TGF-β1基因多态性与左向右分流型先心病的实验研究

发布时间：2018-04-02 14:37

本文选题：左向右分流型先天性心脏病　切入点：心肌纤维化　出处：《中南大学》2012年硕士论文

【摘要】：目的通过检测左向右分流型先天性心脏病患儿血细胞中TGF-β1基因多态性位点+869T/C表达水平,探讨TGF-β1与左向右分流型先天性心脏病心肌纤维化的相关性,以及基因位点+869T/C基因多态性在心肌纤维化过程中的意义。方法随机选择同期行手术治疗或心脏彩超证实有左向右分流型先天性心脏病(室间隔缺损、房间隔缺损、动脉导管未闭)患儿43例,其中男26例、女17例,年龄0.28～8岁,平均1.43±2.65岁。匹配正常健康儿童35例,其中男23例、女12例,年龄0.39～7岁,平均2.51±2.24岁。获得其父母知情同意后晨起空腹采静脉血,提取血细胞中的DNA,通过建立TGF-β1/β-actin实时荧光定量PCR检测方法对临床样本进行检测,比较两组间两个不同等位基因的基因拷贝数分布频率,分析+869T/C基因拷贝数变异与心肌纤维化的关系。结果(1)利用紫外分光光度仪对提取的总DNA进行光度吸收测量,OD26o/OD280在1.7-1.9之间,表明DNA的纯度及浓度在理想范围内；总DNA电泳结果显示：240bp和137bp条带清晰,无拖尾；灰度比大于1,表明DNA完整性好。(2) TGF-β1+869T/C和β-actin的标准曲线线性关系良好,相关系数(R2)均大于0.99。TGF-β1+869T/C标准曲线方程为:Y=-0.2964X+7.8603,R2=0.999; β-actin的标准曲线方程为：Y=-0.2888X+0.9989,R2=0.9989。(3)实验组和对照组+869/β-actin比值分别为0.0881±0.0397(n=43)和0.0436士0.0061(n=35),t=2.632(n=78,p0.05),差异有统计学意义。(4)实验组和对照组三种基因型(TC, TT, CC)实际值和预测值经卡方检验测得p0.05,差异无统计学意义。结论(1)利用TaqMan探针实时荧光定量PCR技术证实人类基因组中TGF-β1基因在左向右分流型先天性心脏病患儿中高表达。(2)左向右分流型先心病组与正常对照组比较不同基因型TGF-β1+869基因位点存在T/C突变。(3)本实验运用的TaqMan探针实时荧光定量PCR法具有高度的特异性和可靠性,是理想的检测基因表达水平的方法。
[Abstract]:Objective to investigate the relationship between TGF- 尾 1 gene polymorphism and myocardial fibrosis in children with left to right shunt congenital heart disease by detecting the expression of TGF- 尾 1 gene polymorphism in blood cells of children with left to right shunt congenital heart disease.And the significance of 869T/C gene polymorphism in myocardial fibrosis.Methods Forty-three children with left to right shunt congenital heart disease (ventricular septal defect, atrial septal defect, patent ductus arteriosus) were randomly selected for surgical treatment or echocardiography, including 26 males and 17 females aged 0.288 years.The average age was 1.43 卤2.65 years old.35 healthy children were matched, including 23 males and 12 females, aged 0.397 years, with an average age of 2.51 卤2.24 years.After obtaining the informed consent of his parents, the venous blood was collected on an empty stomach in the morning to extract the DNA from the blood cells. The clinical samples were detected by real-time fluorescence quantitative PCR assay of TGF- 尾 1 / 尾 -actin, and the frequency of gene copy number distribution of two different alleles between the two groups was compared.To analyze the relationship between 869T/C gene copy number variation and myocardial fibrosis.The results showed that the purity and concentration of DNA were within the ideal range, and the results of total DNA electrophoresis showed that the bands of the total DNA extracted by UV spectrophotometer were clear, and the bands of 137bp and the bands of 137bp were clear and untrailing, and the absorbance of OD26o- / OD280 was between 1.7-1.9, and the results showed that the purity and concentration of DNA were in the ideal range.The grayscale ratio of TGF- 尾 1 and 尾 -actin is higher than 1, which indicates that the standard curve of DNA has good linear relationship with that of 尾 -actin and TGF- 尾 1 869T/C.The actual values and predicted values of TC, TTand CCS were measured by chi-square test (p 0.05), and the difference was not statistically significant.Conclusion: TGF- 尾 1 gene expression in human genome was confirmed by real-time fluorescence quantitative PCR with TaqMan probe in children with left to right shunt congenital heart disease.TGF- 尾 1 869 gene locus has a T / C mutation. (3) the TaqMan probe real-time quantitative PCR method used in this study is highly specific and reliable.It is an ideal method to detect gene expression level.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R725.4

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