【摘要】 目的探讨肿瘤坏死因子α(TNFα)对培养的乳鼠心肌细胞内受磷蛋白(PLB)和肌浆网钙泵蛋白同功酶(SERCA2a)基因表达及细胞内游离钙离子浓度的影响。方法体外培养的乳鼠心肌细胞被随机分成对照组和不同浓度TNFα(1、10、30、50、70μg/L)干预组,用单管一步法逆转录聚合酶链反应(RT-PCR)和Westernblotting技术,观察TNFα对乳鼠心肌细胞PLB和SERCA2a基因表达的影响。采用Fluo-3/Am荧光染色剂负载细胞,在激光共聚焦显微镜下测定TNFα对单个乳鼠心肌细胞内游离钙离子浓度的影响。结果TNFα浓度依赖性增高PLBmRNA和蛋白质的表达,10、30、50、70μg/LTNFα组PLBmRNA表达量分别较对照组增加66%、106%、141%、189%(P<0.05),蛋白质的表达分别较对照组提高30%、48%、73%、114%(P<0.001)。而1μg/LTNFα组与对照组PLBmRNA和蛋白质表达差异均无显著性。TNFα不影响SERCA2a的表达。50μg/LTNFα作用细胞24h,能降低异丙肾上腺素刺激的[Ca2+]i变化幅度的增高,[Ca2+]i变化幅度较对照组减少33%(P<0.01),但对静息状态下[Ca2+]i变化幅度无影响。结论TNFα可增强心肌细胞内PLB的表达,降低心肌细胞内[Ca2+]i的变化,这可能与TNFα对心肌细胞的负性肌力作用有关。更多还原

【Abstract】 Objective To explore the effects of tumor necrosis factor α(TNFα) on the expression of phospholamban(PLB) and sarco(endo) plasmic reticulum Ca2+-ATPase(SERCA2a) and concentration of intracellular free calcium in myocardiocytes. Methods The neonatal rat myocardiocytes were randomly divided into 6 groups: treatment with different concentrations of TNFα(1,10,30,50,and 70 ηg/L, respectively) and without TNFα (control). The mRNA and protein expression of PLB and SERCA2a were detected with one-step reverse transcription-polymerase chain reaction and Western blotting. The changes of intracellular free calcium concentration([Ca2+?]i) in cultured single neonatal rat cardiomyocyte were determined with Fluo-3/AM loading by laser scanning confocal microscopy. Results TNFα significantly increased the expression of PLB mRNA and protein in a dose-dependent fashion. The ratio of PLB/β-actin mRNA in myocardiocytes incubated with 10,30,50,and 70 ηg/L TNFα significantly increased by 66%,106%, 141%, and 189% compared with control (P < 0.05), and protein levels significantly increased by 30%,48%,73%, and 114% respectively compared with control (P < 0.001), but there was no significant difference in PLB mRNA expression between the group treated with 1 μg/L TNFα and control group. TNFα had no effect on the expression of mRNA and protein of SERCA2a. TNFα (50 μg/L) incubated with cell for 24 hours diminished Δ[Ca2+]i of single neonatal rat cardiomyocyte about 33% stimulated by isoproterenol (P < 0.01), but had no effect on Δ[Ca2+]i of cardiomyocyte without isoproterenol stimulation. Conclusion TNFα can increase the expression of PLB and decrease Δ[Ca2+]i in cardiomyocytes, which may be related with its negative inotropic effects on cardicyte.更多还原