漢族散發(fā)法洛四聯(lián)癥患兒GATA6、TBX20基因的表達(dá)研究
發(fā)布時(shí)間：2019-03-15 18:08
【摘要】：目的： 法洛四聯(lián)癥是最常見(jiàn)的紫紺型先天性心臟病,其基本病變包括：室間隔缺損,主動(dòng)脈騎跨,右心室流出道梗阻和右心室肥厚。目前,法洛四聯(lián)癥的病因尚未明確,主要涉及遺傳與環(huán)境等多種因素。GATA6和TBX20是與人類(lèi)心臟發(fā)育相關(guān)的兩個(gè)重要轉(zhuǎn)錄因子,在心臟發(fā)育過(guò)程中發(fā)揮著重要的調(diào)控作用。GATA6基因和TBX20基因的異�？蓪�(dǎo)致多種先天性心臟病發(fā)生。本課題擬研究漢族法洛四聯(lián)癥心肌中GATA6和TBX20的基因表達(dá)情況,從而在基因表達(dá)水平研究GATA6和TBX20與漢族法洛四聯(lián)癥之間的關(guān)系。 方法： 實(shí)驗(yàn)組選取2011年12月至2012年3月在我科確診為法洛四聯(lián)癥并行根治手術(shù)治療的漢族患兒15人,對(duì)照組選取2011年12月至2012年2月在我科確診為室間隔缺損并行修補(bǔ)術(shù)治療的漢族患兒15人。兩組患兒年齡、身高、體重分布相近并且相互匹配。經(jīng)倫理委員會(huì)審核通過(guò)、患兒監(jiān)護(hù)人知情同意后,術(shù)中留取患兒心肌組織。 提取心肌組織總RNA,逆轉(zhuǎn)錄為與RNA互補(bǔ)的cDNA。設(shè)計(jì)并合成特定引物,應(yīng)用實(shí)時(shí)熒光定量聚合酶鏈反應(yīng)技術(shù)對(duì)心肌組織GATA6、TBX20和GAPDH的mRNA目的片段進(jìn)行特異性擴(kuò)增。應(yīng)用兩獨(dú)立樣本t檢驗(yàn)方法比較實(shí)驗(yàn)組與對(duì)照組間GATA6和TBX20的mRNA相對(duì)表達(dá)量。應(yīng)用Bivariate Correlations法對(duì)TBX20mRNA相對(duì)表達(dá)量與TOF患兒肺動(dòng)脈指數(shù)進(jìn)行線性相關(guān)性分析。 固定、包埋心肌組織,應(yīng)用特定抗體對(duì)心肌組織切片進(jìn)行免疫組化染色。鏡下觀察比較實(shí)驗(yàn)組、對(duì)照組間GATA6和TBX20的蛋白表達(dá)水平。 結(jié)果： 在實(shí)時(shí)熒光定量聚合酶鏈反應(yīng)中,實(shí)驗(yàn)組心肌GATA6mRNA表達(dá)水平(0.0543±0.0113)顯著低于對(duì)照組(0.2174±0.0598),P=0.0171；實(shí)驗(yàn)組心肌TBX20mRNA表達(dá)水平(0.0074±0.0015)顯著低于對(duì)照組(0.0229±0.0043),P=0.0034；并且TBX20mRNA的相對(duì)表達(dá)量與對(duì)應(yīng)TOF患兒的肺動(dòng)脈指數(shù)呈線性相關(guān)(P=0.0446,R=0.6782)。免疫組化實(shí)驗(yàn)證實(shí)：法洛四聯(lián)癥患兒心肌GATA6與TBX20蛋白表達(dá)水平均降低。 結(jié)論： 1.在漢族人群心肌組織中,存在GATA6與TBX20基因的表達(dá)產(chǎn)物。 2.無(wú)論是在mRNA水平還是蛋白水平,GATA6基因在漢族法洛四聯(lián)癥患者體內(nèi)表達(dá)水平均降低,提示GATA6基因的表達(dá)異常可能與漢族法洛四聯(lián)癥的發(fā)病機(jī)制有關(guān)。 3.無(wú)論是在mRNA水平還是蛋白水平,TBX20基因在漢族法洛四聯(lián)癥患者體內(nèi)表達(dá)水平均降低,并且TBX20mRNA的相對(duì)表達(dá)量與法洛四聯(lián)癥病人肺動(dòng)脈指數(shù)值線性相關(guān),即：TBX20mRNA相對(duì)表達(dá)量越低,法洛四聯(lián)癥患者的肺動(dòng)脈發(fā)育水平就越低,提示TBX20基因的表達(dá)異�？赡芘c漢族法洛四聯(lián)癥的發(fā)病機(jī)制、特別是漢族法洛四聯(lián)癥患兒的肺動(dòng)脈發(fā)育異常情況有關(guān)。 4.TOF的發(fā)病機(jī)制是一個(gè)涉及多基因共同參與、相互作用與調(diào)控的復(fù)雜過(guò)程。因此,以本研究為基礎(chǔ),未來(lái)擴(kuò)展研究GATA6、TBX20各自上、下游基因在TOF心肌中的表達(dá)情況,將有助于進(jìn)一步梳理相關(guān)分子調(diào)控網(wǎng)絡(luò),完善法洛四聯(lián)癥分子發(fā)病機(jī)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 鹵0.0113) was significantly lower than that in the control group (0.2174 鹵0.0598), P < 0.0171; The expression level of TBX20mRNA in myocardium of the experimental group (0.0074 鹵0.0015) was significantly lower than that of the control group (0.0229 鹵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).
【學(xué)位授予單位】：首都醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R725.4
本文編號(hào)：2440860
[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 鹵0.0113) was significantly lower than that in the control group (0.2174 鹵0.0598), P < 0.0171; The expression level of TBX20mRNA in myocardium of the experimental group (0.0074 鹵0.0015) was significantly lower than that of the control group (0.0229 鹵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).
【學(xué)位授予單位】：首都醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R725.4
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