目的:通过研究肿瘤坏死因子对培养的衰老心肌细胞的线粒体膜电位、胞内钙离子浓度[Ca2+]i的变化,探讨其在心肌细胞损伤中的作用机制。方法:实验于2001-09/2002-09在解放军总医院老年心血管病研究所分子生物学实验室及军事医学科学院电...目的:通过研究肿瘤坏死因子对培养的衰老心肌细胞的线粒体膜电位、胞内钙离子浓度[Ca2+]i的变化,探讨其在心肌细胞损伤中的作用机制。方法:实验于2001-09/2002-09在解放军总医院老年心血管病研究所分子生物学实验室及军事医学科学院电镜中心进行。常规培养心肌细胞。用噻唑兰检测不同浓度肿瘤坏死因子α(1000,3000,5000U/L)对心肌细胞活力的影响。以3000U/L肿瘤坏死因子α刺激心肌细胞,Rhodam ine123和Fluo-3/AM负载后通过共聚焦激光扫描显微镜测量线粒体膜电位和细胞内钙浓度([Ca2+]i)的变化。结果:①噻唑兰法显示3种浓度的肿瘤坏死因子α作用24h后,A值百分率最高,48h后A值百分率下降。②3000U/L肿瘤坏死因子α刺激心肌细胞后,共聚焦扫描图像显示心肌细胞[Ca2+]i升高,作用0,2,12h平均荧光强度分别为29.64±16.33,96.45±29.42,104.52±22.17,有显著性差异(P<0.05)。③3000U/L肿瘤坏死因子α刺激后,激光共聚焦显微镜显示线粒体膜电位在75s内可见荧光强度先稍有降低后升高,未用肿瘤坏死因子α处理组及肿瘤坏死因子α处理后5,10,15,20m in、2及12h的M M P荧光强度分别为134±57,115±43,114±40,112±38,97±28,77±55,34±16,12h后线粒体膜势能下降一半,统计学分析无显著意义。结论:肿瘤坏死因子α能增加[Ca2+]i、降低线粒体膜电位,可能是肿瘤坏死因子α介导心肌细胞损伤的重要机制之一。展开更多
This study examined the current changes of human ether-a-go-go-related gene （hERG） mutation derived from a LQT2 Chinese family with a highly penetrating phenotype. Mutation was identi-fied and site-directed mutagene...This study examined the current changes of human ether-a-go-go-related gene （hERG） mutation derived from a LQT2 Chinese family with a highly penetrating phenotype. Mutation was identi-fied and site-directed mutagenesis was performed to induce the mutation in wild-type （WT） hERG. WT hERG and mutated V535M were cloned and transiently expressed in HEK293 cells. At the 48th and 72nd h after transfection, membrane currents were recorded using whole cell patch-clamp procedures. An A〉G transition at 1605 resulting in replacement of V535M was identified. Compared to WT, V535M mutation significantly decreased tail currents of hERG. At test potential of-40 mV after depolarizing at ＋50 mV, tail current densities were 83.354-7.06 pA/pF in WT and 50.38-4-7.74 pA/pF in V535M respectively （n=20, P〈0.01）. Gating kinetics of bERG revealed that Vl/2 of steady-state inactivation shifted to negative potential in the mutant （V1/2,v535M： -61.814-1.7 mV vs. V1/2, wx： -43.1q-0.71 mV）. The time constant of recovery from inactivation was markedly prolonged in the mutant compared to WT among test potentials. V535M hERG mutation demonstrated markedly decreased tail current densities, which suggests that V535M is a new loss-of-function mutation of hERG channel responsible for LQT2.展开更多
文摘目的:通过研究肿瘤坏死因子对培养的衰老心肌细胞的线粒体膜电位、胞内钙离子浓度[Ca2+]i的变化,探讨其在心肌细胞损伤中的作用机制。方法:实验于2001-09/2002-09在解放军总医院老年心血管病研究所分子生物学实验室及军事医学科学院电镜中心进行。常规培养心肌细胞。用噻唑兰检测不同浓度肿瘤坏死因子α(1000,3000,5000U/L)对心肌细胞活力的影响。以3000U/L肿瘤坏死因子α刺激心肌细胞,Rhodam ine123和Fluo-3/AM负载后通过共聚焦激光扫描显微镜测量线粒体膜电位和细胞内钙浓度([Ca2+]i)的变化。结果:①噻唑兰法显示3种浓度的肿瘤坏死因子α作用24h后,A值百分率最高,48h后A值百分率下降。②3000U/L肿瘤坏死因子α刺激心肌细胞后,共聚焦扫描图像显示心肌细胞[Ca2+]i升高,作用0,2,12h平均荧光强度分别为29.64±16.33,96.45±29.42,104.52±22.17,有显著性差异(P<0.05)。③3000U/L肿瘤坏死因子α刺激后,激光共聚焦显微镜显示线粒体膜电位在75s内可见荧光强度先稍有降低后升高,未用肿瘤坏死因子α处理组及肿瘤坏死因子α处理后5,10,15,20m in、2及12h的M M P荧光强度分别为134±57,115±43,114±40,112±38,97±28,77±55,34±16,12h后线粒体膜势能下降一半,统计学分析无显著意义。结论:肿瘤坏死因子α能增加[Ca2+]i、降低线粒体膜电位,可能是肿瘤坏死因子α介导心肌细胞损伤的重要机制之一。
文摘This study examined the current changes of human ether-a-go-go-related gene （hERG） mutation derived from a LQT2 Chinese family with a highly penetrating phenotype. Mutation was identi-fied and site-directed mutagenesis was performed to induce the mutation in wild-type （WT） hERG. WT hERG and mutated V535M were cloned and transiently expressed in HEK293 cells. At the 48th and 72nd h after transfection, membrane currents were recorded using whole cell patch-clamp procedures. An A〉G transition at 1605 resulting in replacement of V535M was identified. Compared to WT, V535M mutation significantly decreased tail currents of hERG. At test potential of-40 mV after depolarizing at ＋50 mV, tail current densities were 83.354-7.06 pA/pF in WT and 50.38-4-7.74 pA/pF in V535M respectively （n=20, P〈0.01）. Gating kinetics of bERG revealed that Vl/2 of steady-state inactivation shifted to negative potential in the mutant （V1/2,v535M： -61.814-1.7 mV vs. V1/2, wx： -43.1q-0.71 mV）. The time constant of recovery from inactivation was markedly prolonged in the mutant compared to WT among test potentials. V535M hERG mutation demonstrated markedly decreased tail current densities, which suggests that V535M is a new loss-of-function mutation of hERG channel responsible for LQT2.
