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目的观察促红细胞生成素(erythropoietin,EPO)预处理对培养心肌细胞缺氧复氧前后TNFα表达的影响,并进一步研究其可能的NFκB信号机制。方法采用培养乳鼠心肌细胞建立缺氧复氧损伤模型,分为对照组、EPO组[缺氧复氧前24h,培养液中加入终浓度10Uml的重组人促红素(recombinanthumanerythropoietin,RHuEPO)]、EPO+吡咯烷二硫氨基甲酸盐(PDTC)组(缺氧复氧前24h,加入终浓度10Uml的RHuEPO和5μgml的PDTC)和PDTC组(缺氧复氧前24h,加入终浓度5μgml的PDTC)。分别于缺氧复氧损伤前后,以RTPCR和westernblot检测各组心肌细胞TNFα基因表达变化,同时以EMSA检测各组心肌细胞NFκB活性变化。结果缺氧复氧损伤前,各组心肌细胞TNFα基因表达水平差异无统计学意义,均较弱。损伤后各组心肌细胞TNFα基因表达水平较损伤前对照组显著升高(P<0.01),而EPO组TNFα基因表达水平低于其他各组(P<0.01)。缺氧复氧损伤前EPO组NFκB活性高于其他各组(P<0.01),损伤后各组NFκB活性显著高于损伤前对照组(P<0.01),EPO组NFκB活性低于其他各组(P<0.01)。结论EPO预处理抑制缺氧复氧后心肌细胞TNFα基因表达升高,可能与缺氧复氧后NFκB活性升高被抑制有关。EPO预处理可能通过NFκB活化的负反馈机制抑制缺氧复氧后心肌细胞NFκB活性的升高。
Objective To observe the effect of erythropoietin (EPO) preconditioning on the expression of TNFα before and after hypoxia / reoxygenation in cultured cardiomyocytes, and further study the possible mechanism of NFκB signaling. Methods Hypoxia and reoxygenation injury models were established by cultured neonatal rat cardiomyocytes. The rats were divided into control group, EPO group [recombinant human uroeththropoietin (RHuEPO) 10 uml) PDTC group (24h before hypoxia and reoxygenation, adding 10uml final concentration of RHuEPO and 5μgml PDTC) and PDTC group (24h before hypoxia and reoxygenation adding PDTC) . The changes of TNFα mRNA expression in cardiomyocytes were detected by RTPCR and western blot respectively before and after anoxia-reoxygenation injury. At the same time, the changes of NFκB activity were detected by EMSA. Results Before anoxia-reoxygenation injury, TNFα gene expression levels of myocardial cells in each group showed no significant difference, both of which were weaker. After injury, the expression of TNFα mRNA in cardiomyocytes in each group was significantly higher than that in pre-injury control group (P <0.01), while the expression of TNFα gene in EPO group was lower than that in other groups (P <0.01). The activity of NFκB in EPO group was higher than that in other groups before hypoxia and reoxygenation injury (P <0.01). The activity of NFκB in each group was significantly higher than that in pre-injury control group (P <0.01), and the activity of NFκB in EPO group was lower than that in other groups P <0.01). Conclusion EPO pretreatment can inhibit the expression of TNFα gene in cardiomyocytes after hypoxia-reoxygenation, which may be related to the inhibition of NFκB activation after hypoxia-reoxygenation. EPO preconditioning may inhibit the increase of NFκB activity in cardiomyocytes after hypoxia and reoxygenation through the negative feedback mechanism of NFκB activation.