嗎啡預(yù)先給藥對冠脈左前降支結(jié)扎大鼠心肌CGRP的影響

1、去甲腎上腺素誘導(dǎo)心肌細(xì)胞TNF-、NF-B表達(dá)及嗎啡干預(yù)效應(yīng)的研究山西醫(yī)科大學(xué)第二醫(yī)院麻醉科(030001) 呂志敢 郭 政目的 觀察嗎啡（morphine,M）預(yù)先給藥對去甲腎上腺素(norepinepherine,NE)誘導(dǎo)的心肌細(xì)胞腫瘤壞死因子(tumour necrosis factor, TNF-)表達(dá)及心肌細(xì)胞核因子-B(nuclear factor-kappa B,NF-B)活化的影響，探討機(jī)體應(yīng)激反應(yīng)時神經(jīng)源性反應(yīng)介質(zhì)去甲腎上腺素對心肌細(xì)胞表達(dá)TNF-、NF-B產(chǎn)生影響的機(jī)制及嗎啡痛覺干預(yù)在細(xì)胞水平的心肌保護(hù)作用機(jī)制。方法 選用1 3天齡的Sprague-Dawley（SD）大

2、鼠建立體外培養(yǎng)新生大鼠心肌細(xì)胞模型，采用免疫細(xì)胞化學(xué)（IC）染色方法對心肌細(xì)胞進(jìn)行鑒定。第一部分實驗選用36孔培養(yǎng)第4 5天呈亞融合狀態(tài)下細(xì)胞隨機(jī)分為9組，每組4孔：對照組、3h組、6h組、12h組、24h組、NE1組(10-6mol·L-1NE )、NE2組(10-7mol·L-1 NE )、NE3組(10-8 mol·L-1 NE)、 M組。采用酶免疫試驗（EIA）技術(shù)、反轉(zhuǎn)錄PCR（RT-PCR）技術(shù)、免疫細(xì)胞化學(xué)（IC）技術(shù)觀察NE誘導(dǎo)及嗎啡預(yù)先給藥后心肌細(xì)胞內(nèi)TNF-表達(dá)的變化。第二部分實驗選用20孔培養(yǎng)第4 5天呈亞融合狀態(tài)下細(xì)胞隨機(jī)分為5組，每組4孔

3、：對照組、NE1組(10-6mol·L-1NE )、NE2組(10-7mol·L-1 NE )、NE3組(10-8 mol·L-1 NE)、M組。分別采用流式細(xì)胞（FCM）技術(shù)、免疫細(xì)胞化學(xué)（IC）技術(shù)觀察NE誘導(dǎo)后及嗎啡預(yù)先給藥后心肌細(xì)胞內(nèi)NF-B活化的情況。結(jié)果 （1）培養(yǎng)細(xì)胞上清液中加入NE進(jìn)行誘導(dǎo)，3h組、6h組、12h組、24h組、10-6 mol·L-1組、 10-7 mol·L-1組 、10-8 mol·L-1NE組（24小時）均可誘導(dǎo)心肌細(xì)胞TNF-的蛋白和核酸表達(dá)水平較對照組顯著升高（p<0.05），在10-8

4、 mol·L-1 NE、24h組達(dá)高峰。（2）培養(yǎng)細(xì)胞上清液中加入NE進(jìn)行誘導(dǎo)，10-6 mol·L-1、 10-7 mol·L-1 、10-8 mol·L-1NE（24小時）均可誘導(dǎo)心肌細(xì)胞NF-B的激活水平較對照組顯著升高（p<0.05），在10-8 mol·L-1 NE組達(dá)高峰。（3）嗎啡組TNF-的蛋白和核酸表達(dá)水平明顯低于10-8 mol·L-1 基金項目：國家自然科學(xué)基金資助項目（30471656）NE組（p<0.05），NF-B的激活水平低于10-8 mol·L-1 NE組（p<0.05）。結(jié)

5、論 培養(yǎng)細(xì)胞上清中加入去甲腎上腺素可誘導(dǎo)體外培養(yǎng)新生大鼠心肌細(xì)胞TNF-表達(dá)、NF-B激活水平的增高。嗎啡預(yù)先給藥可顯著抑制去甲腎上腺素誘導(dǎo)的體外培養(yǎng)新生大鼠心肌細(xì)胞TNF-、NF-B水平的變化，提示機(jī)體應(yīng)激反應(yīng)時神經(jīng)源性反應(yīng)介質(zhì)去甲腎上腺素對心肌細(xì)胞表達(dá)TNF-、NF-B產(chǎn)生影響，嗎啡可能在細(xì)胞水平發(fā)揮心肌保護(hù)作用?！娟P(guān)鍵詞】乳鼠，心肌細(xì)胞培養(yǎng)，TNF-，NF-B，嗎啡，去甲腎上腺素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