摘要
前言
乌拉地尔是α_1肾上腺素能受体阻滞剂，临床上主要用于高血压治疗中，其机理是阻断突触后α_1受体，使外周阻力明显下降，在中枢通过激活5-HT_(1A)受体的作用，降低延脑心血管调节中枢的交感神经反馈而起降压作用。
5-HT_(1A)受体大量存在于脊髓背角，在疼痛的神经传导中起着重要的作用，不但参与上行神经冲动的传导，也参与下行抑制性冲动传导的作用。
FOS免疫反应阳性细胞的检测是确定神经损伤程度和药物镇痛疗效的有力指标。SP作为初级传入神经末稍释放的物质，对伤害性刺激信息的传递和调控方面，扮演着至关重要的角色，检测SP也是判断镇痛药疗效的重要方法。
本实验通过增加可乐定、枢丹及吗啡等影响因素，以细胞外电生理技术和细胞免疫组织生化技术以及透射电镜来观察乌拉地尔在脊髓镇痛中的对比研究，以探讨其作用机制和相互作用。
实验材料与方法
一、实验材料
1．实验动物：Wistar大白鼠(中国医科大学动物实验中心)
2．主要仪器：图像分析仪(Quantime970，Cambridge Ins)
生理多导记录仪(PM6000，NIHON KOHDEN)
恒温水浴箱(上海医疗仪器厂)
透射电镜(1200EX，USA)
3．主要试剂：乌拉地尔注射液(西安制药厂)
可乐定注射液(北京制药厂)
枢丹注射液(宁波天衡制药厂)
吗啡注射液(沈阳第一制药厂)
FOS—抗及试剂盒(博士德生物工程有限公司)
SP—抗及试剂盒(博士德生物工程有限公司)
二、实验方法
实验动物250一320mg Wistar大白鼠以抽签方式随机分为8组，每组6
只。分别是①正常对照组，实验动物不给以任何刺激，直接在麻醉后开胸灌
注，取脊髓标本;②电刺激生理盐水组，实验动物给以长程电刺激后，开胸灌
注，取脊髓标本;③电刺激乌拉地尔组，实验条件同电刺激生理盐水组;④电
刺激可乐定组，实验条件同电刺激生理盐水组;⑤电刺激枢丹组，实验条件
同电刺激生理盐水组;⑥电刺激乌拉地尔和可乐定复合用药组，实验条件同
电刺激生理盐水组;⑦电刺激乌拉地尔和吗啡复合用药组，实验条件同电刺
激生理盐水组;⑧电刺激乌拉地尔和枢丹复合用药组，实验条件同电刺激生
理盐水组。
方法一:20%氨基甲酸乙脂1 .0扩kg腹腔麻醉下，取正中切口，锐、钝
性剥离至棘突，在外科显微镜下暴露L:_4椎板，以咬合钳去除椎板，小心暴
露L，_4段脊髓，以温盐水纱布覆盖。药物以注射器滴于脊髓表面，暴露游离
坐骨神经，安置刺激电极，在L:水平以钨丝微电极(IMn)沿背根内侧垂直
插人脊髓安置记录电极。设定电刺激参数为周期34Ins，波宽100畔，电压
8v的单次电刺激，数据由PM6000生理多导记录仪记录。
所有数据均由又土s表示，运用配对及组间t检验行统计学分析。
方法二:20%氨基甲酸乙脂1 .0扩kg腹腔麻醉下，行气管插管，潘库澳
铁0.Zm扩kg维持肌松。大白鼠在无菌操作下，沿背部中线Tl，一玩段做切
口，剥离至L:椎板，在外科显微镜下用直径1.5~的球形牙钻在L:根板
钻孔至暴露硬脊膜，以25G针头横向划开硬脊膜，用PO川陀X导管(内经0.
28~，外径0.61~)改良后向尾侧置人约10~，骨水泥封闭创口并固定
导管，逐步缝合软组织，皮下游离至颈部出皮肤并加以固定.动物苏醒后半
小时以2%利多卡因10川以行利多卡因试验，若出现双后肢瘫痪、运动丧
失，表明试验结果阳性，收人饲养笼内饲养，留待2一3天内做药物实验。实
验时由留置导管内缓慢注人药液，暴露游离坐骨神经，将一对不锈钢电极环
绕其上进行电刺激，由DUAL sEN一3205电子刺激仪于给药后10分钟行电
刺激，参数为:单脉冲、间期0.lms、频率IHZ、直流电流lrnA.，时程为10分
钟。半小时后大白鼠开胸，主动脉近端夹闭，远端插管，先以0.9%盐水冲
去血液，再以sood含4%多聚甲醛和0.05%戊二醛的0.lmol\L磷酸缓冲
液(PBS.PH7.3)灌注固定.注毕，立即取肠_5段脊健及脊神经节，置人上述
新鲜固定液中后固定，用于组化及电镜研究。
光镜下将切片上神经细胞内的FOS、SP免疫组织化学阳性信号投射到
图象分析仪(Quantime 970，Combridge Inc)的荧光屏，经假彩色处理和灰度
调节后，按设定程序确定每个免疫组织化学阳性信号的神经元，测量并读数
其积分光密度值。测量结果均以又土s表示。
实验结果
在乌拉地尔的剂量相关性实验中，乌拉地尔0.075mg组与盐水组相
比，其复合动作电位的幅度与时程改变均不明显，P>0.05，差异不显著;乌
拉地尔0.巧mg及0.3mg组与盐水组相比P<0.05，差异显著;但乌拉地尔
0.3mg组与乌拉地尔0.巧mg组相比P>0.05，差异不显著;在乌拉地尔的
对比性研究中，乌拉地尔组与盐水对照组相比，其复合动作电位的幅度与时
程均延长，相关性检验P<0.05，差异显著;可乐定组与盐水对照组相比P<
0.01，差异非常显著，与乌拉地尔组相比P<0.05，差异显著;乌拉地尔和可
乐定组与盐水对照组相比P<0.01，差异非常显著，与乌拉地尔组相比P<
0.05，差异显著，与可乐定组相比P>0.05，差异不显著。枢丹组与盐水对
照组、乌拉地尔组相比P>0.05，
Foreword
Urapidil is a anti - adrenergic alphal receptor agent, usually used to cure hypertension. Its machenism is to block post - synaptic adrenergic alpha receptor and result in the obvious descentment of peripheral vessel resistance. On the other hand, by activating the 5 - HT1A receptor in the central nervous system, urapi-dil decrease the level of sympathetic feedback of cardio - vascular regulation center of brain - stem and depress the high blood pressure.
The dorsum of spinal cord involve in amount of 5 - HT1A receptor, which make an important role in nerves conduction of pain,it takes part in not only the conduction of ascending nerve's pulse but also the conduction of descending nerve's inhibition.
Detection of the FOS immunological reaction is a powerful marker to determine how much the injury of nerves is and the extent of analgesia. SP, as the matter released from termination of primary afferent nerve, take a very important role on the aspect of convection and regulation of harmful irritating information. Detection of the SP immunological reaction is an important method judging the analgesic effect.
This experiment is a contrast research of myelo - analgesia of urapidil with the medium of extracellular electrophysiological technique , biochemistry cellular immunity technique and transmitting electron microscope, by adding influence actor of clonidine, ondansatron and morphine. The purpose of this research is to try to find out it's mechanism and interaction.
Materials
1. Experiment animal:
Wistar's rats 250 -320mg(department of experimental animal, Med Uni)
2. Main instrument:
Image analyzer system ( Quantime970, Cambridge Ins) Multi - ways of physiological recording machine ( PM6000, NIHON KO-HDEN)
Stat -thermometer box( Shanghai medical instrument factory) Transmitted electric telescope(1200 EXs, USA)
3. Primarily medicine:
urapidil (Xian pharmaceutical factory) clonidine( Peking pharmaceutical factory) ondanstron( Hengshui pharmaceutical factory) morphine ( Shenyang first pharmaceutical factory) anti - FOS ( Boster creature co. Ltd) anti - SP ( Boster creature co. Ltd)
Methods
The experimental animal, Wistar' s rats are divided into 8 sets by drawing lots:
(1)normal contrasting group,no any irritating factor is given, drawing the materials of spinal dorsum after instillation; (2)saline group, long - term electricity irritation is given, drawing the materials of spinal dorsum after instillation; (3) urapidil group, long - term electricity irritation is given, drawing the materials of spinal dorsum after instillation ; (4) clonidine group, experimental condition as the saline group; (5) ondanstron group, experimental condition as the saline group; (6) urapidil + clonidine group, experimental condition as the saline group; (7) urapidil + morphine group, experimental condition as the saline group; (8) urapidil + ondanstron group, experimental condition as the saline group.
Method A; Exert the rat into intra - abdominal aneasthesia state under 20%Ethyl urethan 1. 0g/kg . Put a Wistar's rat in prone position,take median incision , detach the subcutaneous tissue to spinous process, expose L1-4 Lamina of
vertebra under microscope, do away with the Lamina of vertebra, reveal L, _4 spinal cord and cover it with warm salt water carbasus. By means of injection syringe , Dribbling the experimental durg onto spinal cord surface, Freeing and exposing sciatic nerve, lining plastic slot insulating the subcutaneous tissue, Placing stimulating electrode on the sciatic nerve;on the other hand, inserting the recording electrode vertically inside the dorsal root with the tungsten filament mi-croelectrode on the level of L1. Setting electrostimulating parameter as cycle -phase 34ms,wave - broadth 100 S, electric voltage 8v and recording the data with the PM6000 multi - physiological instrument.
All data indicates with x s, and exerts pair - matching and inside - bundle t test to statistic analysis.
Metho B: Exert the rat into intra - abdominal aneasthesia state under 20%
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