【作者】 罗萍；

【导师】 卢永昕；

【作者基本信息】 华中科技大学 ， 心血管内科， 2006， 硕士

【摘要】 目的肿瘤坏死因子α(TNF-α)在心力衰竭发生发展中具有重要作用,已证实慢性心力衰竭患者血浆TNF-α水平明显升高。已酮可可碱是一种黄嘌呤衍生物,临床试验证实它可以改善心力衰竭患者的临床状况;动物实验亦报道已酮可可碱作为一种免疫调节剂可以抑制人类单核细胞生成肿瘤坏死因子-α。因此我们假设在心肌细胞中它有相似的作用机制。此实验旨在通过观察已酮可可碱对内毒素诱导新生大鼠心肌细胞生成TNF-α的影响,探讨已酮可可碱对大鼠心肌细胞可能的保护机制。材料与方法取新生大鼠的心肌细胞加以培养,按不同的药物处理分为四组:对照组(A组)、1μg/ml已酮可可碱组(B组)、10μg/ml已酮可可碱组(C组)、100μg/ml已酮可可碱组(D组)。加入生理盐水(A组)和不同浓度的已酮可可碱(B、C、D组)作用一定时间后,再加入内毒素刺激TNF-α生成。作用6小时后,采用酶联免疫吸附法(ELISA)法测定新生大鼠心肌细胞TNF-α的生成,用逆转录聚合酶链式反应(RT-PCR)法检测TNF-αmRNA的表达。结果已酮可可碱干预后,新生大鼠心肌细胞培养液中TNF-α的生成受到不同程度的抑制,且与预处理的已酮可可碱浓度呈正相关。其中1μg/ml已酮可可碱组、10μg/ml已酮可可碱组、100μg/ml已酮可可碱组的TNF-α的生成[(83.37±16.39)pg/ml,(35.43±2.60)pg/ml,(19.72±5.19)pg/ml]都显著低于对照组[(98.50±7.99)pg/ml](P<0.05);已酮可可碱干预后,新生大鼠心肌细胞TNF-αmRNA的表达均受到不同程度的抑制,且与已酮可可碱浓度呈正相关。其中1μg/ml已酮可可碱组、10μg/ml已酮可可碱组、100μg/ml已酮可可碱组心肌细胞TNF-αmRNA表达(0.78±0.02,0.71±0.04,0.62±0.04)都显著低于对照组(0.90±0.02) (P<0.05)。结论已酮可可碱能有效抑制内毒素诱导的新生大鼠心肌细胞产生TNF-α,并能有效减少内毒素诱导的新生大鼠心肌细胞TNF-αmRNA的表达,且这些效应呈剂量依赖性。本实验结果表明已酮可可碱能有效调节TNF-α的生成,而且这一过程至少部分发生在转录水平。这一结果为已酮可可碱应用于治疗心力衰竭提供了实验依据。更多还原

【Abstract】 Objective TNF-αhas played important roles in the development and progression of chronic heart failure. Several lines of reports indicated that the levels of circulating TNF-αwere elevated in patients with congestive heart failure. Pentoxifylline, a xanthin-derived agent, has been showed to improve clinical status in chornic heart failure patients by several clinical trials. Animal studies has showed that pentoxifylline reduced TNF-αproduction in human macrophages as an immunomodulatory agent. Therefore, we studied the effects of pentoxifylline on TNF-αproduction in neonatal rat cardiac myocytes stimulated with lipopplysaccharide to valuate the mechanism underlying pentoxifylline on the protection of rat hearts.Materials and methods Neonatal rats cardiomyocytes were cultured and divided into four groups as follows: control group (A group, cells pretreated with diluent), 1μg/ml pentoxifylline group (B group), 10μg/ml pentoxifylline group (C group) and 100μg/ml pentoxifylline group (D group). Primary cultured cells were pretreated with diluent or various concentration of pentoxifylline for indicated times, then stimulated with lipopplysaccharide for another 6 hours. Then the culture medium and cells were collected for analysis. The level of secreted TNF-αwas measured by enzyme-linked immunosorbent assay (ELISA) and TNF-αmRNA expression was determined by semiquantitative reverse transcriptional polymerase chain reaction(RT-PCR).Results After pretreated with pentoxifylline, TNF-αrelease was significantly decreased in pentoxifylline group. Pentoxifylline reduced TNF–αproduction in a dose dependent manner. With increasing concentration of pentoxifylline (1μg/ml, 10μg/ml, 100μg/ml), TNF-αsecretion showed a liner decrease: (83.37±16.39) pg/ml, (35.43±2.60) pg/ml, (19.72±5.19) pg/ml, lower than control group (98.50±7.99) pg/ml (P<0.05). Pentoxifylline also inhibited TNF-αmRNA expression in neonatal rat cardiomyocytes. There is an inverse relationship between TNF-αmRNA expression and pentoxifylline concentration. Pretreated with different concentration (1μg/ml, 10μg/ml, 100μg/ml ) of pentoxifylline, the TNF-αmRNA expression determined by semi-quantitive RT-PCR was [(0.78±0.02) , (0.71±0.04), (0.62±0.04)], lower than control group (0.90±0.02) (P<0.05).Conclusions Pentoxifylline can effectively inhibit the production of TNF–αand reduced TNF-αmRNA expression in neonatal rat cardiomyocytes stimulated with lipopplysaccharide in a dose-dependent manner. It showed that pentoxifylline reduced TNF–αproduction at least by blocking transcriptional activation, which may provide the experimental basis for the use of pentoxifylline in the therapy of chronic heart failure.更多还原