单纯性先天性心脏病中TBX5基因表达异常的机制  

The mechanism of TBX5 abnormal expression in simple congenital heart disease

作　　者：辛娜[1] 邱广蓉[1] 宫立国[1] 徐小延[1] 孙开来[1] 

机构地区：[1]中国医科大学医学遗传学教研室,沈阳110001

出　　处：《遗传》2009年第4期374-380,共7页Hereditas（Beijing)

基　　金：国家自然科学基金(编号:30070411;30200305;30400485)项目;国家高技术研究发展计划项目(863计划)(编号:Z19-01-01-03);辽宁省教育厅基金项目(编号:202013133;2004C045;20060951)资助

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Tbx5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Tbx5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。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.

关 键 词：先天性心脏病 TBX5 NKX2—5 

分 类 号：R725.4[医药卫生—儿科]