【作者】 李玄英；

【导师】 孙莉；

【作者基本信息】 中国协和医科大学 ， 临床医学， 2008， 博士

【摘要】 吗啡作为阿片类药物的代表,至今仍在缓解中、重度疼痛中占有重要地位,但由于长期应用可导致耐受和成瘾,影响了其在临床中的应用。近年来,针对吗啡耐受机制进行了多方面的研究,建立了在体和体外吗啡耐受模型,并在细胞信号转导方面对吗啡耐受机制的研究取得了一些进展,但由于吗啡耐受机制极其复杂,体外模型获得的结论还难以解释在体模型的一些现象,很多研究尚未得到确切结论,有些结果仍互相矛盾,因此有必要对在体吗啡耐受模型有关受体和基因表达及其调控机制进行深入研究。本研究拟立足于吗啡发挥作用的首要环节—阿片受体来研究吗啡耐受的机制,采用经典吗啡耐受模型、免疫组织化学分析以及荧光定量PCR方法测定μ、δ、κ三种阿片受体在大鼠神经系统的表达,以探讨吗啡耐受情况下三种阿片受体发生变化的主要部位和相互关系,力图为进一步揭示吗啡耐受机制提供实验依据。第一部分吗啡耐受模型的建立选取基础痛阈相近的健康成年雄性Wistar大鼠32只,采用随机数字表将大鼠随机平均分为两组:生理盐水组(NS组,n=16)和硫酸吗啡组(MS组,n=16)。两组大鼠混笼饲养,采用盲法进行给药和痛行为测定。以大鼠热水浴甩尾潜伏期(Tail FlickLateny,TFL)作为痛阈标准。每天两次皮下注射药物(8:00和16:00)并于上午注射前、后30min测定TFL。NS组皮下注射生理盐水1mL/kg,MS组皮下注射吗啡10mg/kg,连续注射7天,第8天早晨各组分别取8只处死,剩余大鼠(分别记为NSN组和MSN组)第8天早晚两次皮下注射纳洛酮0.4mg/kg,第9天亦处死。为了防止烫伤大鼠尾部皮肤,当TFL达到12s时,终止测试并将甩尾潜伏期数值记为12s。观察各组大鼠痛阈变化,以TFL恢复至基础水平作为出现耐受性的标准。结果显示:两组大鼠在观察期体重逐日增加,组间比较体重的增加量无显著性差异。每日注射前测定的痛阈在组间和组内比较无显著性差异,随着吗啡使用次数的增加,MS组用药前痛阈并未发生显著性变化(P＞0.05);MS组在第一天皮下注射吗啡后,痛阈较NS组和基础值明显升高(P＜0.05);随着吗啡用药次数增加,该组大鼠用药后的痛阈逐渐下降,直至第7天用药后痛阈降至基础水平,第9天应用纳络酮后的痛阈与NS组和基础值相比,没有显著性变化。第二部分吗啡耐受大鼠神经系统阿片受体的蛋白表达选取基础痛阈相近的健康成年雄性Wistar大鼠16只,随机平均分为两组:生理盐水组(NS组,n=8)和硫酸吗啡组(MS组,n=8)。给药方法、痛阈测定方法及吗啡耐受判断标准同第一部分,连续注射7天,第8天早晨各组分别取4只处死,剩余大鼠(分别记为NSN组和MSN组)第8天早晚两次皮下注射纳洛酮0.4mg/kg,第9天亦处死。所有大鼠于深麻醉下经灌流固定处死后,取出脑和脊髓组织制作石蜡切片,进行免疫组织化学染色,分析丘脑、下丘脑、蓝斑、海马、中脑导水管周围灰质(PAG)和腰骶段脊髓Ⅰ—Ⅴ板层μ、δ、κ阿片受体的蛋白表达情况。结果显示:吗啡耐受大鼠PAG区域的μ受体和δ受体的蛋白表达明显低于对照组,δ受体在蓝斑区域的表达亦显著低于对照组;κ受体在蓝斑和脊髓背角的表达却显著升高。第三部分吗啡耐受大鼠中枢神经系统阿片受体的mRNA表达选取基础痛阈相近的健康成年雄性Wistar大鼠16只,随机平均分为两组:生理盐水组(NS组,n=8)和硫酸吗啡组(MS组,n=8)。给药方法、痛阈测定方法及吗啡耐受判断标准同第一部分,连续注射7天,第8天早晨各组分别取4只处死,剩余大鼠(分别记为NSN组和MSN组)第8天早晚两次皮下注射纳洛酮0.4mg/kg,第9天亦处死。所有大鼠于深麻醉下断头处死,迅速取出丘脑、下丘脑、蓝斑、海马、PAG、腰骶段脊髓以及背根神经节(DRG),分别提取组织总RNA,并采用荧光定量PCR(real timePCR)方法测定各部位μ、δ、κ阿片受体的mRNA表达情况。结果显示:吗啡耐受大鼠PAG区域的μ受体和δ受体的mRNA表达明显低于对照组,δ受体在蓝斑区域的mRNA表达亦显著低于对照组;κ受体在蓝斑和脊髓背角的mRNA表达均显著升高,但在背根神经节的表达却显著降低。通过本研究,我们得出以下结论:1.吗啡耐受可能是多个阿片受体共同作用的结果;2.吗啡耐受大鼠PAG区域的μ受体呈下调表现;3.吗啡耐受大鼠蓝斑和PAG区域的δ受体呈下调表现;4.吗啡耐受大鼠蓝斑和脊髓背角κ受体呈上调表现,但脊髓背根神经节κ受体明显下调;5.与吗啡耐受相关的部位涉及PAG、蓝斑、脊髓背角以及脊髓背根神经节。更多还原

【Abstract】 Morphine, nowadays, as the most effective opioids, remains a overwhelming drug in the management of moderate or severe pain. But its clinical utility is severely hindered by the development of drug tolerance and physical dependence when administered chronically. Recently, with every effort in the study of opioid tolerance, it has made a great progress in the mechanisms of morphine tolerance. Many kinds of morphine tolerant models were established in vivo or in vitro, and the possible cellular signal trasduction pathways in morphine tolerance were postulated. However, because of the complexity mechanism in morphine tolerance, results from vitro experiments still cannot explain some phenomena in vivo, conclusions from many studies seem inconsistence and need further verification.μ,δ,κare the most common opioid receptors in nervous system which mediate various effects of morphine and were cloned successively. But the role of these three opioid receptors in morphine tolerance and whether the interactions of them are important to morphine tolerance has not been elucidated. This experiment was designed based on the initial component, opioid receptors, to evaluate the changes of three opioid receptors in various regions in nervous system of a classical morphine tolerant rat model by the method of immunohistochemistry and real time PCR, and to provide an experimental evidence to further explore the mechanisms of morphine tolerance.PartⅠEstablishment of Morphine Tolerant Rat Model32 healthy and similar pain threshold adult male Wistar rats were randomly allocated equally to 2 groups. Group NS (n=16), the control group, received a subcutaneous (s.c.) injection of 1ml/kg normal saline (NS) in the nape; group MS (n=16) received a s.c. injection of morphine 10mg/kg. All rats were housed in a same enviroment and received s.c. injection twice a day (8:00 and 16:00) for 7 days. In the morning of the 8th day, 8 control rats and 8 morphine rats were killed. The remaining rats, which named as NSN subgroup and MSN subgroup respectively, received no further morphine injections, bur received 2 injections of naloxone (0.4mg/kg) on the 8th day twice (8:00 and 16:00), and were killed on the 9th day. The tail-flick latency (TFL) as an index of pain threshold was obtained with the rat tail immersed in hot water (52±0.5℃) and was measured in each rat 30 min before and after every morning injection, with the average TFL before the first injection as the baseline value. The cutoff time was 12s to avoid burn of the tail skin. Morphine tolerance was developed, as TFL returned to the baseline level.The rats’ weight which were measured every morning in the observation period increased gradually, and there was no significant changes between two groups. The pain behaviors indicated that TFLs before injection every morning were not significantly different within or between groups. The TFL of Group MS increased significantly than baseline value and Group NS after first morphine injection. But as injection increasing, the average TFL after injection of Group MS shortened gradually and returned to baseline value after the 7th day injection. There were no significant difference in TFL after naloxone injection between groups and baseline value.Part II Protein expression of three opioid receptors in central nervous system of morphine tolerant rats16 healthy and similar pain threshold adult male Wistar rats were randomly allocated equally to 2 groups, named Group NS (n=8) and Group MS (n=8). The drug administration, pain threshold measurement and standard of morphine tolerance are same as Part I . The subcutaneous injection conducted consecutively for 7 days and 4 rats in each group were killed on the 8th morning. The remaining rats, which recorded as NSN subgroup and MSN subgroup, s.c. naloxone 0.4mg/kg twice in the 8th day, and were killed on the 9th morning. All rats were killed with 4℃4% polyformaldehyde perfusion and fixation under deep anesthesia. The brain and spinal cord were anatomised and prepared of paraffin sections for immunohistochemistry (IHC). Analyse protein expression of three opioid receptors in thalamus, hypothalamus, hippocampus, locus ceruleus, periaqueductal gray (PAG) and layer I -V of lumber-sacral spinal cord.The analysis of IHC stain suggested thatμopioid receptor decreased significantly in PAG of morphine tolerant rats.δopioid receptor also had a lower expression in PAG and LC than control group. Butκopioid receptor increased highly in LC and layer I - V of lumber-sacral spinal cord.Part III mRNA expression of three opioid receptors in nervous system of morphine tolerant rats16 healthy and similar pain threshold adult male Wistar rats were randomly allocated equally to 2 groups, named Group NS (n=8) and Group MS (n=8). The drug administration, pain threshold measurement and standard of morphine tolerance are same as Part I . The subcutaneous injection conducted consecutively for 7 days and 4 rats in each group were killed on the 8th morning. The remaining rats, which recorded as NSN subgroup and MSN subgroup, s.c. naloxone 0.4mg/kg twice in the 8th day, and were killed on the 9th morning. All rats were killed decapitation under deep anesthesia. The brain, spinal cord and dorsal root ganglion were anatomised and refrigerated in liquid nitrogen. Extract total RNA of thalamus, hypothalamus, hippocampus, LC, PAG, lumber-sacral spinal cord and dorsal root ganglion. Analyse mRNA expression of three opioid receptors in these regions using real time PCR method.The result of real time PCR indicated that mRNA expression ofμopioid receptor decreased significantly in PAG of morphine tolerant rats.δopioid receptor also had a lower expression in PAG and LC than control group. Thoughκopioid receptor mRNA expression increased highly in LC and lumber-sacral spinal cord, it had a down-regulation in dorsal root ganglion.Based on the results of this study, we may draw the following conclusions:1 .Morphine tolerance may be a result of several opioid receptors coregulation;2.μopioid receptor decreased significantly in PAG of morphine tolerant rats;3.δopioid receptor decreased in PAG and LC of morphine tolerant rats;4.κopioid receptor increased statisticly in LC and lumber-sacral spinal cord, but it had a down-regulation in dorsal root ganglion;5.The involved regions of morphine tolerance should be PAG, LC, spinal cord and dorsal root ganglion.更多还原