论文部分内容阅读
目的探讨首次在国人WPW综合征(PRKAG2心脏综合征)家系中发现的一个新的错义突变PRKAG2 G100S引起心肌肥厚的可能机制。方法使用Invitrogen的GatewayTM重组系统构建含有人全长PRKAG2基因新突变体(PRKAG2 G100S)的重组腺病毒载体;以增强型绿色荧光蛋白(EGFP)为报告基因,将病毒分别感染H9c2大鼠胚胎心肌细胞和SD乳鼠心肌细胞后采用蛋白质印迹方法检测PRKAG2蛋白的表达。重组腺病毒感染H9c2大鼠胚胎心肌细胞48h前后以Rohd-2/AM孵育并测定细胞内游离Ca2+浓度;感染乳鼠心肌细胞48～72h后以过碘酸-雪夫(PAS)法测定细胞内糖原含量。结果重组腺病毒分别感染H9c2大鼠胚胎心肌细胞和SD乳鼠心肌细胞48h后荧光显微镜下可观察到绿色荧光,用PRKAG2单抗能检测到靶蛋白。与野生型PRKAG2和EGFP组比较,重组腺病毒感染H9c2大鼠胚胎心肌细胞48h后突变体PRKAG2 G100S组细胞内游离Ca2+浓度无明显变化;重组腺病毒感染H9c2大鼠胚胎心肌细胞48h前后的细胞内游离Ca2+浓度无明显变化。在重组腺病毒感染乳鼠心肌细胞48h后,PRKAG2 G100S组能检测到明显的心肌细胞内糖原累积。结论 PKRAG2G100S新突变导致心肌肥厚的主要发病机制可能是细胞内糖原累积,Ca2+及其介导的细胞内信号转导途径可能未参与心肌肥厚的发生。
Objective To investigate the possible mechanism of cardiac hypertrophy induced by PRKAG2 G100S, a new missense mutation found in the Chinese family with WPW syndrome (PRKAG2 cardiac syndrome). Methods Recombinant adenoviral vector containing the full-length PRKAG2 gene (PRKAG2 G100S) was constructed by Invitrogen’s GatewayTM recombination system. The enhanced green fluorescent protein (EGFP) was used as the reporter gene to infect H9c2 rat embryonic cardiomyocytes And SD neonatal rat cardiomyocytes were detected by Western blot PRKAG2 protein expression. The recombinant adenovirus was used to infect H9c2 rat embryonic cardiomyocytes 48 h before and after Roh2-A / AM incubation and the intracellular free Ca2 + concentrations were measured. The neonatal rat cardiomyocytes were treated with periodic acid-Schiff (PAS) The original content. Results The recombinant adenovirus were infected with H9c2 rat embryonic cardiomyocytes and SD neonatal rat cardiomyocytes 48h after fluorescence microscopy can be observed under the green fluorescence, PRKAG2 monoclonal antibody can detect the target protein. Compared with the wild-type PRKAG2 and EGFP groups, the intracellular free Ca2 + concentration in the mutant PRKAG2 G100S group was not changed after recombinant adenovirus infection of H9c2 rat embryonic cardiomyocytes 48h. The recombinant adenovirus infected H9c2 rat embryonic cardiomyocytes 48h before and after intracellular Free Ca2 + concentration no significant change. After 48h of recombinant adenovirus-infected neonatal rat cardiomyocytes, the accumulation of glycogen in cardiomyocytes was detected in PRKAG2 G100S group. Conclusion The main pathogenesis of cardiac hypertrophy induced by PKRAG2G100S mutation may be intracellular glycogen accumulation. Ca2 + and its intracellular signal transduction pathways may not participate in the occurrence of cardiac hypertrophy.