酶链反应结合DNA测序技术对上述患儿及对照者的HEY2基因全部外显子及其侧翼序列进行突变检测及单核苷酸多态性(SNP)分析,对所发现的突变位点或单核苷酸多态性位点在患者和健康人中的分布进行比较。【结果】:本组52例患者中,未发现可引起氨基酸序列改变的突变位点,但我们发现了2个新的杂合性突变位点:1例患者的cDNA 621位碱基为A→T杂合性突变,但其编码的氨基酸未发生改变,仍然为丝氨酸,为同义突变,另外,在3例患者的cDNA222位碱基存在一个T→G杂合性突变,也为同义突变,未引起编码氨基酸序列改变,仍然是亮氨酸,该突变也存在于3例对照组中【结论】:HEY2基因可能不是我国法乐四联症患儿的致病基因。更多还原

【Abstract】 Section 1 Screening and Identification of Genes Associated with Human Tetralogy of FallotObjective: To investigated the the molecular mechanism of the development of human Tetralogy of Fallot (TOF). Methods: To investigate the developmental mechanism of human TOF, we compared gene expression profiles of the right ventricular outflow tract myocardium tissues of TOF patients and normally developed fetuses by the DNA microarray technique. With the aid of bioinformatics, we classified the differentially expressed genes and picked out genes which may contribute to the occurrence of TOF. Then the TOF related genes were verified by quantitative real-time PCR and immunohistochemistry in more samples to neutralize the impacts brought by the mismatch of ages and surgical manipulations. Results: Totally, 242 genes were commonly differentially expressed in all the microarray cases. These genes were divided into 13 categories based on the biological process in which they were involved. In the category of developmental related genes, we found the expression level of two conotruncal defect candidate genes CSPG2 and NTRK3 were down-regulated in TOF patients. Ensuring verification procedures confirmed that the expression level of NTRK3 was specifically decreased in TOF patients at both mRNA and protein level, while the expression level of CSPG2 decreased with the increase of age. Conclusions: Our results, in combined with several other previous reports about NTRK3 in animal models, suggested that the insufficient expression of NTRK3 may contribute to the formation of the right ventricular outflow tract defect of human TOF. Section 2 Mutation Screening of HEY2 Gene in Chinese patients with Tetralogy of FallotObjective: To screen the mutation of HEY2 gene in Chinese patients with sporadic tetralogy of Fallot (TOF) .Methods :52 patients with tetralogy of Fallot were selected from Fuwai hospital, After extracting the genomic DNA, the HEY 2 gene was amplified by the means of "touch down polymerase chain reaction"(touch down PCR). After being purified, the PCR products of HEY2 were conducted to the sequencing reaction to investigate whether there were mutations or SNPs or not .Results: No pathogenic mutation was identified in all TOF patients. Only 2 single nucleotide changes including C.222T—G in exon 3 and C.621A—T in exon 5 were found, however both of these 2 heterozygous changes do not alternate the amino acid of the HEY2 protein . Conclusions :The mutation of HEY2 gene might not be associated with tetralogy of Fallot in Chinese population.更多还原