钠通道重叠综合征相关基因SCN5A在H9C2细胞中的表达  

Expression of sodium channel overlap syndrome SCN5A gene in H9C2 cell

作　　者：高洁[1] 史瑞明[1] 吕颖[1] 张军波[1] 马爱群[1] 孙超峰[1] 

机构地区：[1]西安交通大学医学部第一附属医院心血管内科环境与疾病相关基因教育部重点实验室陕西省分子心脏病学重点实验室,陕西西安710061

出　　处：《新乡医学院学报》2015年第11期975-980,共6页Journal of Xinxiang Medical University

基　　金：国家自然科学基金资助项目(编号:30900580)

摘　　要：目的构建表型重叠家系相关基因SCN5A del QKP1507-1509突变型真核表达载体,研究其在H9C2细胞中的表达。方法一步法定点诱变技术构建SCN5A基因del QKP1507-1509突变型真核表达载体,电穿孔方法将野生型和突变型质粒转染至H9C2细胞,应用实时荧光定量聚合酶链反应(RFQ-PCR)及Western blot技术检测其mRNA及蛋白表达情况。结果琼脂糖凝胶电泳法及DNA直接测序法均表明定点突变成功,RFQ-PCR及Western blot结果显示,野生组、突变组和混合组SCN5A基因mRNA相对表达量分别为1.089±0.459、1.011±0.840和1.069±0.492,3组SCN5A基因mRNA相对表达量比较差异无统计学意义(P>0.05)。野生组、突变组和混合组SCN5A蛋白在H9C2细胞的相对表达量分别为3.781±0.221、3.466±0.176和3.714±0.198,3组SCN5A蛋白相对表达量比较差异无统计学意义(P>0.05)。结论成功构建钠通道基因del QKP1507-1509突变型真核表达载体并转染至H9C2细胞,该突变未引起钠通道在细胞膜的异常表达,为进一步功能研究提供了研究基础及方向。Objective To construct the eukaryotic expression vector of del QKP1507-1509 mutant of SCN5 A channel,and to explore the expression of this mutation in H9C2 cells. Methods The eukaryotic expression vector of del QKP1507-1509 mutant of SCN5 A gene was constructed by site-directed mutagenesis method,the wild type and mutational plasmid were transfected into H9C2 cells by electroporation method. The mRNA and protein expressions of the mutant were tested by real-time fluorescent quantitation polymerase chain reaction（ RFQ-PCR） and Western blot analysis respectively. Results Agarose gel electrophoresis and DNA direct sequencing showed that the site-specific mutagenesis was successful. The results of RFQ-PCR and Western blot showed that the relative expression of SCN5 A mRNA in wild group,mutation group and mixed group was1. 089 ＋ 0. 459,1. 011 ＋ 0. 840 and 1. 069 ＋ 0. 492 respectively,and there was no significant difference in the relative expression of SCN5 A mRNA among the three groups（ P〉0. 05）. The relative expression of SCN5 A protein in H9C2 cells in the wild group,mutation group and mixed group was 3. 781 ＋ 0. 221,3. 466 ＋ 0. 176 and 3. 714 ＋ 0. 198 respectively,and there was no significant difference in the relative expression of SCN5 A protein among the three groups（ P〉0. 05）. Conclusions SCN5 A del QKP1507-1509 mutant eukaryotic expression vector is successfully constructed and transfected into H9C2 cells. The mutant does not affect the expression of sodium channel in cell membrane. This provides a basis and direction for further study.

关 键 词：钠通道 SCN5A基因 重叠综合征 H9C2细胞 

分 类 号：Q786[生物学—分子生物学]