中文摘要：

为探讨人类单纯性先天性心脏病患者中TBX5基因表达下调的可能原因，应用变性高效液相色谱（DHPLC）方法检测100例单纯性先天性心脏病患者中TBX5基因上游1200bp调控区的突变情况；应用甲基化敏感性限制性内切酶（MS—RE）法检测50例单纯性先天性心脏病患者和5例非先天性心脏病患者心肌组织TBX5基因启动子区两个CpG岛（转录起始点上游-49— -188bp和-247- -464bp处）的甲基化情况；应用P-match软件预测小鼠乃巧基因上游转录因子Nkx2—5的结合位点，构建Nkx2—5表达载体转染小鼠H9C2（2—1）心肌细胞，RT-PCR及Western blotting检测Tbx5基因表达，凝胶阻滞实验（EMSA）验证Nkx2—5和乃硝基因的作用。结果在100例单纯性先天性心脏病患者中，未检测到TBX5基因上游1200bp调控区突变：非先天性心脏病患者和单纯性先天性心脏病患者在两个CpG岛存在相同的甲基化；小鼠Tbx5基因转录起始点上游- -312— -315bp可能存在Nkx2—5的结合位点，转染Nkx2—5表达载体后Tbx5基因在mRNA及蛋白质水平均有表达增高趋势，Nkx2．5在体外可以与Tbx5基因上游- -312- -315bp序列相结合。以上结果提示TBX5基因调控区突变和两个CpG岛的甲基化不是单纯性先天性心脏病患者心肌组织中TBX5基因表达下调的原因，TBX5基因表达下调可能由于NKX2—5的表达异常引起。

英文摘要：

To explore the mechanism of TBX5 abnormal expression in simple congenital heart disease （CHD）, 100 CHD venous blood, 50 CHD heart tissues, and 5 non-CHD heart tissues were involved in this study. The mutation and methylation in the 1 200 bp region upstream of TBX5 gene were detected by high-performance liquid chromatography （DHPLC） and methylation-sensitive restriction endonuclease （MS-RE）, respectively. The binding site of NKX2-5 to Tbx5 predicted by P-MATCH software was validated by EMSA （Electrophoretic mobility shift assay）. Tbx5 gene expression in mouse cardiac muscle cell H9C2（2-1） transfected with NKX2-5 expression vector was evaluated. No mutation was found in all patients. Both non-CHD and CHD heart tissues had the same methylation in the two CpG islands. Exogenous Nkx2-5 efficiently activated the transcription of the endogenous Tbx5 gene in H9C2 （2-1） cells. EMSA showed that the special binding band appeared when Nkx2-5 existed. These results indicates that the down expression of TBX5 might not be caused by mutation and methylation in the 1 200 bp region upstream of gene, and might be regulated by abnormal expression of NKX2-5 gene in heart muscle of CHD.