TNF-α在乳鼠心肌细胞中诱导HSP70蛋白表达的研究

Tumor Necrosis Factor-a-induced Expression of Heat Shock Prote in 70 in Neonatal Rat Ventricular Cardiomyocytes

热休克蛋白70(HSP70)在细胞修复、存活和维持细胞正常功能方面有着重要作用。作为分子伴侣,它起着心肌保护的作用。已经对重症心脏病人的心肌组织进行了蛋白组学研究,得到了HSP70在心衰病人心肌组织中较正常人心肌组织表达升高的结论,并且在血液中得到了进一步的验证。在进一步的离体细胞实验中用不同剂量的肿瘤坏死因子-alpha(TNF-α)刺激乳鼠心肌细胞,以观察不同时间点HSP70的动态表达情况。培养乳鼠心肌细胞,分别对细胞进行热休克(42℃)、TNF-α和缺血缺氧处理,在不同的时间点收获细胞,以观察HSP70的动态表达情况。用免疫化学、ELISA以及Western blotting的方法对HSP70蛋白进行分析。结果表明,在正常对照细胞中基本没有阳性信号出现,而在经缺血缺氧、热休克(42℃)以及TNF-α处理的细胞中有明显的阳性表达。以上研究首次在乳鼠心肌细胞中证明TNF-α诱导的HSP70表达具有时间和浓度依赖性。通过运用TNF-α对HSP70蛋白表达影响的研究,初步推断HSP70的表达模式,为体内诱导产生HSP70从而发挥心肌保护作用的研究提供一定的理论基础。

Heat shock protein 70 is essential for c el lular recovery,survival and maintenance of cellular function.Heat shock protei n 70 function as molecular chaperons in protein folding and it is a role of card ioprotection against various stresses.Different proteins expression of tissues f rom failing hearts using proueomics have been studied before.HSP70 was found hig hly expressed in the tissues and blood samples from heart failure patients compa ired with non-failing hearts.For exploiting the use of HSP70 as a therapeutic marker,HSP70 expression was studied using Neonatal rat ventricular cardiomyocyt es（NRVM） by teating with TNF-α with various concentrations and time points in vitro.Primary cultures were prepared from neonatal rat ventricular myocytes.Cu ltured cardiomyocytes were incubated with TNF-α at doses of 40 200 and 800U /ml for a dynamic time point as 1,6,12 and 24 hours.NRVM treated with heat sho ck（42℃） were used as positive control.The expression of myocyte HSP70 was quan tified by Western blot analysis and immunohistochemistry using a specific monocl onal antibody.Immunohistochemical analysis revealed HSP70 were nearly not visib le in control cells.However,at 12 h post-hypoxia/SD,HSP70-immunoreactivity （IR） was evident in the cells.The initial demonstration that TNF-alpha exerts c oncentration-and time-dependent effects on the expression of HSP 70 in NRVM w as constinuted.The expression of HSP70 influenced by TNF-α can indicate the p ossibility of a pharmacological approach to HSP70 induction and cardiac protecti on,which may ultimately be of clinical relevance.