| 摘要: |
| 目的 尝试建立类似PRKAG2心脏综合征钠离子通道功能的HEK-293T细胞模型,为探讨PRKAG2心脏综合征心律失常机制奠定基础。方法 分别构建人PRKAG2基因质粒载体和SCN5A基因质粒载体,共转人胚肾细胞(HEK-293T);应用免疫荧光法、Real-time PCR、蛋白质印迹法检测HEK-293T细胞转染结果及基因表达情况。结果 转染48 h后,HEK-293T细胞在细胞免疫荧光显微镜下可见红色荧光和绿色荧光。Real-time PCR及蛋白印迹结果证实:单独转染SCN5A组、SCN5A+PRKAG2组、SCN5A+G100S组、SCN5A+R302Q组SCN5A基因及蛋白均有表达,而在空白对照组无表达,差异有统计学意义(P<0.05);SCN5A+PRKAG2组PRKAG2基因及蛋白高表达,而空白对照组、SCN5A组、SCN5A+R302Q组、SCN5A+G100S组表达量较低,差异有统计学意义(P<0.05)。结论 成功建立类似PRKAG2心脏综合征钠离子通道的HEK-293T细胞模型,为后续研究奠定了基础。 |
| 关键词: PRKAG综合征 心脏肌细胞 钠通道 人胚肾细胞 共转染 |
| DOI：10.3724/SP.J.1008.2014.01243 |
| 投稿时间：2014-03-11修订日期：2014-10-10 |
| 基金项目:国家自然科学基金(81170092). |
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| Establishing HEK-293T cell model carrying human sodium channel and PRKAG2 gene |
| WANG Jun1,2,ZHENG Xing1* |
(1. Department of Cardiology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China;
2. Department of Cardiology, The Fifth People's Hospital of Dalian, Dalian 116000, Liaoning, China
*Corresponding authors) |
| Abstract: |
| Objective To establish a HEK-293T cell model with sodium channel function similar to that in PRKAG2 syndrome. Methods Vectors of PRKAG2 gene and SCN5A gene were constructed and were used to co-transfect HEK-293T cells. Immunofluorescence, real-time PCR and Western blotting analysis were used to examine the transfection results and the expression of interested mRNA and protein. Results Imunofluorescence findings showed red fluorescence and green fluorescence in HEK-293T cells 48 hours after co-transfection. Real-time PCR and Western blotting analysis showed SCN5A expression in pure SCN5A group, SCN5A + wild-type group, SCN5A + R302Q mutant group, and SCN5A + G100S mutant group, but not in the blank control group, showing significant significance(P<0.05). Expression of PRKAG2 gene and protein in SCN5A+PRKAG2 group was significantly higher than those in the blank control group, pure SCN5A group, SCN5A+R302Q, and SCN5A+G100S groups (P<0.05) . Conclusion We have successfully established a HEK-293T cell model with sodium channel function similar to that in PRKAG2 syndrome, paving a way for future study. |
| Key words: PRKAG2 syndrome cardiac myocytes sodium channels HEK-293T cells co-transfection |
