吗啡预先给药对冠脉左前降支结扎大鼠心肌CGRP的影响

1、去甲肾上腺素诱导心肌细胞TNF-、NF-B表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-)表达及心肌细胞核因子-B(nuclear factor-kappa B,NF-B)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-、NF-B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。方法 选用1 3天龄的Sprague-Dawley（SD）大

2、鼠建立体外培养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组(10-6mol·L-1NE )、NE2组(10-7mol·L-1 NE )、NE3组(10-8 mol·L-1 NE)、 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-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分为5组，每组4孔

3、：对照组、NE1组(10-6mol·L-1NE )、NE2组(10-7mol·L-1 NE )、NE3组(10-8 mol·L-1 NE)、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-B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、12h组、24h组、10-6 mol·L-1组、 10-7 mol·L-1组 、10-8 mol·L-1NE组（24小时）均可诱导心肌细胞TNF-的蛋白和核酸表达水平较对照组显著升高（p<0.05），在10-8

4、 mol·L-1 NE、24h组达高峰。（2）培养细胞上清液中加入NE进行诱导，10-6 mol·L-1、 10-7 mol·L-1 、10-8 mol·L-1NE（24小时）均可诱导心肌细胞NF-B的激活水平较对照组显著升高（p<0.05），在10-8 mol·L-1 NE组达高峰。（3）吗啡组TNF-的蛋白和核酸表达水平明显低于10-8 mol·L-1 基金项目：国家自然科学基金资助项目（30471656）NE组（p<0.05），NF-B的激活水平低于10-8 mol·L-1 NE组（p<0.05）。结

5、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-表达、NF-B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-、NF-B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-、NF-B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-，NF-B，吗啡，去甲肾上腺素The expression of TNF-、NF-B induced by norepinepherine in cultured neonatal rat cardiomyocytes and the int

6、ervention effects of MorphineLV Zhi-gan, GUO Zheng, Department of Anaesthesia, The Second Hospital of Shanxi Medica lUniversity, Taiyuan 030001，ChinaAbstractObjective The aim of the study was to investigate the effects of morphine preconditioning on TNF-、NF-B expression induced by norepinepherine in

7、 cultured neonatal rat cardiomyocytes to explore the mechanism of myocardial protective effects of morphine. Methods （1）The model of original cultured cardiomyocytes of SD rat was established and to observe and identify neonatal rat cardiomyocytes with morphology and immunocytochemical assay method

8、under a reverse microscope. （2）36 holes rat cardiomyocytes were randomly divided into 9 groups: the control group; 3h group; 6h group; 12h group; 24h group; NE1 group(10-6 mol·L-1); NE2 group(10-7mol·L-1); NE3 group(10-8 mol·L-1) ;MOR group. Cells were pre-administrated 10-5mol·L

9、-1 MOR before NE 30 minutes, respectively. The cells was cultured as scheduled to process for TNF-enzyme immunometric assay (EIA) ，for TNF-mRNA Semi-quantitative examination using RT-PCR technique,for TNF-immunocyto- chemistry in order to observe the change of TNF-induced by norepinepherine. （3）20 h

10、oles rat cardiomyocytes were randomly divided into 5 groups: the control group; NE1 group(10-6 mol·L-1); NE2 group(10-7mol·L-1); NE3 group(10-8 mol·L-1) ; MOR group. Cells were pre-administrated 10-5mol·L-1 MOR before NE 30 minutes, respectively.The cells was cultured as schedule

11、d to process for NF-B using Flow Cytometry (FCM) technique，for NF-B immunocyto- chemistry in order to observe the change ofNF-B induced by norepinepherine. Results （1）The results showed the differential changes in the levels of TNF- and TNF-mRNA in the NE group as compared with the baseline in the c

12、ontrol group (p<0.05), the levels of TNF-and TNF-mRNA in cultured cardiomyocytes were significantly elevated after NE, and peaked in NE3 group,with 10-8 mol·L-1NE for 24 hours. （2）The expression and activation of NF-B increased significantly in NE group as compared with the baseline in the c

13、ontrol group, the levels of NF-B in cultured cardiomyocytes were significantly elevated after NE, and peaked in NE3 group,with 10-8 mol·L-1NE for 24 hours.（3） The expression of TNF- and TNF-mRNA decreased significantly in MOR group as compared with NE3 group （4）The expression and activation of NF-B decreased significantly in MOR group as compared with NE3 group.Conclusion （1） The NE could cause increase on TNF-expression、NF-B activation in cultured cardiomyocytes. （2） Preconditioning treatment with morphine could weaken levels of TNF-expression、NF-B activation induced b