汉族散发法洛四联症患儿GATA6、TBX20基因的表达研究

发布时间：2019-03-15 18:08

【摘要】：目的：法洛四联症是最常见的紫绀型先天性心脏病,其基本病变包括：室间隔缺损,主动脉骑跨,右心室流出道梗阻和右心室肥厚。目前,法洛四联症的病因尚未明确,主要涉及遗传与环境等多种因素。GATA6和TBX20是与人类心脏发育相关的两个重要转录因子,在心脏发育过程中发挥着重要的调控作用。GATA6基因和TBX20基因的异常可导致多种先天性心脏病发生。本课题拟研究汉族法洛四联症心肌中GATA6和TBX20的基因表达情况,从而在基因表达水平研究GATA6和TBX20与汉族法洛四联症之间的关系。方法：实验组选取2011年12月至2012年3月在我科确诊为法洛四联症并行根治手术治疗的汉族患儿15人,对照组选取2011年12月至2012年2月在我科确诊为室间隔缺损并行修补术治疗的汉族患儿15人。两组患儿年龄、身高、体重分布相近并且相互匹配。经伦理委员会审核通过、患儿监护人知情同意后,术中留取患儿心肌组织。提取心肌组织总RNA,逆转录为与RNA互补的cDNA。设计并合成特定引物,应用实时荧光定量聚合酶链反应技术对心肌组织GATA6、TBX20和GAPDH的mRNA目的片段进行特异性扩增。应用两独立样本t检验方法比较实验组与对照组间GATA6和TBX20的mRNA相对表达量。应用Bivariate Correlations法对TBX20mRNA相对表达量与TOF患儿肺动脉指数进行线性相关性分析。固定、包埋心肌组织,应用特定抗体对心肌组织切片进行免疫组化染色。镜下观察比较实验组、对照组间GATA6和TBX20的蛋白表达水平。结果：在实时荧光定量聚合酶链反应中,实验组心肌GATA6mRNA表达水平(0.0543±0.0113)显著低于对照组(0.2174±0.0598),P=0.0171；实验组心肌TBX20mRNA表达水平(0.0074±0.0015)显著低于对照组(0.0229±0.0043),P=0.0034；并且TBX20mRNA的相对表达量与对应TOF患儿的肺动脉指数呈线性相关(P=0.0446,R=0.6782)。免疫组化实验证实：法洛四联症患儿心肌GATA6与TBX20蛋白表达水平均降低。结论： 1.在汉族人群心肌组织中,存在GATA6与TBX20基因的表达产物。 2.无论是在mRNA水平还是蛋白水平,GATA6基因在汉族法洛四联症患者体内表达水平均降低,提示GATA6基因的表达异常可能与汉族法洛四联症的发病机制有关。 3.无论是在mRNA水平还是蛋白水平,TBX20基因在汉族法洛四联症患者体内表达水平均降低,并且TBX20mRNA的相对表达量与法洛四联症病人肺动脉指数值线性相关,即：TBX20mRNA相对表达量越低,法洛四联症患者的肺动脉发育水平就越低,提示TBX20基因的表达异常可能与汉族法洛四联症的发病机制、特别是汉族法洛四联症患儿的肺动脉发育异常情况有关。 4.TOF的发病机制是一个涉及多基因共同参与、相互作用与调控的复杂过程。因此,以本研究为基础,未来扩展研究GATA6、TBX20各自上、下游基因在TOF心肌中的表达情况,将有助于进一步梳理相关分子调控网络,完善法洛四联症分子发病机制。
[Abstract]:Aim: tetralogy of Fallot (Tetralogy of Fallot) is the most common cyanotic congenital heart disease. Its basic pathological changes include ventricular septal defect (VSD), aortic treadmill, right ventricular outflow tract obstruction (RVO) and right ventricular hypertrophy (RVH). At present, the etiology of tetralogy of Fallot is not clear, mainly related to genetic and environmental factors. GATA6 and TBX20 are two important transcription factors related to human heart development. The abnormality of GATA 6 gene and TBX20 gene may lead to many congenital heart diseases. The purpose of this study was to study the gene expression of GATA6 and TBX20 in myocardium of Han nationality with tetralogy of Fallot, and to study the relationship between GATA6, TBX20 and tetralogy of Fallot in Han nationality at the level of gene expression. Methods: from December 2011 to March 2012, 15 children of Han nationality who were diagnosed as tetralogy of Fallot and treated by radical operation in our department were selected in the experimental group. From December 2011 to February 2012, 15 children of Han nationality who were diagnosed as ventricular septal defect (VSD) and repaired in our department were selected in the control group. The age, height and weight distribution of the two groups were similar and matched each other. After examination and approval by the ethics committee and informed consent of the guardian, myocardial tissue was retained during the operation. Extraction of Total RNA, reverse Transcriptase from Myocardial tissue as complementary cDNA. with RNA Specific primers were designed and synthesized, and the specific mRNA fragments of GATA6,TBX20 and GAPDH were amplified by real-time fluorescence quantitative polymerase chain reaction (FQ-PCR). The mRNA expressions of GATA6 and TBX20 were compared between the experimental group and the control group by two independent sample t-test. Bivariate Correlations method was used to analyze the linear correlation between the relative expression of TBX20mRNA and pulmonary artery index in children with TOF. The myocardial tissue was fixed and embedded, and the myocardial tissue sections were stained by immunohistochemical staining with specific antibodies. The protein expression levels of GATA6 and TBX20 in the experimental group and the control group were observed and compared under microscope. Results: in real-time fluorescence quantitative polymerase chain reaction (FQ-PCR), the level of myocardial GATA6mRNA expression in the experimental group (0.0543 卤0.0113) was significantly lower than that in the control group (0.2174 卤0.0598), P < 0.0171; The expression level of TBX20mRNA in myocardium of the experimental group (0.0074 卤0.0015) was significantly lower than that of the control group (0.0229 卤0.0043), and the relative expression of TBX20mRNA was linearly correlated with the pulmonary artery index of the corresponding TOF children (P = 0.0446, R = 0.6782). Immunohistochemistry showed that the expression level of GATA6 and TBX20 protein in myocardium of tetralogy of Fallot was lower than that of control group. Conclusions: 1. The expression products of GATA6 and TBX20 genes were found in the myocardium of Han nationality. 2. No matter at the level of mRNA or protein, the expression level of GATA6 gene in the patients with tetralogy of Fallot was decreased, suggesting that the abnormal expression of GATA6 gene might be related to the pathogenesis of tetralogy of Fallot in Han nationality. 3. No matter at mRNA level or protein level, the expression level of TBX20 gene in tetralogy of Fallot patients of Han nationality was decreased, and the relative expression of TBX20mRNA was linearly correlated with pulmonary artery index of tetralogy of Fallot patients, that is, the lower the relative expression of TBX20mRNA, the lower the expression level of TBX20mRNA gene in the patients with tetralogy of Fallot. The lower the level of pulmonary artery development in patients with tetralogy of Fallot suggests that the abnormal expression of TBX20 gene may be related to the pathogenesis of tetralogy of Fallot in Han nationality especially in children with tetralogy of Fallot in Han nationality. The pathogenesis of 4.TOF is a complex process involving multi-gene interaction and regulation. Therefore, on the basis of this study, the future expansion study on the expression of GATA6,TBX20 upstream and downstream genes in TOF myocardium will be helpful to further comb the related molecular regulatory networks and improve the molecular pathogenesis of tetralogy of Fallot (Tetralogy of Fallot).
【学位授予单位】：首都医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R725.4

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