吗啡预先给药对冠脉左前降支结扎大鼠心肌CGRP的影响

吗啡预先给药对冠脉左前降支结扎大鼠心肌CGRP的影响 麻醉学基础研究 去甲肾上腺素诱导心肌细胞TNF-α、NF-κB达及吗啡干 预效应的研究 山西医科大学第二医院麻醉科(030001) 吕志敢 郭 政 目的 观察吗啡(morphine,M)预先给药对去甲肾上腺素(norepinepherine,NE)诱导的心肌细胞肿瘤坏死因子(tumour necrosis factor, TNF-α)表达及心肌细胞核因子-κB(nuclear factor-kappa B,NF-κB)活化的影响，探讨机体应激反应时神经源性反应介质去甲肾上腺素对心肌细胞表达TNF-α、NF-κB产生影响的机制及吗啡痛觉干预在细胞水平的心肌保护作用机制。 ~ 3天龄的Sprague-Dawley(SD)大鼠建立体外培养方法 选用1 新生大鼠心肌细胞模型，采用免疫细胞化学(IC)染色方法对心肌细胞进行鉴定。 第一部分实验选用36孔培养第4 ~ 5天呈亚融合状态下细胞随机分为9组，每组4孔:对照组、3h组、6h组、12h组、24h组、NE1 -6-1 -7-1 -8-1 组(10mol?LNE)、NE2组(10mol?L NE)、NE3组(10 mol?LNE)、 M组。采用酶免疫试验(EIA)技术、反转录PCR(RT-PCR)技术、免疫细胞化学(IC)技术观察NE诱导及吗啡预先给药后心肌细胞内TNF-α表达的变化。 第二部分实验选用20孔培养第4 ~ 5天呈亚融合状态下细胞随机 -6-1 分为5组，每组4孔:对照组、NE1组(10mol?LNE)、NE2组 -7-1 -8-1 (10mol?L NE)、NE3组(10 mol?LNE)、M组。分别采用流式细胞(FCM)技术、免疫细胞化学(IC)技术观察NE诱导后及吗啡预先给药后心肌细胞内NF-κB活化的情况。 结果 (1)培养细胞上清液中加入NE进行诱导，3h组、6h组、 -6-1-7-1-8-112h组、24h组、10 mol?L组、 10 mol?L组 、10 mol?LNE组(24小时)均可诱导心肌细胞TNF-α的蛋白和核酸表达水平较对照 -8-1组显著升高(p<0.05)，在10 mol?L NE、24h组达高峰。 -6-1-7(2)培养细胞上清液中加入NE进行诱导，10 mol?L、 10 -1-8-1mol?L 、10 mol?LNE(24小时)均可诱导心肌细胞NF-κB的激活 -8-1水平较对照组显著升高(p<0.05)，在10 mol?L NE组达高峰。 -8-1(3)吗啡组TNF-α的蛋白和核酸表达水平明显低于10 mol?L 基金项目:国家自然科学基金资助项目(30471656) 1 麻醉学基础研究 -8-1NE组(p<0.05)，NF-κB的激活水平低于10 mol?L NE组 (p<0.05)。 结论 培养细胞上清中加入去甲肾上腺素可诱导体外培养新生大 鼠心肌细胞TNF-α表达、NF-κB激活水平的增高。吗啡预先给药可 显著抑制去甲肾上腺素诱导的体外培养新生大鼠心肌细胞TNF-α、 NF-κB水平的变化，提示机体应激反应时神经源性反应介质去甲肾上 腺素对心肌细胞表达TNF-α、NF-κB产生影响，吗啡可能在细胞水 平发挥心肌保护作用。 【关键词】乳鼠，心肌细胞培养，TNF-α，NF-κB，吗啡，去甲肾上 腺素 The expression of TNF-α、NF-κB induced by norepinepherine in cultured neonatal rat cardiomyocytes and the intervention effects of Morphine LV Zhi-gan, GUO Zheng, Department of Anaesthesia, The Second Hospital of Shanxi Medica lUniversity, Taiyuan 030001，China Abstract Objective The aim of the study was to investigate the effects of morphine preconditioning on TNF-α、NF-κB expression induced by norepinepherine in cultured neonatal rat cardiomyocytes to explore the mechanism of myocardial protective effects of morphine. (1)The model of original cultured cardiomyocytes of Methods SD rat was established and to observe and identify neonatal rat cardiomyocytes with morphology and immunocytochemical assay method under a reverse microscope. (2)36 holes rat cardiomyocytes were randomly divided into 9 groups: the control group; 3h group; 6h -6-1group; 12h group; 24h group; NE1 group(10 mol?L); NE2 -7-1-8-1group(10mol?L); NE3 group(10 mol?L) ;MOR group. Cells were -5-1pre-administrated 10mol?L MOR before NE 30 minutes, respectively. The cells was cultured as scheduled to process for TNF-αenzyme immunometric assay (EIA) ，for TNF-αmRNA Semi-quantitative examination using RT-PCR technique,for TNF-αimmunocyto- chemistry 2 麻醉学基础研究 in order to observe the change of TNF-αinduced by norepinepherine. (3)20 holes rat cardiomyocytes were randomly divided into 5 groups: -6-1-7-1the control group; NE1 group(10 mol?L); NE2 group(10mol?L); -8-1NE3 group(10 mol?L) ; MOR group. Cells were pre-administrated -5-110mol?L MOR before NE 30 minutes, respectively.The cells was cultured as scheduled to process for NF-κB using Flow Cytometry (FCM) technique，for NF-κB immunocyto- chemistry in order to observe the change ofNF-κB induced by norepinepherine. Results (1)The results showed the differential changes in the levels of TNF-α and TNF-αmRNA in the NE group as compared with the baseline in the control group (p<0.05), the levels of TNF-αand TNF- αmRNA in cultured cardiomyocytes were significantly elevated after NE, -8-1and peaked in NE3 group,with 10 mol?LNE for 24 hours. (2)The expression and activation of NF-κB increased significantly in NE group as compared with the baseline in the control group, the levels of NF-κB in cultured cardiomyocytes were significantly elevated after NE, and peaked -8-1in NE3 group,with 10 mol?LNE for 24 hours.(3) The expression of TNF-α and TNF-αmRNA decreased significantly in MOR group as compared with NE3 group (4)The expression and activation of NF-κB decreased significantly in MOR group as compared with NE3 group. Conclusion (1) The NE could cause increase on TNF-α expression、NF-κB activation in cultured cardiomyocytes. (2) Preconditioning treatment with morphine could weaken levels of TNF-α expression、NF-κB activation induced by NE in cultured cardiomyocytes, which might imply that morphine might play an important myocardial preservation role in cell levels. 【Key words】 Neonatal rat, Cardiomyocyte Culture, TNF-α,NF-κB, morphine, Norepinepherine. 3