肿瘤坏死因子α对心肌细胞线粒体和膜电位、胞内钙离子浓度的影响

Influence of tumor necrosis factor alpha on the changes of mitochondrial membrane potential and cystolic calcium concentration in cardiac myocytes

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。

AIM： By researching the influence of tumor necrosis factor （TNF） on the changes of mitoehondrial membrane potential and cystolic calcium concen-tration [Ca^2＋]i in aging cardiac myoeytes, to investigate the effective mechanism in injured cardiac myoeytes. METHODS： This experiment was conducted in the laboratory of molecular biology, Institute of Geriatric Cardiovascular Disease, General Hospital of Chinese PLA and Electron Microscope Center, Chinese Academy of Military Medical Sciences from September 2001 to September 2002. The cardiac myoeytes were cultured routinely. The influenoe of TNF-α （1000, 3 000,5 000 U/L） at different concentration on activity of cardiac myoeytes was observed with MTT method. The 3 000 U/L TNFα was used to stimulate cardiac myacytes. After the load of Rodamine123 and Fluo-3/ AM, the changes of mitoehondrial membrane potential and [Ca^2＋]i were measured with confoeal scanning microscope. RESULTS：① The MTT method showed that the 3 types of TNFα after affecting for 24 hours, percentage of A value was the highest, and after 48 hours the percentage of A value decreased. ② After 3 000 U/L TNF-α stimulating on cardiac myoeytes, the confoeal scanning results showed the [Ca^2＋]i of cardiac myoeytes increased. The average fluorescence concentration was 29.64±16.33,96.45±29.42,104.52±22.17,respectively after affecting for 0, 2 and 12 hours with significant difference （P〈0.05）. ③ After stimulating with 3 000 U/L TNF-α, the confoeal scanning results showed the fluoroscence intensity of mitoehondrial membrane potential decreased slightly, and then increased within 75 s. MMP fluorescence intensity in the without TNF-α disposal group and after 5, 10, 15, 20 minutes, 2 and 12 hours disposal with TNF-α was 134±57,115±43,114±40,112±38,97±28, 77±55,34±16, respectively. Potential energy of mltochondrion membrane decreased by a half after 12 hours, and the statistics analysis had insignificant meanings. CONCLUSION： TNF-α can increase [Ca^2＋]i and decrease mitochondrial membrane potential, which can be one of important mechanisms of cardiac myocyte injury mediated by TNF-α.