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芬太尼和电针对慢性炎性痛大鼠脊髓背角．．．．．．
时间：2010-08-24 09:09:38 来源： 作者：
| The influence of fentanyl and electro-acupuncture on the expression of OFQ mRNA and BDNF mRNA in spinal dorsal horn of chronic inflammatory rats YuanYuan, Yu Yonghao, Wang Guolin Department of Anesthesiology, Tianjin Medical University General Hospital, Tianjin 300052 Objective: We observed the influence of electro-acupuncture (EA), fentanyl and their combined application on the expression of OFQ mRNA and BDNF mRNA in spinal dorsal horn in order to investigate the mechanism of EA analgesia. Methods: In our study, CFA induced unilateral arthritic SD rats were used to establish a chronic inflammatory pain model. 30 rats were divided into 5 groups randomly, 6 rats in each group. They were control group, simple inflammatory group, EA group, fentanyl group and combined application group. We selected the method of hot plate to examine the variation of score of hyperesthesia and RT-PCR to examine the variation of expression of OFQ mRNA and BDNF mRNA in spinal dorsal horn. Results: The expression of OFQ mRNA and BDNF mRNA in spinal dorsal horn of the simple inflammatory group was significantly higher than that of the control group(P<0.05); the score of hyperesthesia of the EA group, fentanyl group and combined application group was significantly higher than that of the simple inflammatory group(P<0.05); the expression of OFQ mRNA and BDNF mRNA in spinal dorsal horn of the EA group, fentanyl group and combined application group was significantly lower than that of the simple inflammatory group(P<0.05); the score of hyperesthesia of the combined application group was significantly higher than that of the EA group(P<0.05); the expression of OFQ mRNA and BDNF mRNA in spinal dorsal horn of the combined application group was significantly lower than that of the EA group(P<0.05). Conclusion: EA can strengthen the analgesia effect of fentanyl . OFQ and BDNF play roles in the process of electro-acupuncture analgesia. Keywords: Electro-acupuncture; Fentanyl; Analgesic mechanism; Orphanin FQ; Brain derived neurotrophic factor 几千年来的实践证明针刺镇痛是一种有效的镇痛方法，在临床广泛应用。但针刺镇痛存在镇痛不全、耐受及个体差异大等缺点。一些实验表明某些药物与电针合用，可以弥补针刺效应的不足。本课题从临床应用出发，选择临床常用镇痛药芬太尼与电针合用，观察其镇痛效果并对其机制进行深入探讨，为临床慢性疼痛治疗寻求新的途径。 1 材料与方法 1．1 实验动物与分组 体重为220±20g健康成年雌性SD大鼠30只，随机分成5组。 实验动物分组及相应的处理如下： A 正常组：蛛网膜下隙置管（第1天），导管内注入生理盐水10μl（第4，5，6，7，8，9，10天） B 单纯致炎组：蛛网膜下隙置管，右踝关节腔内注射50μlCFA致痛（第1天）后，导管内注入生理盐水10μl（第4，5，6，7，8，9，10天） C 电针处理组：蛛网膜下隙置管，右踝关节腔内注射50μlCFA致痛（第1天）后，导管内注入生理盐水10μl（第4，5，6，7，8，9，10天），电针处理（第4，7天） D 芬太尼处理组：蛛网膜下隙置管，右踝关节腔内注射50μlCFA致痛（第1天）后，导管内注入芬太尼1μg（10μl）（第4，5，6，7，8，9，10天） E电针+芬太尼处理组：蛛网膜下隙置管，右踝关节腔内注射50μlCFA致痛（第1天）后，电针处理（第4，7天），导管内注入芬太尼1ug（10μl）（第4，5，6，7，8，9，10天） 第11天，腹腔内注射水合氯醛（0.375ml/100g）将大鼠麻醉，在无菌条件下，取大鼠脊髓L4-6节段，迅速置于-80℃冰箱内。 1．2 检测方法 (1)行为学实验（热板法） 以完成置管、CFA致炎为第1天，从第4天到第10天，每天9：00AM 开始，在室温安静环境中，分别测定大鼠双后肢缩脚潜伏期,连续测定3次，每次间隔10min，求得其平均值作为测定结果。根据痛敏分数=致炎侧缩脚潜伏期―对照侧缩脚潜伏期，计算痛敏分数。 (2) RT-PCR法检测组织中OFQ和BDNFmRNA的表达情况 用TRIZOl抽提脊髓组织总RNA. 反转录:30℃15min, 42℃30min, 5℃5min. PCR: 按照mRNA Selective PCR Ver.1.1试剂盒说明书操作，分两个反应体系分别对300bp β–actin和OFQmRNA及300bp β–actin和BDNFmRNA进行扩增。 300bp β–actin引物序列：上游序列：5’－CAT CTC TTG CTC GAA GTC CA－3’下游序列：5’－ATC ATG TTT GAG ACC TTC AAC A－3’扩增片段为300bp； OFQ引物序列：上游序列：5’－TCC CAC GGC TGC ACC ATG AAA A－3’下游序列：5’－GGC TCC TGG CTA CAC ATT AC－3’扩增片段为576bp； BDNF引物序列：上游序列：5’－ATC GAA GAG CTG CTG GAT GAG－3’下游序列：5’－GTA GTT CGG CAT TGC GAG TTC－3’扩增片段为434bp. 各反应体系的总体积均为50.0ul. 热循环参数： PCR:OFQ90℃ 预变性4min，85℃变性1min，58℃退火1min，72℃延伸1.5min，30个循环后72℃延伸4min. BDNF90℃ 预变性4min，85℃变性1min，55℃退火1min，72℃延伸1.5min，30个循环后72℃延伸4min.RT-PCR扩增产物琼脂糖凝胶电泳。 (3) 统计学处理 本实验结果中所有数据均以平均数±标准差（ ±s）表示，各组间实验结果比较均采用单因素方差分析，应用SPSS10.0统计软件进行统计学处理，以P<0.05为具有统计学差异。 2 结果 2．1 行为学实验： (1)单纯电针组，单纯给药组，针药合用组与单纯致炎组同一天相比，痛敏分数显著性升高（P﹤0.05）。 (2)针药合用组与单纯电针组同一天相比，痛敏分数显著性升高（P﹤0.05）。（见表1） 目前，BDNF在脊髓水平的作用机制还不清楚。大量的实验证实BDNF的作用有一些重要的特征。第一，BDNF对突触传递的易化作用需要功能性的突触后的NMDA受体的参与[5,6]，而且在神经系统的其它部位，包括脊髓的运动神经元[7]和海马[8,9]，BDNF对长时程突触传递的修饰作用也是NMDA受体依赖性的。第二，细胞内钙离子浓度的增加对BDNF发挥作用是必需的。第三，BDNF作用的细胞内转导途径包括磷脂酶C及其下游蛋白激酶C的活性改变。这一途径激活导致了包括NMDA受体在内的一系列膜蛋白的磷酸化以及突触传递效能的持续性改变。有实验证实脊髓trkB受体被激活时能增加NMDA受体开放的时间及反应性，诱导并维持中枢敏化[10]。因此，研究BDNF在痛觉调制中的作用，以及针药合用对其的影响，将会为临床疼痛的治疗探索新的途径。 参考文献 1 Cox BM, Chavkin C, Christie MJ, et al. The IUPHAR compendium of receptor characterization and classification. IUPHAR Media, 2002,23: 321–333. 2 Mogil J,Pasternak G. The molecular and behavioral pharmacology of the orphanin FQ/ nociceptin peptide and receptor family. Pharmacol,2001,53: 381–415. 3 Kabirullah Lutfy, Imran Khaliq. Orphanin FQ/nociceptin blocks cocaine-induced behavioral sensitization in rats.Psychopharmacology, 2002, 164:168-176. 4 Zhu Zhao Wen, Friess Helmut, Wang Li, etc. Brain-derived neurotrophic factor(BDNF) is up regulated and associated with pain in chronic pancreatitis. Digestive diseases and sciences, 2001,46:1633-1639. 5 Bardoni R. Excitatory synaptic transmission in neonatal dorsal horn, NMDA and ATP receptors. News Physiol. Sci,2001,16: 95–100. 6 Bardoni R, Magherini PC, MacDermott AB. Activation of NMDA receptors drives action potentials in superficial dorsal horn from neonatal rats. Neuroreport, 2000,11:1721–1727. 7 Arvanian VL, Mendell LM. Acute modulation of synaptic transmission to motoneurons by BDNF in the neonatal rat spinal cord. Neurosci, 2001,14:1800–1808. 8 Manabe, T. Does BDNF have pre- or postsynaptic targets? Science,2002, 295: 1651–1653. 9 Kovalchuk Y, Hans E, Kafitz K. Postsynaptic induction of BDNF-mediated long-term potentiation. Science,2002, 295: 1729–1734. 10 Garraway, Sandra M, Petruska. BDNF sensitizes the response of lamine Ⅱ neurons to high threshold primary afferent inputs. research report ,2003,18:2467-2476. |
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