中药止痛贴镇痛机制研究
摘要
目的：癌痛作为不同于炎性痛和神经病理性痛的一种特有的疼痛，是临床癌症患者晚期最常并发的疼痛，常见于乳腺癌、肺癌、前列腺癌晚期等，严重影响癌症患者的生活质量，并在一定程度上缩短患者的寿命，因而受到广泛重视。
西医现有治疗措施的局限性及毒副作用，使45%的癌症患者的疼痛不能得到有效控制。中药止痛贴经实验研究及临床应用，均证明其治疗疼痛有确切的疗效，其选用延胡索、红花、马钱子、青风藤、桃仁等药物，具有辛散温通、通络散结、活血化瘀、消肿止痛等功效。但其作用机制尚不清楚，本研究通过建立骨癌痛模型，应用行为学、影像学、病理学和免疫组化技术等方法探讨中药止痛贴对抗癌痛的作用及可能机制。
材料与方法：
1．大鼠骨癌痛模型的建立
方法：选择雌性Wistar大鼠178只，随机分为6组，分别为正常对照组（28只），假手术组、模型组、中药止痛贴组、吗啡组、扶他林组，每组各30只。造模方法：模型组、中药止痛贴组、吗啡组、扶他林组共120只，按照Medhurst等的方法制作胫骨癌痛模型，大鼠麻醉后仰卧位捆绑，从左侧胫骨上部切开皮肤，分离皮下组织直至暴露胫骨，使用牙科手转在胫骨上部打孔，后用10μl微量注射器向骨髓腔内缓慢注入Walker256肿瘤细胞液5μl（肿瘤细胞约5×105），再向其中注入空气1μl，然后以无菌骨腊封堵针孔，局部用庆大霉素冲洗，创口涂用红霉素软膏。假手术组，接种相同容量的PBS液，其余操作同模型组。正常对照组不予任何处理。
检测指标：一般状态观察、疼痛行为学检测（包括术前和术后的自发痛行为观察、机械性触诱发痛及热痛过敏的缩爪阈值检测）、影像学检查（局部Ｘ线摄片）、病理组织学检查（取材局部骨组织切片，进行苏木素-伊红染色）。其中机械性触诱发痛及热痛过敏的缩爪阈值分别在术前和术后每周测定。影像学检测和病理组织学检测可以观察骨质破坏情况。通过以上指标的结果最终判定造模是否成功，以及药物镇痛治疗是否有效。
2．中药止痛贴镇痛机制研究
方法：选择雌性Wistar大鼠178只，随机分为正常对照组、假手术组、模型组、中药止痛贴组、吗啡组、扶他林组，每组30只。造模大鼠左侧胫骨上段骨髓腔注入5μl（肿瘤细胞约5×105）。假手术组：左侧胫骨上段骨髓腔注入等量PBS液；正常对照组：不予任何处置。从术后第7天开始各组按实验方案分别给予中药止痛贴、吗啡、扶他林进行治疗干预。术前测定6组大鼠手术侧后爪机械性触诱发痛及热痛过敏的缩爪阈值，术后1～21d每周（术后7、14、21d）在测定手术侧后肢机械触性诱发痛及热痛过敏的缩爪阈值后，各取至少8只大鼠，使用4％多聚甲醛灌流，取大鼠脊髓L4-6节段，采用免疫组织化学方法检测以下指标在骨癌痛大鼠腰段脊髓中的分布及组织定位：
（1）受体及受体效应酶：NMDAR2B（N-甲基-D-天冬氨酸2B受体）、NK1（神经激肽1受体）、ACⅧ（腺苷酸环化酶Ⅷ）、COX-2（环氧化酶2）。
（2）细胞内信号转导途径：PKA（蛋白激酶A）、pCaMKⅡ（磷酸化钙调蛋白激酶Ⅱ）、pERK（磷酸化细胞外信号调节激酶）、p-p38（磷酸化p38）。
（3）核内转录与蛋白翻译表达：pCREB（磷酸化cAMP反应元件结合蛋白）、Fos蛋白。
（4）骨癌痛特征性改变的检测指标： GFAP（星形胶质细胞胶质源性酸性纤维蛋白）。
3．统计学方法
成组数据用mean±s表示，n代表样本数。组间比较用方差分析(Two-way RepeatedMeasures ANOVA)，同组间不同时间点数据比较用方差分析(One-way ANOVA)，当P＜0.05时认为差别存在统计学意义。各组率的比较用X2检验，两组间比较，经X2分割计算，P＜0.005时认为差别存在统计学意义。应用的统计分析软件为SPSS11.0。
结果：
1．大鼠骨癌痛模型的建立
结果：（1）疼痛行为学观察：①自发痛行为观察：造模后大鼠术侧肢体负重能力随骨癌的进展逐渐降低，至骨癌后期，术侧肢体处于持续的蜷缩状态，病鼠可表现为拖足行走。对侧肢体、正常对照组及假手术组未见此现象出现。正常对照组在整个实验观察期内双侧后肢均未观察到明显缩足行为。假手术组术侧术后3d开始有缩足行为，与基础值相比有差异（P＜0.05），但1周后基本消失。
模型组术侧术后3d开始有缩足行为，与假手术组相比无差异（P＞0.05），7d左右自发缩足次数明显增多，且缩足累计时间在观察期内逐次延长，术后第3、7、10、14d观察到的缩足时间分别为45.94±20.78s，95.32±24.92s，156.39±23.52s，237.72±27.51s，与正常对照组和假手术组比较差异有统计学意义（P＜0.05）。
②机械性触诱发痛（MWT）及热痛过敏（PWT）的缩爪阈值：术后1～6d，模型组大鼠术侧后爪的机械性触诱发痛和热痛过敏缩爪阈值与正常对照组比较无统计学差异（P>0.05）；术后14～21d，模型组大鼠手术侧后爪的机械性触诱发痛和热痛过敏缩爪阈值均较正常对照组降低（P＜0.05）。
（2）影像学检查：Ｘ光片显示，模型组大鼠术侧胫骨出现明显的骨质破坏，其破坏程度与造模时间程正相关。模型组大鼠在术后第7d仅见注射部位骨髓腔内密度稍低，未见明确的骨质破坏区；术后第14d观察到局部骨密度不均，并出现少量骨皮质缺失；术后第21d可见骨质破坏程度明显加重，骨皮质缺失更加明显，甚至发生骨折。假手术组术侧、正常对照组及模型组对侧后肢在术后第7、14、21d均未观察到骨质破坏现象发生。
（3）病理组织学检查：术后第7、14、21d分别取材，将术侧后肢胫骨接种局部做石蜡切片，经HE染色显示，术后7d，胫骨骨髓腔内肿瘤细胞生长活跃，但骨皮质、骨小梁完整，未见明显骨质破坏；14d骨髓腔内大量肿瘤细胞浸润，骨小梁破坏，骨皮质出现缺失、变薄；21d骨皮质严重缺损，髓腔内充满肿瘤细胞，且穿破骨皮质向外生长，侵犯周围肌肉组织。而假手术组、正常对照组骨质完好、组织无破坏。
经多方面、多角度评价，确认骨癌痛造模成功。
2．药物镇痛效用评价
结果：机械性触诱发痛（MWT）和热痛过敏（PWT）缩爪阈值：术后1～6d，造模各组大鼠术侧后爪的MWT和PWT与正常对照组、假手术组和基础值比较无统计学差异（P＞0.05）；术后7d开始，模型组、中药止痛贴组、吗啡组、扶他林组开始下降，与正常对照组比较差异存在统计学意义（p＜0.05），模型组至术后21d持续下降（p＜0.05）。吗啡组和扶他林组术后14d可见增高（p＞0.05），术后21d又出现降低（p＜0.05），而正常对照组和假手术组与基础值比较差异无统计学意义（p＞0.05）。中药止痛贴组于术后14～21d可见MWT和PWT增高（p＜0.05）。正常对照组、假手术组与基础值比较无统计学差异（p＞0.05）。
3．中药止痛贴镇痛机制研究
结果：免疫组化显示，（1）NMDAR2B、NK1、ACⅧ、COX-2、PKA、pCaMKⅡ、pERK、p-p38、pCREB、Fos、GFAP检测，模型组绝大多数在术后14～21d各种指标的免疫反应阳性神经元细胞或阳性细胞比值逐步升高，与正常对照组、假手术组比较存在统计学差异（p＜0.005）。（2）吗啡组在干预早期（7d）可见NMDAR2B、NK1、COX-2、PKA、pCaMKⅡ、pCREB、Fos阳性神经元比值下降，与正常对照组、假手术组比较差异无统计学意义（p＞0.005），但随着用药时间的延长，干预14d又见升高，与正常对照组和假手术组比较差异存在统计学意义（P＜0.005）。ACⅧ在吗啡干预早期即见增高，并持续至干预结束，与正常对照组和假手术组比较差异存在统计学意义（p＜0.005）。pERK、p-p38在吗啡干预早期即见下降，并持续至干预结束，与正常对照组及假手术组比较无统计学差异（p＞0.005）。GFAP在吗啡干预早期即出现升高，直至干预晚期，与正常对照组比较差异有统计学意义（p＜0.005）。（3）扶他林组药物干预7d，可见NMDAR2B、PKA、pCaMKⅡ、pERK、p-p38、pCREB、Fos阳性神经元比值下降，与正常对照组和假手术组比较差异无统计学意义（p＞0.005），但之后出现缓慢升高，与正常对照组、假手术组比较存在统计学意义（p＜0.005）。NK1在扶他林干预早期即见其阳性神经元比值增高，并持续至扶他林干预结束，与正常对照组合假手术组比较差异存在统计学意义（p＜0.005）。ACⅧ和COX-2在扶他林干预早期即见其阳性神经元比值降低，并持续至扶他林干预结束，与正常对照组合假手术组比较差异无统计学意义（p＞0.005）。GFAP在扶他林干预早期即出现升高，直至干预晚期，与正常对照组比较差异有统计学意义（p＜0.005）。（4）中药止痛贴（CM）组于药物干预7d即可见NMDAR2B、ACⅧ、COX-2、PKA、pCREB、Fos、GFAP指标下降，直至干预14d结束，与正常对照组和假手术组比较差异无统计学意义（p＞0.005）。pCaMKⅡ、pERK、p-p38在中药止痛贴干预7d时，其阳性神经元比值仍然较高，与正常对照组和假手术组比较差异存在统计学意义（p＜0.005），干预14d才开始出现下降，与正常对照组和假手术组比较差异无统计学意义（p＞0.005）。NK1则表现为早期下降，后期增高（p＜0.005）。
结论：
1．中药止痛贴一方面通过影响NMDA和NK1受体的活性，进而阻断细胞内信号转导途径，减少核内转录与翻译，另一方面通过抑制星形胶质细胞活化，减少兴奋性神经调质和前炎症介质的释放来抑制中枢敏化的形成，从而发挥镇痛作用。
2．吗啡在干预早期可以抑制NMDA和NK1受体的活性，阻断细胞内信号转导途径，减少核内转录与翻译，后期次作用则由抑制表现为激活。此外，其干预早期即可激活星形胶质细胞，从而导致其镇痛效果逐渐减弱，并出现痛阈降低的现象，表现为痛觉异常和痛觉过敏。
3．非甾体类镇痛消炎药物表现为抑制NMDA受体和COX-2的活性，阻断细胞内信号转导途径，减少核内转录与翻译，此外，其干预早期即可激活星形胶质细胞，因而随着病情的发展，效果逐渐减弱，其镇痛作用有限。
Purpose：Cancer pain is a special pain that differ from the inflammatory andneuropathic pain, it is the most accompanied by pain in advanced cancer patientsclinically, common in advanced breast cancer, lung cancer, prostate cancer, andseriously affecting the quality of life of cancer patients, to some extent inpatients with shortened life span, so it has been paid attention to.
The limitations and side effects exist in Western medicine treatment cannot effectively controlled about45%of the patients with cancer pain. Chinesemedicine analgesic plaster in the treatment of pain has exact curative effect,the selection of Corydalis, safflower, nux vomica, Caulis, walnuts and otherdrugs, role in Xin bulk temperature, Tongluo Sanjie, promoting blood circulationand removing blood stasis, detumescence acetanilide wait. But its mechanism ofaction is unclear, this research established a model of bone cancer pain, andreseach the effects and possible mechanisms of of Chinese medicine analgesicplaster on cancer pain by behavioral science, imaging, pathology andimmunohistochemistry method.
Material and method：
1.Establishment of bone cancer pain model in rat
Material: Wistar rats(female),Walker256,Morphine hydrochloride injection(Northeast Pharmaceutical Group, Shenyang Pharmaceutical Co., Ltd), Voltarencream (Beijing Novartis Pharmaceutical Co., Ltd.), Chinese medicine pain paste(homemade), Bone wax (Nantong Hua Likang Medical Supplies Co., Ltd.)
Methods:178female Wistar rats were randomly divided into6groups,respectively, into normal control group, sham operation group, model group,Chinese medicine analgesic plaster group, morphine group, Votalin group, with 30rats in each group. The method of making model: model group, Chinese medicineanalgesic plaster group, morphine group, Votalin group120in total, Models oftibial cancer pain were made according to the method described by Medhurst etal.Rats were incised the skin from the left side of the upper tibia, separatedthe subcutaneous tissue until exposed tibia, then use the dental hand turn topunch in the upper tibia, using the10μl micro syringe into the marrow cavityand slowly infusing Walker256tumor cell liquid5μl (about5×105tumor cells),then injecting air1μl, and sealing pinholes with aseptic bone wax, flushingthe wound with gentamicin, coated with erythromycin ointment. Sham operationgroup, the same operateing with the same amount of PBS fluid as model group.Not disposed of the Normal control group.
Detection of indexes: the general state observation, pain behavior detection(including preoperative and postoperative spontaneous pain behaviorobservation, mechanical allodynia and thermal hyperalgesia in the paw withdrawalthreshold detection), imaging (partial X ray), histopathological examination(based on local bone tissue slices, for hematoxylin-eosin staining). Themechanical allodynia and thermal hyperalgesia in the paw withdrawal thresholdin the preoperative and postoperative are determined weekly. Imagingexamination and histopathological detection can be observed in bone destruction.The above indicators of outcome can determined the mold whether success, as wellas analgesic drug treatment is effective.
2. Study on the analgesic mechanism of Chinese medicine analgesic plaster
Material: Anti-of NMDAR1(Wuhan Boster Biological Engineering Co.,Ltd.),Anti-of COX2(Wuhan Boster Biological Engineering Co., Ltd), Anti-PKA(Wuhan Boster Biological Engineering Co., Ltd.), Anti-GFAP (Wuhan BosterBiological Engineering Co., Ltd.), Anti-NK2R,(Beijing Boaosen biologicalTechnology Co., Ltd), Anti-ADCY8,(Beijing Boaosen biological Technology Co.,Ltd), Anti-phosphors-CAMKK2,(Beijing Boaosen biological technology Co., Ltd), The Anti-phospho-of ERK1/2(the Beijing Boaosen bio-technology Co., Ltd),.Anti-phospho-MAPK14(Thr180/Tyr182)(the Beijing Boaosen bio-technology Co.,Ltd.), Anti-phospho-CREB-1(Ser133)(the Beijing Boaosen bio-technology Co.,Ltd.), Anti-c-fos in (the Beijing Boaosen bio-technology Co., Ltd.), DABconcentrate (the Beijing Boaosen Biotechnology Co., Ltd), Type SABCimmunohistochemical staining kit (Wuhan Boster Biological Engineering Co.,Ltd)，Universal DAB staining kit (the Beijing Boaosen Biotechnology Co., Ltd),
Methods:178female Wistar rats were randomly divided into6groups,respectively, into normal control group, sham operation group, model group,Chinese medicine analgesic plaster group, morphine group, Votalin group, with24rats in each group. Infusing Walker256tumor cell liquid5μl (about5×105tumor cells). Sham operation group injected the same amount PBS liquid intothe left tibia bone marrow cavity, no intervention in normal control group. Fromthe seventh postoperative day in each group according to the experimental schemewere given Chinese medicine analgesic plaster, morphine, Votalin, applying fora therapeutic intervention. Preoperative determination of six groups of ratswith lateral MWT and PWT, Postoperative1～21d determinated weekly (after7,14,21days). first of all take6～8rats,4%paraformaldehyde was used toperfuse the rat, takeing the rat spinal cord L4-6segment, then usingimmunohistochemistry technology to observe the positive product distributionand change of the following indicator in the rat lumbar spinal cord of bone cancerpain:
(1) receptor and effector enzymes: NMDAR2B (N-methyl-D-aspartic acidreceptor2B), NK1(neurokinin1receptor), AC VIII (adenylate cyclase VIII),COX-2(COX2).
(2) the intracellular signal transduction pathways: PKA (protein kinase A),pCaMK II (the phosphorylation of calmodulin kinase II), pERK (phosphorylationof extracellular signal regulated kinase, p-p38(phosphorylation of p38).
(3) nuclear transcription and Translation: pCREB (phosphorylation of cAMPresponse element binding protein), and Fos protein.
(4) bone cancer pain feature indicator: GFAP (astrocytic glial fibrillaryacidic protein).
3．statistical methods
Group method of data using mean±s, n represents the number of samples.Groups were compared using analysis of variance (Two-way Repeated MeasuresANOVA), the same group at different time data were compared using analysis ofcovariance (One-way ANOVA), when P<0.05think difference of statisticalsignificance. Each rate compared with X2testing, comparison between two groups,the X2segmentation computing, P<0.005, think difference of statisticalsignificance. Application of statistical analysis software is SPSS11.0.
Results：
1．Establishment of the rat model of bone cancer pain
Results:(1) pain behavior observation:①spontaneous pain behaviorobservation: the model of rats after the corresponding-side limb weight-bearingability with bone cancer progressed gradually reduced, to bone cancer laterperiod, the corresponding-side limb is in persistent crouch state, disease ratcan be manifested as dragging feet. On the side of the body of sham operationgroup, normal control group showed no this phenomenon.
Normal control group during the period of whole experimental observation,the hind limbs were not observed flinching behavior. In Sham operation group,the side of postoperation started flinching behavior only after postoperation3d, comparing with baseline differences (P<0.05), but after1weeks, thedifference disappeared.
Model group lateral postoperative3d started flinching behavior, comparedwith sham operation group had no difference (P>0.05),7d flinch flinchingfrequency increased significantly, and total time in the observation period gradually extend, after3,7,10,14d observed in the paw withdrawal timerespectively45.94±20.78s,95.32±24.92s,156.39±23.52s,237.72±27.51s,compared with the normal control group and sham operation groups had significantdifference (P<0.05).
②Mechanical allodynia (MWT) and thermal hyperalgesia (PWT) of the pawwithdrawal threshold: after1～6d, the rats of model group in side clawmechanical allodynia and thermal hyperalgesia paw withdrawal threshold comparedwith the normal control group showed no significant difference (P>0.05); after14～21d the rats in the model group, operation and claw mechanical allodyniaand thermal hyperalgesia paw withdrawal threshold was lower than that in normalgroup (P<0.05).
(2)X-ray shows, model group rats with lateral tibial marked bone destruction,the damage degree and moulding process is related to time. The rats in the modelgroup in the postoperative7d only injection site intra bone marrow density isslightly low, no clear area of bone destruction; after14d observed local bonedensity is uneven, and the emergence of a small number of cortical bone loss;after the21d visible bone destruction degree aggravate, cortical bone loss morevisible, even fracture. Sham operation group, normal control group and modelgroup in the contralateral hind at postoperative7,14,21d were not observed inbone destruction phenomenon.
(3)Histopathological examination after7,14,21d respectively, material,operation hind tibiae vaccination side partial paraffin sections, stained withHE,7d after operation, the tibial bone marrow cavity tumor cell growth and active,but the cortical bone, intact trabecular bone, no obvious damage of bone marrowcavity;14d large number of tumor cells infiltration, trabecular bone destruction,bone cortex is missing, thinning;21d cortical bone defect, medullary cavityfilled with tumor cells, and perforation of cortical bone outward growth,invasion of the surrounding muscle tissue. While the Sham operation group, normal control group with no intact tissue damage.
By the multifaceted, multi-angle evaluation, we can confirm the bone cancerpain model is success.2．The evaluation of drug analgesia effectiveness
Results: mechanical allodynia(MWT) and thermal hyperalgesia (PWT) pawwithdrawal threshold: after1～6d, model rats with lateral claw MWT and PWT innormal control group, sham operation group and basic value comparison showedno significant difference (P>0.05); from postoperative7d, model group, Chinesemedicine analgesic plaster group, morphine group, Votalin group began todecrease, as compared with the normal control group differences werestatistically significant (P<0.05), model group21d after operation (P<0.05)continued to decline. The morphine group and Votalin group after14d visibleincreased (P>0.05),21d after surgery and decreased (P<0.05), and the normalcontrol group and sham operation group compared with baseline differences werenot statistically significant (P>0.05). Chinese medicine analgesic plastergroup after14～21d became MWT and PWT increased (P <0.05). Normal control group,sham operation group and the basic values of no significant difference (P>0.05)..3. Study on the analgesic mechanism of Chinese medicine analgesic plaster
Results: immunohistochemistry showed,(1) NMDAR2B, NK1, AC Ⅷ, COX-2,PKA,pCaMK Ⅱ, pERK, p-p38, pCREB, Fos, GFAP detection, model group most after14～21d various indicators of immune reaction positive neurons or positive cell ratiogradually increased, and the normal control group sham operation group, therewas significant difference (P<0.005).(2) the morphine group in earlyintervention (7d), NK1, COX-2, NMDAR2B PKA, pCaMK Ⅱ, pCREB, Fos positiveneurons were decreased, and the normal control group, sham operation groupcompare difference not to have statistical significance (P>0.005), but withthe long time use, the intervention of14d again escalated, and normal controlgroup and sham operation group had statistical significance (P<0.005). AC VIII in morphine intervention early to see increased, and continues through the endof the intervention, and the normal control group and sham operation group hadstatistical significance (P<0.005). PERK, p-p38in morphine intervention earlysign of decline, and continues through the intervention ended, and the normalcontrol group and sham operation group, no significant difference (P>0.005).GFAP in morphine intervention occurs early intervention increased, until late,compared with the control group the differences were statistically significant(P<0.005).(3) Votalin group drug intervention in7d, NMDAR2B, PKA, visible pCaMKⅡ, pERK, p-p38, pCREB, Fos positive neurons were decreased, and the normalcontrol group and sham operation group compare difference not to have statisticalsignificance (P>0.005), but after a slow increase, and the normal control group,sham operation group compared with the presence of statistical significance(P<0.005). NK1in applying intervention in the early to see its positive neuronsincreased ratio, and continues through the Votalin intervention ended, andnormal control fake operation group had statistical significance (P<0.005). ACⅧand COX-2in applying intervention early to see its positive neurons wereincreased, and continues through the Votalin intervention ended, and normalcontrol combination of sham operation group compare difference not to havestatistical significance (P>0.005). GFAP in applying intervention occurs earlyintervention increased, until late, compared with the control group thedifferences were statistically significant (P<0.005).(4) Chinese medicineanalgesic plaster group (CM) on drug intervention is visible on NMDAR2B,7d,COX-2, PKA, AC ⅧpCREB, Fos, GFAP index decreased until the end of14d,intervention, and normal control group and sham operation group comparedifference not to have statistical significance (P>0.005). PCaMK Ⅱ, pERK,p-p38in pain medicine paster on7d, its positive neurons ratio remains high,and the normal control group and sham operation group had statisticalsignificance (P<0.005), the intervention of14d began to decline, and the normal control group and sham operation group difference was not statisticallysignificant (P>0.005). NK1manifestations of early decline, later increased(P<0.005).
Conclusion：
1．On one hand, traditional Chinese analgesic plaster can influence the effectof NMDA and NK1receptor activity, thereby blocking intracellular signaltransduction pathways, reduced transcription and translation, on the other handby inhibiting the activation of astrocytes, a decrease in excitatoryneuromodulator and pro-inflammatory mediators are released to inhibition ofcentral sensitization formation, thus its analgesic effect.
2．During early intervention of morphine, it can suppress NMDA and NK1receptoractivity, blocking intracellular signal transduction pathways, reducedtranscription and translation, than follow its inhibiting role is activation.In addition, the early intervention can activated astrocytes, which leads toits analgesic effect gradually weakened, and the emergence of the phenomenonof reduced pain threshold, manifested as allodynia and hyperalgesia.
3．Nonsteroidal anti-inflammatory and analgesic drug showed the inhibitory roleon NMDA receptors and COX-2activity, blocking intracellular signal transductionpathways, reduced transcription and translation, moreover, its intervention inearly activated astrocytes, and as the disease progresses, the effect decreasedgradually, and the analgesic effect is limited.
西医现有治疗措施的局限性及毒副作用，使45%的癌症患者的疼痛不能得到有效控制。中药止痛贴经实验研究及临床应用，均证明其治疗疼痛有确切的疗效，其选用延胡索、红花、马钱子、青风藤、桃仁等药物，具有辛散温通、通络散结、活血化瘀、消肿止痛等功效。但其作用机制尚不清楚，本研究通过建立骨癌痛模型，应用行为学、影像学、病理学和免疫组化技术等方法探讨中药止痛贴对抗癌痛的作用及可能机制。
材料与方法：
1．大鼠骨癌痛模型的建立
方法：选择雌性Wistar大鼠178只，随机分为6组，分别为正常对照组（28只），假手术组、模型组、中药止痛贴组、吗啡组、扶他林组，每组各30只。造模方法：模型组、中药止痛贴组、吗啡组、扶他林组共120只，按照Medhurst等的方法制作胫骨癌痛模型，大鼠麻醉后仰卧位捆绑，从左侧胫骨上部切开皮肤，分离皮下组织直至暴露胫骨，使用牙科手转在胫骨上部打孔，后用10μl微量注射器向骨髓腔内缓慢注入Walker256肿瘤细胞液5μl（肿瘤细胞约5×105），再向其中注入空气1μl，然后以无菌骨腊封堵针孔，局部用庆大霉素冲洗，创口涂用红霉素软膏。假手术组，接种相同容量的PBS液，其余操作同模型组。正常对照组不予任何处理。
检测指标：一般状态观察、疼痛行为学检测（包括术前和术后的自发痛行为观察、机械性触诱发痛及热痛过敏的缩爪阈值检测）、影像学检查（局部Ｘ线摄片）、病理组织学检查（取材局部骨组织切片，进行苏木素-伊红染色）。其中机械性触诱发痛及热痛过敏的缩爪阈值分别在术前和术后每周测定。影像学检测和病理组织学检测可以观察骨质破坏情况。通过以上指标的结果最终判定造模是否成功，以及药物镇痛治疗是否有效。
2．中药止痛贴镇痛机制研究
方法：选择雌性Wistar大鼠178只，随机分为正常对照组、假手术组、模型组、中药止痛贴组、吗啡组、扶他林组，每组30只。造模大鼠左侧胫骨上段骨髓腔注入5μl（肿瘤细胞约5×105）。假手术组：左侧胫骨上段骨髓腔注入等量PBS液；正常对照组：不予任何处置。从术后第7天开始各组按实验方案分别给予中药止痛贴、吗啡、扶他林进行治疗干预。术前测定6组大鼠手术侧后爪机械性触诱发痛及热痛过敏的缩爪阈值，术后1～21d每周（术后7、14、21d）在测定手术侧后肢机械触性诱发痛及热痛过敏的缩爪阈值后，各取至少8只大鼠，使用4％多聚甲醛灌流，取大鼠脊髓L4-6节段，采用免疫组织化学方法检测以下指标在骨癌痛大鼠腰段脊髓中的分布及组织定位：
（1）受体及受体效应酶：NMDAR2B（N-甲基-D-天冬氨酸2B受体）、NK1（神经激肽1受体）、ACⅧ（腺苷酸环化酶Ⅷ）、COX-2（环氧化酶2）。
（2）细胞内信号转导途径：PKA（蛋白激酶A）、pCaMKⅡ（磷酸化钙调蛋白激酶Ⅱ）、pERK（磷酸化细胞外信号调节激酶）、p-p38（磷酸化p38）。
（3）核内转录与蛋白翻译表达：pCREB（磷酸化cAMP反应元件结合蛋白）、Fos蛋白。
（4）骨癌痛特征性改变的检测指标： GFAP（星形胶质细胞胶质源性酸性纤维蛋白）。
3．统计学方法
成组数据用mean±s表示，n代表样本数。组间比较用方差分析(Two-way RepeatedMeasures ANOVA)，同组间不同时间点数据比较用方差分析(One-way ANOVA)，当P＜0.05时认为差别存在统计学意义。各组率的比较用X2检验，两组间比较，经X2分割计算，P＜0.005时认为差别存在统计学意义。应用的统计分析软件为SPSS11.0。
结果：
1．大鼠骨癌痛模型的建立
结果：（1）疼痛行为学观察：①自发痛行为观察：造模后大鼠术侧肢体负重能力随骨癌的进展逐渐降低，至骨癌后期，术侧肢体处于持续的蜷缩状态，病鼠可表现为拖足行走。对侧肢体、正常对照组及假手术组未见此现象出现。正常对照组在整个实验观察期内双侧后肢均未观察到明显缩足行为。假手术组术侧术后3d开始有缩足行为，与基础值相比有差异（P＜0.05），但1周后基本消失。
模型组术侧术后3d开始有缩足行为，与假手术组相比无差异（P＞0.05），7d左右自发缩足次数明显增多，且缩足累计时间在观察期内逐次延长，术后第3、7、10、14d观察到的缩足时间分别为45.94±20.78s，95.32±24.92s，156.39±23.52s，237.72±27.51s，与正常对照组和假手术组比较差异有统计学意义（P＜0.05）。
②机械性触诱发痛（MWT）及热痛过敏（PWT）的缩爪阈值：术后1～6d，模型组大鼠术侧后爪的机械性触诱发痛和热痛过敏缩爪阈值与正常对照组比较无统计学差异（P>0.05）；术后14～21d，模型组大鼠手术侧后爪的机械性触诱发痛和热痛过敏缩爪阈值均较正常对照组降低（P＜0.05）。
（2）影像学检查：Ｘ光片显示，模型组大鼠术侧胫骨出现明显的骨质破坏，其破坏程度与造模时间程正相关。模型组大鼠在术后第7d仅见注射部位骨髓腔内密度稍低，未见明确的骨质破坏区；术后第14d观察到局部骨密度不均，并出现少量骨皮质缺失；术后第21d可见骨质破坏程度明显加重，骨皮质缺失更加明显，甚至发生骨折。假手术组术侧、正常对照组及模型组对侧后肢在术后第7、14、21d均未观察到骨质破坏现象发生。
（3）病理组织学检查：术后第7、14、21d分别取材，将术侧后肢胫骨接种局部做石蜡切片，经HE染色显示，术后7d，胫骨骨髓腔内肿瘤细胞生长活跃，但骨皮质、骨小梁完整，未见明显骨质破坏；14d骨髓腔内大量肿瘤细胞浸润，骨小梁破坏，骨皮质出现缺失、变薄；21d骨皮质严重缺损，髓腔内充满肿瘤细胞，且穿破骨皮质向外生长，侵犯周围肌肉组织。而假手术组、正常对照组骨质完好、组织无破坏。
经多方面、多角度评价，确认骨癌痛造模成功。
2．药物镇痛效用评价
结果：机械性触诱发痛（MWT）和热痛过敏（PWT）缩爪阈值：术后1～6d，造模各组大鼠术侧后爪的MWT和PWT与正常对照组、假手术组和基础值比较无统计学差异（P＞0.05）；术后7d开始，模型组、中药止痛贴组、吗啡组、扶他林组开始下降，与正常对照组比较差异存在统计学意义（p＜0.05），模型组至术后21d持续下降（p＜0.05）。吗啡组和扶他林组术后14d可见增高（p＞0.05），术后21d又出现降低（p＜0.05），而正常对照组和假手术组与基础值比较差异无统计学意义（p＞0.05）。中药止痛贴组于术后14～21d可见MWT和PWT增高（p＜0.05）。正常对照组、假手术组与基础值比较无统计学差异（p＞0.05）。
3．中药止痛贴镇痛机制研究
结果：免疫组化显示，（1）NMDAR2B、NK1、ACⅧ、COX-2、PKA、pCaMKⅡ、pERK、p-p38、pCREB、Fos、GFAP检测，模型组绝大多数在术后14～21d各种指标的免疫反应阳性神经元细胞或阳性细胞比值逐步升高，与正常对照组、假手术组比较存在统计学差异（p＜0.005）。（2）吗啡组在干预早期（7d）可见NMDAR2B、NK1、COX-2、PKA、pCaMKⅡ、pCREB、Fos阳性神经元比值下降，与正常对照组、假手术组比较差异无统计学意义（p＞0.005），但随着用药时间的延长，干预14d又见升高，与正常对照组和假手术组比较差异存在统计学意义（P＜0.005）。ACⅧ在吗啡干预早期即见增高，并持续至干预结束，与正常对照组和假手术组比较差异存在统计学意义（p＜0.005）。pERK、p-p38在吗啡干预早期即见下降，并持续至干预结束，与正常对照组及假手术组比较无统计学差异（p＞0.005）。GFAP在吗啡干预早期即出现升高，直至干预晚期，与正常对照组比较差异有统计学意义（p＜0.005）。（3）扶他林组药物干预7d，可见NMDAR2B、PKA、pCaMKⅡ、pERK、p-p38、pCREB、Fos阳性神经元比值下降，与正常对照组和假手术组比较差异无统计学意义（p＞0.005），但之后出现缓慢升高，与正常对照组、假手术组比较存在统计学意义（p＜0.005）。NK1在扶他林干预早期即见其阳性神经元比值增高，并持续至扶他林干预结束，与正常对照组合假手术组比较差异存在统计学意义（p＜0.005）。ACⅧ和COX-2在扶他林干预早期即见其阳性神经元比值降低，并持续至扶他林干预结束，与正常对照组合假手术组比较差异无统计学意义（p＞0.005）。GFAP在扶他林干预早期即出现升高，直至干预晚期，与正常对照组比较差异有统计学意义（p＜0.005）。（4）中药止痛贴（CM）组于药物干预7d即可见NMDAR2B、ACⅧ、COX-2、PKA、pCREB、Fos、GFAP指标下降，直至干预14d结束，与正常对照组和假手术组比较差异无统计学意义（p＞0.005）。pCaMKⅡ、pERK、p-p38在中药止痛贴干预7d时，其阳性神经元比值仍然较高，与正常对照组和假手术组比较差异存在统计学意义（p＜0.005），干预14d才开始出现下降，与正常对照组和假手术组比较差异无统计学意义（p＞0.005）。NK1则表现为早期下降，后期增高（p＜0.005）。
结论：
1．中药止痛贴一方面通过影响NMDA和NK1受体的活性，进而阻断细胞内信号转导途径，减少核内转录与翻译，另一方面通过抑制星形胶质细胞活化，减少兴奋性神经调质和前炎症介质的释放来抑制中枢敏化的形成，从而发挥镇痛作用。
2．吗啡在干预早期可以抑制NMDA和NK1受体的活性，阻断细胞内信号转导途径，减少核内转录与翻译，后期次作用则由抑制表现为激活。此外，其干预早期即可激活星形胶质细胞，从而导致其镇痛效果逐渐减弱，并出现痛阈降低的现象，表现为痛觉异常和痛觉过敏。
3．非甾体类镇痛消炎药物表现为抑制NMDA受体和COX-2的活性，阻断细胞内信号转导途径，减少核内转录与翻译，此外，其干预早期即可激活星形胶质细胞，因而随着病情的发展，效果逐渐减弱，其镇痛作用有限。
Purpose：Cancer pain is a special pain that differ from the inflammatory andneuropathic pain, it is the most accompanied by pain in advanced cancer patientsclinically, common in advanced breast cancer, lung cancer, prostate cancer, andseriously affecting the quality of life of cancer patients, to some extent inpatients with shortened life span, so it has been paid attention to.
The limitations and side effects exist in Western medicine treatment cannot effectively controlled about45%of the patients with cancer pain. Chinesemedicine analgesic plaster in the treatment of pain has exact curative effect,the selection of Corydalis, safflower, nux vomica, Caulis, walnuts and otherdrugs, role in Xin bulk temperature, Tongluo Sanjie, promoting blood circulationand removing blood stasis, detumescence acetanilide wait. But its mechanism ofaction is unclear, this research established a model of bone cancer pain, andreseach the effects and possible mechanisms of of Chinese medicine analgesicplaster on cancer pain by behavioral science, imaging, pathology andimmunohistochemistry method.
Material and method：
1.Establishment of bone cancer pain model in rat
Material: Wistar rats(female),Walker256,Morphine hydrochloride injection(Northeast Pharmaceutical Group, Shenyang Pharmaceutical Co., Ltd), Voltarencream (Beijing Novartis Pharmaceutical Co., Ltd.), Chinese medicine pain paste(homemade), Bone wax (Nantong Hua Likang Medical Supplies Co., Ltd.)
Methods:178female Wistar rats were randomly divided into6groups,respectively, into normal control group, sham operation group, model group,Chinese medicine analgesic plaster group, morphine group, Votalin group, with 30rats in each group. The method of making model: model group, Chinese medicineanalgesic plaster group, morphine group, Votalin group120in total, Models oftibial cancer pain were made according to the method described by Medhurst etal.Rats were incised the skin from the left side of the upper tibia, separatedthe subcutaneous tissue until exposed tibia, then use the dental hand turn topunch in the upper tibia, using the10μl micro syringe into the marrow cavityand slowly infusing Walker256tumor cell liquid5μl (about5×105tumor cells),then injecting air1μl, and sealing pinholes with aseptic bone wax, flushingthe wound with gentamicin, coated with erythromycin ointment. Sham operationgroup, the same operateing with the same amount of PBS fluid as model group.Not disposed of the Normal control group.
Detection of indexes: the general state observation, pain behavior detection(including preoperative and postoperative spontaneous pain behaviorobservation, mechanical allodynia and thermal hyperalgesia in the paw withdrawalthreshold detection), imaging (partial X ray), histopathological examination(based on local bone tissue slices, for hematoxylin-eosin staining). Themechanical allodynia and thermal hyperalgesia in the paw withdrawal thresholdin the preoperative and postoperative are determined weekly. Imagingexamination and histopathological detection can be observed in bone destruction.The above indicators of outcome can determined the mold whether success, as wellas analgesic drug treatment is effective.
2. Study on the analgesic mechanism of Chinese medicine analgesic plaster
Material: Anti-of NMDAR1(Wuhan Boster Biological Engineering Co.,Ltd.),Anti-of COX2(Wuhan Boster Biological Engineering Co., Ltd), Anti-PKA(Wuhan Boster Biological Engineering Co., Ltd.), Anti-GFAP (Wuhan BosterBiological Engineering Co., Ltd.), Anti-NK2R,(Beijing Boaosen biologicalTechnology Co., Ltd), Anti-ADCY8,(Beijing Boaosen biological Technology Co.,Ltd), Anti-phosphors-CAMKK2,(Beijing Boaosen biological technology Co., Ltd), The Anti-phospho-of ERK1/2(the Beijing Boaosen bio-technology Co., Ltd),.Anti-phospho-MAPK14(Thr180/Tyr182)(the Beijing Boaosen bio-technology Co.,Ltd.), Anti-phospho-CREB-1(Ser133)(the Beijing Boaosen bio-technology Co.,Ltd.), Anti-c-fos in (the Beijing Boaosen bio-technology Co., Ltd.), DABconcentrate (the Beijing Boaosen Biotechnology Co., Ltd), Type SABCimmunohistochemical staining kit (Wuhan Boster Biological Engineering Co.,Ltd)，Universal DAB staining kit (the Beijing Boaosen Biotechnology Co., Ltd),
Methods:178female Wistar rats were randomly divided into6groups,respectively, into normal control group, sham operation group, model group,Chinese medicine analgesic plaster group, morphine group, Votalin group, with24rats in each group. Infusing Walker256tumor cell liquid5μl (about5×105tumor cells). Sham operation group injected the same amount PBS liquid intothe left tibia bone marrow cavity, no intervention in normal control group. Fromthe seventh postoperative day in each group according to the experimental schemewere given Chinese medicine analgesic plaster, morphine, Votalin, applying fora therapeutic intervention. Preoperative determination of six groups of ratswith lateral MWT and PWT, Postoperative1～21d determinated weekly (after7,14,21days). first of all take6～8rats,4%paraformaldehyde was used toperfuse the rat, takeing the rat spinal cord L4-6segment, then usingimmunohistochemistry technology to observe the positive product distributionand change of the following indicator in the rat lumbar spinal cord of bone cancerpain:
(1) receptor and effector enzymes: NMDAR2B (N-methyl-D-aspartic acidreceptor2B), NK1(neurokinin1receptor), AC VIII (adenylate cyclase VIII),COX-2(COX2).
(2) the intracellular signal transduction pathways: PKA (protein kinase A),pCaMK II (the phosphorylation of calmodulin kinase II), pERK (phosphorylationof extracellular signal regulated kinase, p-p38(phosphorylation of p38).
(3) nuclear transcription and Translation: pCREB (phosphorylation of cAMPresponse element binding protein), and Fos protein.
(4) bone cancer pain feature indicator: GFAP (astrocytic glial fibrillaryacidic protein).
3．statistical methods
Group method of data using mean±s, n represents the number of samples.Groups were compared using analysis of variance (Two-way Repeated MeasuresANOVA), the same group at different time data were compared using analysis ofcovariance (One-way ANOVA), when P<0.05think difference of statisticalsignificance. Each rate compared with X2testing, comparison between two groups,the X2segmentation computing, P<0.005, think difference of statisticalsignificance. Application of statistical analysis software is SPSS11.0.
Results：
1．Establishment of the rat model of bone cancer pain
Results:(1) pain behavior observation:①spontaneous pain behaviorobservation: the model of rats after the corresponding-side limb weight-bearingability with bone cancer progressed gradually reduced, to bone cancer laterperiod, the corresponding-side limb is in persistent crouch state, disease ratcan be manifested as dragging feet. On the side of the body of sham operationgroup, normal control group showed no this phenomenon.
Normal control group during the period of whole experimental observation,the hind limbs were not observed flinching behavior. In Sham operation group,the side of postoperation started flinching behavior only after postoperation3d, comparing with baseline differences (P<0.05), but after1weeks, thedifference disappeared.
Model group lateral postoperative3d started flinching behavior, comparedwith sham operation group had no difference (P>0.05),7d flinch flinchingfrequency increased significantly, and total time in the observation period gradually extend, after3,7,10,14d observed in the paw withdrawal timerespectively45.94±20.78s,95.32±24.92s,156.39±23.52s,237.72±27.51s,compared with the normal control group and sham operation groups had significantdifference (P<0.05).
②Mechanical allodynia (MWT) and thermal hyperalgesia (PWT) of the pawwithdrawal threshold: after1～6d, the rats of model group in side clawmechanical allodynia and thermal hyperalgesia paw withdrawal threshold comparedwith the normal control group showed no significant difference (P>0.05); after14～21d the rats in the model group, operation and claw mechanical allodyniaand thermal hyperalgesia paw withdrawal threshold was lower than that in normalgroup (P<0.05).
(2)X-ray shows, model group rats with lateral tibial marked bone destruction,the damage degree and moulding process is related to time. The rats in the modelgroup in the postoperative7d only injection site intra bone marrow density isslightly low, no clear area of bone destruction; after14d observed local bonedensity is uneven, and the emergence of a small number of cortical bone loss;after the21d visible bone destruction degree aggravate, cortical bone loss morevisible, even fracture. Sham operation group, normal control group and modelgroup in the contralateral hind at postoperative7,14,21d were not observed inbone destruction phenomenon.
(3)Histopathological examination after7,14,21d respectively, material,operation hind tibiae vaccination side partial paraffin sections, stained withHE,7d after operation, the tibial bone marrow cavity tumor cell growth and active,but the cortical bone, intact trabecular bone, no obvious damage of bone marrowcavity;14d large number of tumor cells infiltration, trabecular bone destruction,bone cortex is missing, thinning;21d cortical bone defect, medullary cavityfilled with tumor cells, and perforation of cortical bone outward growth,invasion of the surrounding muscle tissue. While the Sham operation group, normal control group with no intact tissue damage.
By the multifaceted, multi-angle evaluation, we can confirm the bone cancerpain model is success.2．The evaluation of drug analgesia effectiveness
Results: mechanical allodynia(MWT) and thermal hyperalgesia (PWT) pawwithdrawal threshold: after1～6d, model rats with lateral claw MWT and PWT innormal control group, sham operation group and basic value comparison showedno significant difference (P>0.05); from postoperative7d, model group, Chinesemedicine analgesic plaster group, morphine group, Votalin group began todecrease, as compared with the normal control group differences werestatistically significant (P<0.05), model group21d after operation (P<0.05)continued to decline. The morphine group and Votalin group after14d visibleincreased (P>0.05),21d after surgery and decreased (P<0.05), and the normalcontrol group and sham operation group compared with baseline differences werenot statistically significant (P>0.05). Chinese medicine analgesic plastergroup after14～21d became MWT and PWT increased (P <0.05). Normal control group,sham operation group and the basic values of no significant difference (P>0.05)..3. Study on the analgesic mechanism of Chinese medicine analgesic plaster
Results: immunohistochemistry showed,(1) NMDAR2B, NK1, AC Ⅷ, COX-2,PKA,pCaMK Ⅱ, pERK, p-p38, pCREB, Fos, GFAP detection, model group most after14～21d various indicators of immune reaction positive neurons or positive cell ratiogradually increased, and the normal control group sham operation group, therewas significant difference (P<0.005).(2) the morphine group in earlyintervention (7d), NK1, COX-2, NMDAR2B PKA, pCaMK Ⅱ, pCREB, Fos positiveneurons were decreased, and the normal control group, sham operation groupcompare difference not to have statistical significance (P>0.005), but withthe long time use, the intervention of14d again escalated, and normal controlgroup and sham operation group had statistical significance (P<0.005). AC VIII in morphine intervention early to see increased, and continues through the endof the intervention, and the normal control group and sham operation group hadstatistical significance (P<0.005). PERK, p-p38in morphine intervention earlysign of decline, and continues through the intervention ended, and the normalcontrol group and sham operation group, no significant difference (P>0.005).GFAP in morphine intervention occurs early intervention increased, until late,compared with the control group the differences were statistically significant(P<0.005).(3) Votalin group drug intervention in7d, NMDAR2B, PKA, visible pCaMKⅡ, pERK, p-p38, pCREB, Fos positive neurons were decreased, and the normalcontrol group and sham operation group compare difference not to have statisticalsignificance (P>0.005), but after a slow increase, and the normal control group,sham operation group compared with the presence of statistical significance(P<0.005). NK1in applying intervention in the early to see its positive neuronsincreased ratio, and continues through the Votalin intervention ended, andnormal control fake operation group had statistical significance (P<0.005). ACⅧand COX-2in applying intervention early to see its positive neurons wereincreased, and continues through the Votalin intervention ended, and normalcontrol combination of sham operation group compare difference not to havestatistical significance (P>0.005). GFAP in applying intervention occurs earlyintervention increased, until late, compared with the control group thedifferences were statistically significant (P<0.005).(4) Chinese medicineanalgesic plaster group (CM) on drug intervention is visible on NMDAR2B,7d,COX-2, PKA, AC ⅧpCREB, Fos, GFAP index decreased until the end of14d,intervention, and normal control group and sham operation group comparedifference not to have statistical significance (P>0.005). PCaMK Ⅱ, pERK,p-p38in pain medicine paster on7d, its positive neurons ratio remains high,and the normal control group and sham operation group had statisticalsignificance (P<0.005), the intervention of14d began to decline, and the normal control group and sham operation group difference was not statisticallysignificant (P>0.005). NK1manifestations of early decline, later increased(P<0.005).
Conclusion：
1．On one hand, traditional Chinese analgesic plaster can influence the effectof NMDA and NK1receptor activity, thereby blocking intracellular signaltransduction pathways, reduced transcription and translation, on the other handby inhibiting the activation of astrocytes, a decrease in excitatoryneuromodulator and pro-inflammatory mediators are released to inhibition ofcentral sensitization formation, thus its analgesic effect.
2．During early intervention of morphine, it can suppress NMDA and NK1receptoractivity, blocking intracellular signal transduction pathways, reducedtranscription and translation, than follow its inhibiting role is activation.In addition, the early intervention can activated astrocytes, which leads toits analgesic effect gradually weakened, and the emergence of the phenomenonof reduced pain threshold, manifested as allodynia and hyperalgesia.
3．Nonsteroidal anti-inflammatory and analgesic drug showed the inhibitory roleon NMDA receptors and COX-2activity, blocking intracellular signal transductionpathways, reduced transcription and translation, moreover, its intervention inearly activated astrocytes, and as the disease progresses, the effect decreasedgradually, and the analgesic effect is limited.
引文
[1]Wall PD, Melzack R. Textbook of pain,4th. Philadelphia：Churchill-Livingstone，1999,220-228.
[2]孙忠实，朱珠.新一代非甾体类镇痛消炎药—罗非昔布.世界药品信息，2001，2：68-70.
[3]Aas A T, Tonnessen T I, Brun A, et al. Growth inhibition of rat glioma cellsin vitro and in vivo by aspirin[J]. Neurooncol,1995,24（2）:171-180.
[4]Nicolas Voilley, Jande Weille, Julien Mamet, et al. Nonsteroidanti-inflammatory drugs inhibit both the activity and the inflammation-inducedexpression of acid sensing ion channels in nociceptors[J]. Neuroscience,2001,21（20）:8026-8033.
[5]Vane JR, Bakhle YS, Botting RM. Cyclooxygenases1and2[J]. Annu Rev PharmacolToxicol,1998,38:97-120.
[6]Yamamoto T, Nozaki-Taguchi N. The role of cyclooxygenase-1and-2in the ratformalin test[J]. Anesth Analg,2002,94:962-967.
[7]Beiche F, Brune K, Geisslinger G, et al. Expression of cyclooxygenase isoformsin the rat spinal cord and their regulation during adjuvant-induced arthritis[J].Inflamm Res,1998,47:482-487.
[8]Hinz B， Brune K. Cyclooxygenase-2:10years later[J]. J Pharmacol Exp Ther,2002,300:367-375.
[9]Chandrasekharan NV, Dai H, Roos KL, et al. COX-3, a cyclooxygenase-1variantinhibited by acetaminophen and other analgesic/antipyretic drugs: cloning,structure, and expression[J]. Proc Natl Acad Sci USA,2002,99:13926-13931.
[10]Schwab JM, Brechtel K, Nguyen TD, et al. Persistent accumulation ofcyclooxygenase-1(COX-1) expressing microglia/macrophages and upregulation byendothelium following spinal cord injury[J]. J Neuroimmunol,2000,111:122-130.
[11] Zhang Y et al. Morphine: a protective or destructive role in neurons?Neuroscientist,2008,14:561-570.
[12] Henriksen G et al. Imaging of opioid receptors in the central nervous system.Brain,2008,131:1171-1196.
[13] Janecka A et al. Opioid receptors and their ligands. Curr Top Med Chem,2004,4:1-17.
[14]Nestler EJ,George KA.Molecular and cellular basis of addiction. Sience,1997,278(3):58-64.
[15]Nestler EJ.Molecular mechanisims of drug addicetion.JNerurosci,1992,12:2439-2450.
[16]Nestler EJ.Under siege:the brain on opiates.Neuron,1996,16:897-900.
[17]徐美林，陈崇宏.药物依赖性的神经递质－信号转导机制的研究进展.海峡药学杂志,2004,16(3):1-6.
[18]程金伟,李玲,高胜利.G亚基介导的MAPK信号转导通路在吗啡依赖中的作用.中华临床医药,2004,5(14):10-14.
[19]张瑞岭,郝伟.阿片依赖与脑基因表达.中国药物依赖性杂志.2002,11(4):241-244.
[20]Hayward MD,Duman RS,Nestler EJ.Induction of the c-fos protooncogene duringopiate withdrawal in the locus coeruleus and other regions of rats brain.BrainRes,1990,525:256-266.
[21]Nye HE,Nestler EJ.Induction of chronic Fos-related antigens in rat brainby chronic morphine dependence.Mol Pharmacol,1996,49:636-645.
[22]Widnekk KL,Self DW,Lane SB,et al.Regulation of CREB exression:in vivoevidence foe a functional role in morphine action in the nucleus accumbens.JPharmacol Exp,1996,276(1):306-315.
[23]Georges F,Stinus L,Le Moine C.mapping of c-fos gene exression in the brainduring morphine dependence and precipitated withdrawal,and phenotypeidentification of the striatal neurons involved.Eur JNeurosci,2000,12(2):4475-4486.
[24]Torres G,Horoith JM.Drugs of abuse and brain gene expression.PsychosomaticMed,1999,61:630-650.
[25]Lane-Ladd SB,Pineda J,Broundy VA,et al.CREB(cAMP response element–bindingprotein)in the locus coeruleus:biochemical,physiological.and bihacioralevidence for a role in opiate dependence.J Neurosc1997,17(20):7890-901.
[26]梁建辉,叶向锋,郑继旺.钙调素与吗啡依赖.中国药物依赖性杂志,2001,10(1):10-12.
[27]Leza,JC,Lizasoain I,San-Martin-Clark O,et al.Morphine-induced changes incerebral and cerebellar nitric oxide synthase activity.Eur JPharmacol,1995,285:95-99.
[28]邹冈主编.基础神经药理学.第2版.北京：科学出版社，1999.
[29]卢建，余应年，徐仁宝主编.受体、信号转导系统与疾病.山东科学技术出版社.2001
[30]Chen L,Huang LY.Sustained potentiation of NMDA receptor-mediated glutamateresponses through activation of protein kinase C by a muopioid.Neuron,1991,7(2):319-326.
[31]胡晓东，李凤源.阿片类对钙通道的影响.中国药物依赖性杂志，1999，8（3）：169-171.
[32]Connor M,Henderson G,δ-andμ-opioid rceptor mobilization of intracellularcalcium in SH-SY5Y human neuroblastoma cells.Br J Pharmacol,1996,117:333-340.
[33]Spencer RJ,Jin W,Thayer SA,et al.Mobilization of Ca2＋from intracellularstores in teansfected neuron cells by activation of mutiple opoioid receptorsubtypes.Biochem Pharmacol,1997,54:809-818.
[34]Smart D,Smith G.,Lambert SG.μ-opioid receptor stimulation ofinositol(1,4,5)triphosphate formation via spertusis toxinsensitive G protein.JNeurochem,1994,62:1009-1014.
[35]Li LY,Chang KJ.The stimulatory effect of opioids on mitogen-activatedprotein kinase in Chinese hamster ovary cells transfected to express mu-opioidreceptors.Mol Pharmacol,1996,50(3):599-602.
[36]Gutstein HB,Rubie EA,Mansour A,et al.Opioid effects on mitogen-activatedprotein kinase signaling cascades.Anesthesiology,1997,87(5):1118-1126.
[37]Belcheva MM,Coscia CJ.Diversity of G protein-coupled receptor signalingpathways to ERK/MAP kinase.Neurosignals,2002,11:34-44.
[38]Belcheva MM,Szucs M,Wang D,et al.Mu-opioid receptor-mediated ERKactivationinvovles calmodulin dependent receptors transactivation.J BioChem,2001,276:33847-33853.
[39]Lynch ME. Antidepressants as analgesics：a review of randomized controlledtrials. J Psychiatry Neurosci,2001,26:30-36.
[40]Ansari A.The efficacy of newer antidepressants in the treatment of chronicpain： a review of current literature Harv Rev Psychiatry,2000,7:257-277.
[41]Stracke H， Meyer UE， SchumacherHE， et al. Mexiletine in the treatmentof diabetic neuropathy. Diabetes Care,1992,15:1550-1555.
[42]Christersen D，Gantron M，Cruilbaud G，et al. Effect of gabapentin andlamotrigine on mechanical allodynia-like behaviour in a rat model of trigeminalneuropathic pain. Pain，2001,93:147-153.
[43]Sindruy SH，Andersen G，Madsen C，et al. Tramadol relieves pain and allodyniain polyneuropathy： a randomized，double-blind， controlled， trialPain,1999,87:85-90.
[44]Tallarida RJ. Drug synergism：its detection and applications. J PharmacalExp Ther，2001,298:865-872.
[45]Wolfe F，Smythe HA，Yunus MB，et al. The American College of Rheumatology1990Criteria for Classification of Fibromyalgia： Report of the MulticenterCriteria Committee. Arthritis Rheum,1990,33:160-172.
[1]Russell K, Portenoy DP, Paul Jaeobsen. Breakthrough Pain: characteristicsand impact in patients with cancer Pain. Pain,1999,81(1999):129-134.
[2]Mercadante S. Malignant bone pain:pathophysiology andtreatment.Pain.1997:69:1-18.
[3]Schwei MJ, Honore P, Rogers SD, et al. Neurochemical and cellularrecoganization of the spinal cord in a murine model of bone cancer pain. J.Neurosci.1999,19(24):10886-10897.
[4]Medhurst SJ, Walker K, Bowes M, et al. A rat model of bone cancer pain. Pain.2002,96(1-2):129-140.
[5]Mao-Ying QL, Zhao J, Dong ZQ, et al. A rat model of bone cancer pain inducedby intra-tibia inoculation of Walker256mammary gland carcinoma cells.BiochemBiophys Res Commun,2006,345(4):1292-1298.
[6]Zhang RX, Liu B, Wang L, et al. Spinal glial activation in a new rat modelof bone cancer pain produced by prostate cancer cell inoculation of thetibia.Pain,2005,118(1-2):125-136.
[7]聂远，刘淑俊，邸立军.癌痛及其对癌症患者生活质量影响的调查[J].中华肿瘤杂志，2000，22（5）：432-434.
[8]战祥毅，王文萍，高奉等.中药止痛巴布贴镇痛效果实验.辽宁中医药大学学报，2011，13（7）：45-46.
[9]Petralia RS,Yokotani N,Wenthold RJ.Light and electron microscopedistribution of the NMDA receptor subunit NMDAR1in the rat nervous system usinga selective anti-peptide antibody.J Neurosci,1994,14:667-696.
[10]Moussaoui SM, Hemans E, Mathieu AM, et al. Polyclonal antibodies againstthe rat NK-1receptor. Characterization and localization in the spinal cord.NeuroReport,1992,3:1073-1076.
[11]Brown JL, Liu H, Maggio JE, et al. Morphological characterization ofsubstance P receptor-immunoreactive neurons caudalis. J Comp Neurol,1995,356:327-344.
[12]Piercey MF, Dobry PKL, Schroeder LA, et al. Behavioral evidence thatsubstance P may be a spinal cord sensory neurotransmitter. Brain Res.1981,210:407-412.
[13]KORZUS E.The relation of transcription to memory formation. Acta BiochimPol,2003,50:775-882.
[14]Pang XY, Liu T, Jiang F, et al. Activation of spinal ERK signaling pathwaycontributes to pain-related response induced by scorpion Buthus martensi Karchvenom. Toxicon,2008,51:994-1007.
[15]Ji RR. Peripheral and centralmechanisms of inflammatory pain, with emphasisonMAP kinases. CurrDrugTargets Inflamm Allergy,2004,3∶299-303.
[16]ObataK, NoguchiK. MAPK activation in nociceptive neurons and painhypersensitivity. Life Sci.2004,74∶2643~2653
[17]张健，聂鑫，冯智英.鞘内注射阿米替林对福尔马林致痛大鼠行为学及脊髓Fos表达的影响[J].山东医药，2007，47（23）：26-27.
[18]Mercadante S.Recent progress in the pharmacotherapy of cancer pain.ExpertRev Anticancer Ther,2001,1(3):487-494
[19]Mantyh PW,Clohisy DR,Koltzenburg M,et al.Molecular mechanisms of cancerpain.Nat Rev Cancer,2002,2(3):201-209
[20]Saad F,Perrotte P,Benard F,et al.Bone health in men with prostatecancer:diagnostic and therapeutic considerations.Can J Urol,2005,12suppl2:9-15
[21]Ahn H,Mousavi P,Roth S,et al.Stability of the metastatic spine pre and postvertebroplasty.J Spinal Disord Tech,2006,19(3):178-182
[22]Wacnik PW,Eikmeier LJ,Ruggles TR,et al.Functional interactions betweentumor and peripheral nerve:morphology,algogen identification,and behavioralcharacterrization of a new murine model of cancer pain.J Neurosci,2001,21(23):9355-9366
[23]Wacnik PW,Kehl LJ,Trempe TM,et al.Tumor implantation in mouse humerus evokesmovement-related hyperalgesia exceeding that evoked by intramuscularcarrageenan.Pain,2003,101(1-2):175-186
[24]Fox A,Medhurst S,Courade JP,et al.Anti-hyperalgesic activity of the cox-2inhibitor lumiracoxib in a model of bone cancer pain in therat.Pain,2004,107(1-2):33-40
[25]项红兵，安珂，杨辉，等.骨癌痛模型和相关疼痛病理机制研究进展.临床麻醉学杂志，2005，21（5）：350-352
[26]张瑛，韩济生，王韵.癌症痛的神经生物学机制研究进展·生理科学进展,2004,35(3):224-228
[27]胡卫,陈涛.鼠移植性肝癌模型研究进展.河南肿瘤学杂志，2003,16(6):463-466
[28]Simpkins H,Lehman JM,Mazurkiewicz JE,et al.A morphological and phenotypicanalysis of Walker256cells. Cancer Res,1991,51(4):1334-1338.
[29]施波，傅招娣，雷小虹，等.153Sm-乙二胺四亚甲基膦酸对Walker256癌荷瘤大鼠骨侵袭骨溶解的抑制作用.中华肿瘤杂志，1997，19（6）：423-426
[30]Luger NM.,Mach DB,Sevcik MA et al.Bone Cancer Pain:From Model to Mechanismto Therapy.Journal of Pain and Symptom Management,2005,29(5):32-46
[31]Manthyh PW,Clohisy DR,Koltzenburg M,et al.Molecular mechanisms of cancerpain.Nature Rev(Cancer)2002,2(3):201-209
[32]Sarlak A,Gundes H,Ozkurkcugil C,et al.Solitary calcaneal metastasis insuperficial bladder carcinoma.Int J Clin Pract,2000,54(10):681-682
[33]鞠大宏，侯依泽.针刺镇痛的中医机理探讨.中国中医基础医学杂志,1998.4（增刊）：162
[34]李民，王维栋，李敏.浅谈虚性疼痛的治疗.甘肃中医学院学报,1994.11（1）：12
[35]杨晋翔，赵进喜.疼痛性疾病中医现代治疗学.学苑出版社,2001：18-23
[36]李园，吴晓秀，李佩文．中医对癌痛的认识及治疗概况[J]．中日友好医院学报，2002,16(5-6)：335．
[37]Khan MY,Mathur KB.Molecular biology of opioid receptors: recent advances[J].Neuroimmunomodulation,1997,4(5,6):285-297.
[38]Minarri M, et al. Mol Brain Res,1995;30:203
[39]VanVlien BJ,VanRiswijk AL,Warden G,et al.A daplive changes in the numberof Gs and Gi Proteins underlie adenylylcyin sensitization in morphine-treatedrat striatal neurons [J].Eur J pharmacol,1993,245(1):23-29.
[40]Wolfgang M,Stephan H.Opioidergic modulatin of Voltage-activated K+currentsin magnocellular neurons of the supraoptic neuron in rat [J].Jneurophysiol,1999,81(4):1617-1625.
[41]Manui HK.G protein.Implications for psychiatry.Am Psychiatry,1992,149:746-760.
[42]Attali B,Vogel Z.Long-term opiate exposure leads to reduction of the alphaI-1subunit of GTP-binding protein.J Neurochem,1989,53:1636-1639.
[43]Vogel Z,Barg J,Attali B,et al.Differential effect of mu,delta,and kappaligands on G protein alpha subunits in cultured brain cells.J NeurosciRes,1990,27:106-111.
[44]Baker AK, Hoffmann VL, Meert TF. Dextromethorphan and ketamine potentiatethe antinociceptive effects of mu-but not delta-or kappa-opioid agonists in amouse model of acute pain.Pharmacol Biochem Behav,2002,74:73-86.
[45]TKoch,AW idera, K Bartzsch,et al.Receptor endocytosis counteracts thedevelopment of opioid tolerance[J].Mo.l Pharmacol，2005,67:280-287
[46]NaritaM, SuzukiM,NaritaM,etal.Mu-opioid receptor internalization-dependent and independent mechanisms of the development of tolerance tomu-opioidreceptor agonists: Comparison between etorphine andmorphine[J].Neurosc,i2006,138(2):609-619
[47]Kow LM,Commons KG,Ogawa S,et al.Potentiation of the excitatior action ofNMDA in ventrolateral periaqueductal gray by theμ-opioid receptor agonist,DAMGO.Brain Res,2002,935:87-102.
[48]Aicher SA,Goldberg A,Sharma S.Co-localization of mu opioid receptor andN-methyl-D-aspartate receptor in the trigeminal dorsal horn.JPain,2002,3:203-210.
[49]Trujillo KA,Akil H.Inhibition of morphine tolerance and dependence by theNMDA receptor antagonist MK-801.Science,1991,251:85-87.
[50]Zhu H,Ho IK.NMDA-R1antisense oligonucleotide attenuates withdrawal signsfrom morphine.Eur J Pharmacol,1998,352:151-156.
[51]Miyamoto Y,Yamada K,Nagai T,et al.Behavioural adaptations to addictivedrugs in mice lacking the NMDA receptor epsilon1subunit.Eur JNeurosci,2004,19:151-158.
[52]Dunbar S,Yaksh TL.Concurrent spinal infusion of MK801blocks spinaltolerance and dependence induced by chronic intrathecal morphine in therat.Anesthesiology,1996,84:1177-1788.
[53]Tannenbaum H, Bombardier C, Davis P, Russell AS. An evidence-based approachto prescribing nonsteroidal antiinflammatory drugs. Third Canadian ConsensusConference. J Rheumatol2006;33:140-157.
[54]Cashman JN. The mechanisms of action of NSAIDs in analgesia. Drugs.1996;52Suppl5:13-23.
[55]Moussaoui SM, Hemans E, Mathieu AM, et al. Polyclonal antibodies againstthe rat NK-1receptor. Characterization and localization in the spinal cord.NeuroReport,1992,3:1073-1076.
[56]Brown JL, Liu H, Maggio JE, et al. Morphological characterization ofsubstance P receptor-immunoreactive neurons caudalis. J Comp Neurol,1995,356:327-344.
[57]Piercey MF, Dobry PKL, Schroeder LA, et al. Behavioral evidence thatsubstance P may be a spinal cord sensory neurotransmitter. Brain Res.1981,210:407-412.
[58]Margherita G, Arianna CR, Elisa B, et al. Substance P-inducedcyclooxygenase-2expression in human umbilical vein endothelial cells. BritishJournal of Pharmacology.2006,147:681–689.
[59]M Gallicchio, E Benetti, AC Rosa, et al. Tachykinin receptor modulation ofcyclooxygenase-2expression in human polymorphonuclear leucocytes. BritishJournal of Pharmacology.2009,156:486–496.
[60]Seybold VS，Jia YP，Abraharns LG. Cyclo-oxygenase-2contributes to centralsensitization in rats peripheral inflammation. Pain，2003,105:47-55.
[61]Rabassda X. Nimesalide, A selective cyclooxygenase-2inhibitoranti-inflammatory drug. Drugs Today,1996,32(suppl D):1.
[62]Willingale HL, Gardiner NJ, Melynlont N, et al. Prostanoids synthesized bycyclo-oxygenase-2insoforms in rats spinal cord and their contribution to thedevelopment of neuronal hyperexcitability. Br J Pharmacol.1997,122:1593-1604.
[63]Yaksh TL，Dirig DM，Conway CM，et al. The acute antihyperalgesic action ofnonsteroidal, anti-inflammatory drugs and release of spinal prostaglandin E2is medisted by the inhibition of constitutive spinal cyclooxygenase-2(COX-2)but not COX-1. J Neurosci，2001,21:5847-5853.
[64]李准民，郭曲练，邹望远，等.鞘内注射吗啡对福尔马林炎性疼痛大鼠脊髓背角环氧化酶-2表达的影响.中国麻醉学杂志，2006，26（9）：834-837.
[65]Cashman JN. The mechanisms of action of NSAIDs in analgesia. Drugs.1996;52Suppl5:13-23.
[66]Sharma SK, Klee WA, Nirenberg M,et al. Dual regulation of adenylate cyclaseaccounts for narcotic dependence and tolerance. Proc Natl Acad Sci USA,1975,72:3092-3096
[67]Musacchio JM, Greenspan DL. The adenylate cyclase rebound response tonaloxone in the NG108-15cells: effects of etorphine and other opiates.Neuropharmacology,1986,25:833-837
[68]Naito K, Kuriyama K. Effect of morphine administration on adenyl cyclaseand3',5'~cyclic nucleotide phosphodiesterase activites in the brain. JapanJ Pharmacol,1973,23:274-276
[69]Duman RS, Tallman JF, Nestler EJ,et al. Acute and chronic opiate-regulationof adenylate cyclase in brain:specific effects in locus coeruleus. J PharmacolExp Ther,1988,46:1033-1039
[70]Dolphin AC. Nucleotide binding proteins in signal transduction and disease.TINS,1987,10:53-57.
[71]黄丽妹,朱建梁,孙敏.哺乳动物腺苷酸环化酶亚型的研究进展[J].国际病理科学与临床杂志.2011,31(2):161-166.
[72]Mene P, Simonson MS, Dunn MJ. Eicosanoids, mesangial contraction, andintracellular signal transduction. Tohoku J Exp Med.1992Jan;166(1):57-73.
[73]Soderling TR.CaM-kinases:modulators of synaptic plasticity[J].Curr OpinNeurobiol,2000,10(3):375-380.
[74]Choi SS, Seo YJ, Shim EJ. Involvement of phosphorylated Ca2+/calmodulin-dependentprotein kinase II and phosphorylated extracellularsignal-regulatedprotein in the mouse formalin pain mode.l Brain Res,2006,1108:28~38.
[75]Xia Z, Storm DR. The role of calmodulin as a signal integrator for synapticplasticity. NatRevNeu rosc,i2005,6:267~276.
[76]Kennedy MB.Signal-processing machines at the postsynaptic density[J].Science,2000,290(5492):750-754.
[77]Malenka RC,Nicoll RA.Long-term potentiation--a decade of progress[J].Science,1999,285(5435):1870-1874.
[78]Lou L,Zhou T,Wang P,et al.Modulation of Ca2＋/calmodulin-dependent proteinkinase II activity by acute and chronic morphine adminstration in rathippozampus:differential regulation of alpha and beta isoform. MolPharmacol,1999,55(3):557-563
[79]Chang L，Karin M．Mammalian MAP kinase signalling cascades[J］．Nature，2001，410(6824):37-40．
[80]Svensson CI,Fitzsimmons B,Aziz S.Spinal p38β isoform mediates tissueinjury-induced hyperalgesia and spinal sensitization.J Neurochem,2005,92：1508–1520.
[81]Medhurst SJ，Walker K，Bowes M，et al.A rat model of bone cancer pain.Pain，2002，96：129-140.
[82]Tsuda M,Mizokoshi A,Shigemoto-mogami Y,et al.Activation of p38mitogen-activated protein kinase in spinal hyperactive microglia contributes to painhypersensitivity following peripheral nerve injury.Glia,2004,89：89–95.
[83]Svensson CI,Marsala M,Westerlund A，et al.Activation of p38mitogen-activated protein kinase in spinal microglia is a critical link ininflammation-induced spinal pain processing.J Neurochem，2003，86：1534–1544.
[84]Hua XY,Svensson CI,Matsui T,et al.Intrathecal minocycline attenuatesperipheral inflammation-induced hyperalgesia by inhibiting p38MAPK in spinalmicroglia. European Journal of Neuroscience,2005,22：2431-2440.
[85]Jin SX,Zhuang ZY,Woolf CJ,et al.p38mitogen-activated protein kinase isactivated after a spinal nerve ligation in spinal cord microglia and dorsal rootganglion neurons and contributes to the generation of neuropathic pain.JNeurosci,2003,23：4017–4022.
[86]Li LY, Chang KJ. The stimulatory effect of opioids on mitogen-activatedprotein kinase in Chinese hamster ovary cells transfected to express mu-opioidreceptors.Mol Pharmacol.1996,50(3):599-602
[87]Shehla W and Daniel D. NSAID Inhibition of Prostate Cancer Cell MigrationIs Mediated by Nag-1Induction via the p38MAPK-p75NTR Pathway. Mol Cancer Res.2010.8(12):1656–1664
[88]Caivano M, Cohen P. Role of mitogen-activated protein kinase cascades inmediating lipopolysaccharide-stimulated induction of cyclooxygenase-2and IL-1beta in RAW264macrophages.J Immunol.2000.164(6):3018-3025
[89]Oshima T, Miwa H, Joh T. Aspirin induces gastric epithelial barrierdysfunction by activating p38MAPK via claudin-7. Am J Physiol Cell Physiol.2008.295(3):C800-806.
[90]Marie K, Lonze BE, Ginty DD. Function and regulation of CREB familytranscription factors in the nervous system.Neuron,2002,35605~623.
[91]林雪梅,王泉石,朱琳等.福尔马林致痛对大鼠行为学及脊髓NO、Fos的影响[J].四川大学学报〔医学版),2003,34(3):523-526.
[92]傅强，王新华，张宏，等. CREB1蛋白在大鼠急、慢性吗啡依赖和戒断中的表达变化.[J]解放军医学杂志，2006，31（9）：887-889.
[93]Stevenson MA, Zhao MJ, Asea A,et al..Salicylic acid and aspirin inhibitthe activity of RSK2kinase and repress RSK2-dependent transcription of cyclicAMP response element binding protein-and NF-kappa B-responsive genes. J Immunol.1999.163(10):5608-5616.
[94]HonoréP,Buritova J,Besson J M. Intraplantar Morphine Depresses Spinal c-FosExpression Induced by Carrageenin Inflammation but not by Noxious Heat[J]. BrJ Pharmacol,1996,118(3):671-680.
[95]Hiroshi N,Simons C T,Cuellar J M,et al.Opioid Modulation of Scratching andSpinal c-fos Expression Evoked by Intradermal Serotonin[J].JNeurosci,2003,23(34):10784-10790.
[96]Honore，P.，Luger，N.M.，Sabino，M.A.C.，et al. steoProtegerin blocks boneeancer-induced skeletal destruction，skeletal Pain and Pain-relatedneuroehemical reorganization of the spinal Nature Medicine,2000,6:521-528.
[97]Vasudeva Raghavendra,Joyce A.DeLeo.The role of astrocytes and microglia inpersistent pain.Advances in Molecular and Cell Biology,2004,31：951–966.
[98]Wieseler-Frank J，Maier SF，Watkins LR，et al.Glial activation andpathological pain.Neurochemistry International，2004，45：389–395.
[99]Zhang RX,Liu B,Wang L,et al.Spinal glial activation in a new rat model ofbone cancer pain produced by prostate cancer cell inoculation of thetibia.Pain,2005,118(1-2)：125-136.
[100]Watkins LR,Maier SF.Gliaia novel drug discovery target for clinicalpain.Nat Rev Drug Discov,2003,2：973-985.
[101]陈敏，项红兵.胶质细胞参与疼痛中枢敏感化的机制[J].中国临床康复，2005，9（45）：127-129.
[102]Schwei MJ,Honore P,Rogers SD,et al.Neurochemical and cellularreorganization of the spinal cord in a murine model of bone cancer pain.JNeurosci，1999,19：10886-10897.
[103]Honore P.，Rogers SD，Sehwei MJ，et al. Murine models of inflammatory，neuropathie and cancer Pain eaeh generates a unique se of neurochemieal changesin the spinal cord and sensory neurons. Neuroscience,2000,98:585-598.
[104]Zhang RX，Liu B，Wang LB，et al. Spinal glial activation in a new rat modelof bone cancer pain produced by prostate cancer cell inoculation of the tibia.Pain,2005,118:125-136.
[105]Watkins RW, Milligan ED, Maier SF.Spinal cord glial new player in pain.Pain,2001,93(3):201-205.
[106]Watkins LR, Milligan ED, Maier SF.Glia activation:a driving force forpathological pain.Trends Neurosci,2001,24(8):450-455.
[107]Song P,Zhao ZQ.The involvement of glial cells in the development of morphinetolerance.Neurosci Res,2001,39(3):281-286.
[108]Hauser KF,Stiene MA,Mattson MP,et al.mu-Opioid receptor–induced Ca2+mobilization and astroglial development morphine inhibits DNA synthesis andstimulates cellular hypertrophy through a Ca2+-dependent mechanism. BrainRes.1996,720(1-2):191-203.
[109]张博爱，马兴荣，贾延劼，等.尼美舒利对大鼠的脑保护作用[J].郑州大学学报（医学版），2006，41（3）：483-486.
[110]WakitaH, TomimotoH, Akiguchi I, et al. Protective effect of cyclosporineAon whiter matter change in the rat brain after chronic cerebral hypoperfusion.
Stroke,1995,26(8):1415
[2]孙忠实，朱珠.新一代非甾体类镇痛消炎药—罗非昔布.世界药品信息，2001，2：68-70.
[3]Aas A T, Tonnessen T I, Brun A, et al. Growth inhibition of rat glioma cellsin vitro and in vivo by aspirin[J]. Neurooncol,1995,24（2）:171-180.
[4]Nicolas Voilley, Jande Weille, Julien Mamet, et al. Nonsteroidanti-inflammatory drugs inhibit both the activity and the inflammation-inducedexpression of acid sensing ion channels in nociceptors[J]. Neuroscience,2001,21（20）:8026-8033.
[5]Vane JR, Bakhle YS, Botting RM. Cyclooxygenases1and2[J]. Annu Rev PharmacolToxicol,1998,38:97-120.
[6]Yamamoto T, Nozaki-Taguchi N. The role of cyclooxygenase-1and-2in the ratformalin test[J]. Anesth Analg,2002,94:962-967.
[7]Beiche F, Brune K, Geisslinger G, et al. Expression of cyclooxygenase isoformsin the rat spinal cord and their regulation during adjuvant-induced arthritis[J].Inflamm Res,1998,47:482-487.
[8]Hinz B， Brune K. Cyclooxygenase-2:10years later[J]. J Pharmacol Exp Ther,2002,300:367-375.
[9]Chandrasekharan NV, Dai H, Roos KL, et al. COX-3, a cyclooxygenase-1variantinhibited by acetaminophen and other analgesic/antipyretic drugs: cloning,structure, and expression[J]. Proc Natl Acad Sci USA,2002,99:13926-13931.
[10]Schwab JM, Brechtel K, Nguyen TD, et al. Persistent accumulation ofcyclooxygenase-1(COX-1) expressing microglia/macrophages and upregulation byendothelium following spinal cord injury[J]. J Neuroimmunol,2000,111:122-130.
[11] Zhang Y et al. Morphine: a protective or destructive role in neurons?Neuroscientist,2008,14:561-570.
[12] Henriksen G et al. Imaging of opioid receptors in the central nervous system.Brain,2008,131:1171-1196.
[13] Janecka A et al. Opioid receptors and their ligands. Curr Top Med Chem,2004,4:1-17.
[14]Nestler EJ,George KA.Molecular and cellular basis of addiction. Sience,1997,278(3):58-64.
[15]Nestler EJ.Molecular mechanisims of drug addicetion.JNerurosci,1992,12:2439-2450.
[16]Nestler EJ.Under siege:the brain on opiates.Neuron,1996,16:897-900.
[17]徐美林，陈崇宏.药物依赖性的神经递质－信号转导机制的研究进展.海峡药学杂志,2004,16(3):1-6.
[18]程金伟,李玲,高胜利.G亚基介导的MAPK信号转导通路在吗啡依赖中的作用.中华临床医药,2004,5(14):10-14.
[19]张瑞岭,郝伟.阿片依赖与脑基因表达.中国药物依赖性杂志.2002,11(4):241-244.
[20]Hayward MD,Duman RS,Nestler EJ.Induction of the c-fos protooncogene duringopiate withdrawal in the locus coeruleus and other regions of rats brain.BrainRes,1990,525:256-266.
[21]Nye HE,Nestler EJ.Induction of chronic Fos-related antigens in rat brainby chronic morphine dependence.Mol Pharmacol,1996,49:636-645.
[22]Widnekk KL,Self DW,Lane SB,et al.Regulation of CREB exression:in vivoevidence foe a functional role in morphine action in the nucleus accumbens.JPharmacol Exp,1996,276(1):306-315.
[23]Georges F,Stinus L,Le Moine C.mapping of c-fos gene exression in the brainduring morphine dependence and precipitated withdrawal,and phenotypeidentification of the striatal neurons involved.Eur JNeurosci,2000,12(2):4475-4486.
[24]Torres G,Horoith JM.Drugs of abuse and brain gene expression.PsychosomaticMed,1999,61:630-650.
[25]Lane-Ladd SB,Pineda J,Broundy VA,et al.CREB(cAMP response element–bindingprotein)in the locus coeruleus:biochemical,physiological.and bihacioralevidence for a role in opiate dependence.J Neurosc1997,17(20):7890-901.
[26]梁建辉,叶向锋,郑继旺.钙调素与吗啡依赖.中国药物依赖性杂志,2001,10(1):10-12.
[27]Leza,JC,Lizasoain I,San-Martin-Clark O,et al.Morphine-induced changes incerebral and cerebellar nitric oxide synthase activity.Eur JPharmacol,1995,285:95-99.
[28]邹冈主编.基础神经药理学.第2版.北京：科学出版社，1999.
[29]卢建，余应年，徐仁宝主编.受体、信号转导系统与疾病.山东科学技术出版社.2001
[30]Chen L,Huang LY.Sustained potentiation of NMDA receptor-mediated glutamateresponses through activation of protein kinase C by a muopioid.Neuron,1991,7(2):319-326.
[31]胡晓东，李凤源.阿片类对钙通道的影响.中国药物依赖性杂志，1999，8（3）：169-171.
[32]Connor M,Henderson G,δ-andμ-opioid rceptor mobilization of intracellularcalcium in SH-SY5Y human neuroblastoma cells.Br J Pharmacol,1996,117:333-340.
[33]Spencer RJ,Jin W,Thayer SA,et al.Mobilization of Ca2＋from intracellularstores in teansfected neuron cells by activation of mutiple opoioid receptorsubtypes.Biochem Pharmacol,1997,54:809-818.
[34]Smart D,Smith G.,Lambert SG.μ-opioid receptor stimulation ofinositol(1,4,5)triphosphate formation via spertusis toxinsensitive G protein.JNeurochem,1994,62:1009-1014.
[35]Li LY,Chang KJ.The stimulatory effect of opioids on mitogen-activatedprotein kinase in Chinese hamster ovary cells transfected to express mu-opioidreceptors.Mol Pharmacol,1996,50(3):599-602.
[36]Gutstein HB,Rubie EA,Mansour A,et al.Opioid effects on mitogen-activatedprotein kinase signaling cascades.Anesthesiology,1997,87(5):1118-1126.
[37]Belcheva MM,Coscia CJ.Diversity of G protein-coupled receptor signalingpathways to ERK/MAP kinase.Neurosignals,2002,11:34-44.
[38]Belcheva MM,Szucs M,Wang D,et al.Mu-opioid receptor-mediated ERKactivationinvovles calmodulin dependent receptors transactivation.J BioChem,2001,276:33847-33853.
[39]Lynch ME. Antidepressants as analgesics：a review of randomized controlledtrials. J Psychiatry Neurosci,2001,26:30-36.
[40]Ansari A.The efficacy of newer antidepressants in the treatment of chronicpain： a review of current literature Harv Rev Psychiatry,2000,7:257-277.
[41]Stracke H， Meyer UE， SchumacherHE， et al. Mexiletine in the treatmentof diabetic neuropathy. Diabetes Care,1992,15:1550-1555.
[42]Christersen D，Gantron M，Cruilbaud G，et al. Effect of gabapentin andlamotrigine on mechanical allodynia-like behaviour in a rat model of trigeminalneuropathic pain. Pain，2001,93:147-153.
[43]Sindruy SH，Andersen G，Madsen C，et al. Tramadol relieves pain and allodyniain polyneuropathy： a randomized，double-blind， controlled， trialPain,1999,87:85-90.
[44]Tallarida RJ. Drug synergism：its detection and applications. J PharmacalExp Ther，2001,298:865-872.
[45]Wolfe F，Smythe HA，Yunus MB，et al. The American College of Rheumatology1990Criteria for Classification of Fibromyalgia： Report of the MulticenterCriteria Committee. Arthritis Rheum,1990,33:160-172.
[1]Russell K, Portenoy DP, Paul Jaeobsen. Breakthrough Pain: characteristicsand impact in patients with cancer Pain. Pain,1999,81(1999):129-134.
[2]Mercadante S. Malignant bone pain:pathophysiology andtreatment.Pain.1997:69:1-18.
[3]Schwei MJ, Honore P, Rogers SD, et al. Neurochemical and cellularrecoganization of the spinal cord in a murine model of bone cancer pain. J.Neurosci.1999,19(24):10886-10897.
[4]Medhurst SJ, Walker K, Bowes M, et al. A rat model of bone cancer pain. Pain.2002,96(1-2):129-140.
[5]Mao-Ying QL, Zhao J, Dong ZQ, et al. A rat model of bone cancer pain inducedby intra-tibia inoculation of Walker256mammary gland carcinoma cells.BiochemBiophys Res Commun,2006,345(4):1292-1298.
[6]Zhang RX, Liu B, Wang L, et al. Spinal glial activation in a new rat modelof bone cancer pain produced by prostate cancer cell inoculation of thetibia.Pain,2005,118(1-2):125-136.
[7]聂远，刘淑俊，邸立军.癌痛及其对癌症患者生活质量影响的调查[J].中华肿瘤杂志，2000，22（5）：432-434.
[8]战祥毅，王文萍，高奉等.中药止痛巴布贴镇痛效果实验.辽宁中医药大学学报，2011，13（7）：45-46.
[9]Petralia RS,Yokotani N,Wenthold RJ.Light and electron microscopedistribution of the NMDA receptor subunit NMDAR1in the rat nervous system usinga selective anti-peptide antibody.J Neurosci,1994,14:667-696.
[10]Moussaoui SM, Hemans E, Mathieu AM, et al. Polyclonal antibodies againstthe rat NK-1receptor. Characterization and localization in the spinal cord.NeuroReport,1992,3:1073-1076.
[11]Brown JL, Liu H, Maggio JE, et al. Morphological characterization ofsubstance P receptor-immunoreactive neurons caudalis. J Comp Neurol,1995,356:327-344.
[12]Piercey MF, Dobry PKL, Schroeder LA, et al. Behavioral evidence thatsubstance P may be a spinal cord sensory neurotransmitter. Brain Res.1981,210:407-412.
[13]KORZUS E.The relation of transcription to memory formation. Acta BiochimPol,2003,50:775-882.
[14]Pang XY, Liu T, Jiang F, et al. Activation of spinal ERK signaling pathwaycontributes to pain-related response induced by scorpion Buthus martensi Karchvenom. Toxicon,2008,51:994-1007.
[15]Ji RR. Peripheral and centralmechanisms of inflammatory pain, with emphasisonMAP kinases. CurrDrugTargets Inflamm Allergy,2004,3∶299-303.
[16]ObataK, NoguchiK. MAPK activation in nociceptive neurons and painhypersensitivity. Life Sci.2004,74∶2643~2653
[17]张健，聂鑫，冯智英.鞘内注射阿米替林对福尔马林致痛大鼠行为学及脊髓Fos表达的影响[J].山东医药，2007，47（23）：26-27.
[18]Mercadante S.Recent progress in the pharmacotherapy of cancer pain.ExpertRev Anticancer Ther,2001,1(3):487-494
[19]Mantyh PW,Clohisy DR,Koltzenburg M,et al.Molecular mechanisms of cancerpain.Nat Rev Cancer,2002,2(3):201-209
[20]Saad F,Perrotte P,Benard F,et al.Bone health in men with prostatecancer:diagnostic and therapeutic considerations.Can J Urol,2005,12suppl2:9-15
[21]Ahn H,Mousavi P,Roth S,et al.Stability of the metastatic spine pre and postvertebroplasty.J Spinal Disord Tech,2006,19(3):178-182
[22]Wacnik PW,Eikmeier LJ,Ruggles TR,et al.Functional interactions betweentumor and peripheral nerve:morphology,algogen identification,and behavioralcharacterrization of a new murine model of cancer pain.J Neurosci,2001,21(23):9355-9366
[23]Wacnik PW,Kehl LJ,Trempe TM,et al.Tumor implantation in mouse humerus evokesmovement-related hyperalgesia exceeding that evoked by intramuscularcarrageenan.Pain,2003,101(1-2):175-186
[24]Fox A,Medhurst S,Courade JP,et al.Anti-hyperalgesic activity of the cox-2inhibitor lumiracoxib in a model of bone cancer pain in therat.Pain,2004,107(1-2):33-40
[25]项红兵，安珂，杨辉，等.骨癌痛模型和相关疼痛病理机制研究进展.临床麻醉学杂志，2005，21（5）：350-352
[26]张瑛，韩济生，王韵.癌症痛的神经生物学机制研究进展·生理科学进展,2004,35(3):224-228
[27]胡卫,陈涛.鼠移植性肝癌模型研究进展.河南肿瘤学杂志，2003,16(6):463-466
[28]Simpkins H,Lehman JM,Mazurkiewicz JE,et al.A morphological and phenotypicanalysis of Walker256cells. Cancer Res,1991,51(4):1334-1338.
[29]施波，傅招娣，雷小虹，等.153Sm-乙二胺四亚甲基膦酸对Walker256癌荷瘤大鼠骨侵袭骨溶解的抑制作用.中华肿瘤杂志，1997，19（6）：423-426
[30]Luger NM.,Mach DB,Sevcik MA et al.Bone Cancer Pain:From Model to Mechanismto Therapy.Journal of Pain and Symptom Management,2005,29(5):32-46
[31]Manthyh PW,Clohisy DR,Koltzenburg M,et al.Molecular mechanisms of cancerpain.Nature Rev(Cancer)2002,2(3):201-209
[32]Sarlak A,Gundes H,Ozkurkcugil C,et al.Solitary calcaneal metastasis insuperficial bladder carcinoma.Int J Clin Pract,2000,54(10):681-682
[33]鞠大宏，侯依泽.针刺镇痛的中医机理探讨.中国中医基础医学杂志,1998.4（增刊）：162
[34]李民，王维栋，李敏.浅谈虚性疼痛的治疗.甘肃中医学院学报,1994.11（1）：12
[35]杨晋翔，赵进喜.疼痛性疾病中医现代治疗学.学苑出版社,2001：18-23
[36]李园，吴晓秀，李佩文．中医对癌痛的认识及治疗概况[J]．中日友好医院学报，2002,16(5-6)：335．
[37]Khan MY,Mathur KB.Molecular biology of opioid receptors: recent advances[J].Neuroimmunomodulation,1997,4(5,6):285-297.
[38]Minarri M, et al. Mol Brain Res,1995;30:203
[39]VanVlien BJ,VanRiswijk AL,Warden G,et al.A daplive changes in the numberof Gs and Gi Proteins underlie adenylylcyin sensitization in morphine-treatedrat striatal neurons [J].Eur J pharmacol,1993,245(1):23-29.
[40]Wolfgang M,Stephan H.Opioidergic modulatin of Voltage-activated K+currentsin magnocellular neurons of the supraoptic neuron in rat [J].Jneurophysiol,1999,81(4):1617-1625.
[41]Manui HK.G protein.Implications for psychiatry.Am Psychiatry,1992,149:746-760.
[42]Attali B,Vogel Z.Long-term opiate exposure leads to reduction of the alphaI-1subunit of GTP-binding protein.J Neurochem,1989,53:1636-1639.
[43]Vogel Z,Barg J,Attali B,et al.Differential effect of mu,delta,and kappaligands on G protein alpha subunits in cultured brain cells.J NeurosciRes,1990,27:106-111.
[44]Baker AK, Hoffmann VL, Meert TF. Dextromethorphan and ketamine potentiatethe antinociceptive effects of mu-but not delta-or kappa-opioid agonists in amouse model of acute pain.Pharmacol Biochem Behav,2002,74:73-86.
[45]TKoch,AW idera, K Bartzsch,et al.Receptor endocytosis counteracts thedevelopment of opioid tolerance[J].Mo.l Pharmacol，2005,67:280-287
[46]NaritaM, SuzukiM,NaritaM,etal.Mu-opioid receptor internalization-dependent and independent mechanisms of the development of tolerance tomu-opioidreceptor agonists: Comparison between etorphine andmorphine[J].Neurosc,i2006,138(2):609-619
[47]Kow LM,Commons KG,Ogawa S,et al.Potentiation of the excitatior action ofNMDA in ventrolateral periaqueductal gray by theμ-opioid receptor agonist,DAMGO.Brain Res,2002,935:87-102.
[48]Aicher SA,Goldberg A,Sharma S.Co-localization of mu opioid receptor andN-methyl-D-aspartate receptor in the trigeminal dorsal horn.JPain,2002,3:203-210.
[49]Trujillo KA,Akil H.Inhibition of morphine tolerance and dependence by theNMDA receptor antagonist MK-801.Science,1991,251:85-87.
[50]Zhu H,Ho IK.NMDA-R1antisense oligonucleotide attenuates withdrawal signsfrom morphine.Eur J Pharmacol,1998,352:151-156.
[51]Miyamoto Y,Yamada K,Nagai T,et al.Behavioural adaptations to addictivedrugs in mice lacking the NMDA receptor epsilon1subunit.Eur JNeurosci,2004,19:151-158.
[52]Dunbar S,Yaksh TL.Concurrent spinal infusion of MK801blocks spinaltolerance and dependence induced by chronic intrathecal morphine in therat.Anesthesiology,1996,84:1177-1788.
[53]Tannenbaum H, Bombardier C, Davis P, Russell AS. An evidence-based approachto prescribing nonsteroidal antiinflammatory drugs. Third Canadian ConsensusConference. J Rheumatol2006;33:140-157.
[54]Cashman JN. The mechanisms of action of NSAIDs in analgesia. Drugs.1996;52Suppl5:13-23.
[55]Moussaoui SM, Hemans E, Mathieu AM, et al. Polyclonal antibodies againstthe rat NK-1receptor. Characterization and localization in the spinal cord.NeuroReport,1992,3:1073-1076.
[56]Brown JL, Liu H, Maggio JE, et al. Morphological characterization ofsubstance P receptor-immunoreactive neurons caudalis. J Comp Neurol,1995,356:327-344.
[57]Piercey MF, Dobry PKL, Schroeder LA, et al. Behavioral evidence thatsubstance P may be a spinal cord sensory neurotransmitter. Brain Res.1981,210:407-412.
[58]Margherita G, Arianna CR, Elisa B, et al. Substance P-inducedcyclooxygenase-2expression in human umbilical vein endothelial cells. BritishJournal of Pharmacology.2006,147:681–689.
[59]M Gallicchio, E Benetti, AC Rosa, et al. Tachykinin receptor modulation ofcyclooxygenase-2expression in human polymorphonuclear leucocytes. BritishJournal of Pharmacology.2009,156:486–496.
[60]Seybold VS，Jia YP，Abraharns LG. Cyclo-oxygenase-2contributes to centralsensitization in rats peripheral inflammation. Pain，2003,105:47-55.
[61]Rabassda X. Nimesalide, A selective cyclooxygenase-2inhibitoranti-inflammatory drug. Drugs Today,1996,32(suppl D):1.
[62]Willingale HL, Gardiner NJ, Melynlont N, et al. Prostanoids synthesized bycyclo-oxygenase-2insoforms in rats spinal cord and their contribution to thedevelopment of neuronal hyperexcitability. Br J Pharmacol.1997,122:1593-1604.
[63]Yaksh TL，Dirig DM，Conway CM，et al. The acute antihyperalgesic action ofnonsteroidal, anti-inflammatory drugs and release of spinal prostaglandin E2is medisted by the inhibition of constitutive spinal cyclooxygenase-2(COX-2)but not COX-1. J Neurosci，2001,21:5847-5853.
[64]李准民，郭曲练，邹望远，等.鞘内注射吗啡对福尔马林炎性疼痛大鼠脊髓背角环氧化酶-2表达的影响.中国麻醉学杂志，2006，26（9）：834-837.
[65]Cashman JN. The mechanisms of action of NSAIDs in analgesia. Drugs.1996;52Suppl5:13-23.
[66]Sharma SK, Klee WA, Nirenberg M,et al. Dual regulation of adenylate cyclaseaccounts for narcotic dependence and tolerance. Proc Natl Acad Sci USA,1975,72:3092-3096
[67]Musacchio JM, Greenspan DL. The adenylate cyclase rebound response tonaloxone in the NG108-15cells: effects of etorphine and other opiates.Neuropharmacology,1986,25:833-837
[68]Naito K, Kuriyama K. Effect of morphine administration on adenyl cyclaseand3',5'~cyclic nucleotide phosphodiesterase activites in the brain. JapanJ Pharmacol,1973,23:274-276
[69]Duman RS, Tallman JF, Nestler EJ,et al. Acute and chronic opiate-regulationof adenylate cyclase in brain:specific effects in locus coeruleus. J PharmacolExp Ther,1988,46:1033-1039
[70]Dolphin AC. Nucleotide binding proteins in signal transduction and disease.TINS,1987,10:53-57.
[71]黄丽妹,朱建梁,孙敏.哺乳动物腺苷酸环化酶亚型的研究进展[J].国际病理科学与临床杂志.2011,31(2):161-166.
[72]Mene P, Simonson MS, Dunn MJ. Eicosanoids, mesangial contraction, andintracellular signal transduction. Tohoku J Exp Med.1992Jan;166(1):57-73.
[73]Soderling TR.CaM-kinases:modulators of synaptic plasticity[J].Curr OpinNeurobiol,2000,10(3):375-380.
[74]Choi SS, Seo YJ, Shim EJ. Involvement of phosphorylated Ca2+/calmodulin-dependentprotein kinase II and phosphorylated extracellularsignal-regulatedprotein in the mouse formalin pain mode.l Brain Res,2006,1108:28~38.
[75]Xia Z, Storm DR. The role of calmodulin as a signal integrator for synapticplasticity. NatRevNeu rosc,i2005,6:267~276.
[76]Kennedy MB.Signal-processing machines at the postsynaptic density[J].Science,2000,290(5492):750-754.
[77]Malenka RC,Nicoll RA.Long-term potentiation--a decade of progress[J].Science,1999,285(5435):1870-1874.
[78]Lou L,Zhou T,Wang P,et al.Modulation of Ca2＋/calmodulin-dependent proteinkinase II activity by acute and chronic morphine adminstration in rathippozampus:differential regulation of alpha and beta isoform. MolPharmacol,1999,55(3):557-563
[79]Chang L，Karin M．Mammalian MAP kinase signalling cascades[J］．Nature，2001，410(6824):37-40．
[80]Svensson CI,Fitzsimmons B,Aziz S.Spinal p38β isoform mediates tissueinjury-induced hyperalgesia and spinal sensitization.J Neurochem,2005,92：1508–1520.
[81]Medhurst SJ，Walker K，Bowes M，et al.A rat model of bone cancer pain.Pain，2002，96：129-140.
[82]Tsuda M,Mizokoshi A,Shigemoto-mogami Y,et al.Activation of p38mitogen-activated protein kinase in spinal hyperactive microglia contributes to painhypersensitivity following peripheral nerve injury.Glia,2004,89：89–95.
[83]Svensson CI,Marsala M,Westerlund A，et al.Activation of p38mitogen-activated protein kinase in spinal microglia is a critical link ininflammation-induced spinal pain processing.J Neurochem，2003，86：1534–1544.
[84]Hua XY,Svensson CI,Matsui T,et al.Intrathecal minocycline attenuatesperipheral inflammation-induced hyperalgesia by inhibiting p38MAPK in spinalmicroglia. European Journal of Neuroscience,2005,22：2431-2440.
[85]Jin SX,Zhuang ZY,Woolf CJ,et al.p38mitogen-activated protein kinase isactivated after a spinal nerve ligation in spinal cord microglia and dorsal rootganglion neurons and contributes to the generation of neuropathic pain.JNeurosci,2003,23：4017–4022.
[86]Li LY, Chang KJ. The stimulatory effect of opioids on mitogen-activatedprotein kinase in Chinese hamster ovary cells transfected to express mu-opioidreceptors.Mol Pharmacol.1996,50(3):599-602
[87]Shehla W and Daniel D. NSAID Inhibition of Prostate Cancer Cell MigrationIs Mediated by Nag-1Induction via the p38MAPK-p75NTR Pathway. Mol Cancer Res.2010.8(12):1656–1664
[88]Caivano M, Cohen P. Role of mitogen-activated protein kinase cascades inmediating lipopolysaccharide-stimulated induction of cyclooxygenase-2and IL-1beta in RAW264macrophages.J Immunol.2000.164(6):3018-3025
[89]Oshima T, Miwa H, Joh T. Aspirin induces gastric epithelial barrierdysfunction by activating p38MAPK via claudin-7. Am J Physiol Cell Physiol.2008.295(3):C800-806.
[90]Marie K, Lonze BE, Ginty DD. Function and regulation of CREB familytranscription factors in the nervous system.Neuron,2002,35605~623.
[91]林雪梅,王泉石,朱琳等.福尔马林致痛对大鼠行为学及脊髓NO、Fos的影响[J].四川大学学报〔医学版),2003,34(3):523-526.
[92]傅强，王新华，张宏，等. CREB1蛋白在大鼠急、慢性吗啡依赖和戒断中的表达变化.[J]解放军医学杂志，2006，31（9）：887-889.
[93]Stevenson MA, Zhao MJ, Asea A,et al..Salicylic acid and aspirin inhibitthe activity of RSK2kinase and repress RSK2-dependent transcription of cyclicAMP response element binding protein-and NF-kappa B-responsive genes. J Immunol.1999.163(10):5608-5616.
[94]HonoréP,Buritova J,Besson J M. Intraplantar Morphine Depresses Spinal c-FosExpression Induced by Carrageenin Inflammation but not by Noxious Heat[J]. BrJ Pharmacol,1996,118(3):671-680.
[95]Hiroshi N,Simons C T,Cuellar J M,et al.Opioid Modulation of Scratching andSpinal c-fos Expression Evoked by Intradermal Serotonin[J].JNeurosci,2003,23(34):10784-10790.
[96]Honore，P.，Luger，N.M.，Sabino，M.A.C.，et al. steoProtegerin blocks boneeancer-induced skeletal destruction，skeletal Pain and Pain-relatedneuroehemical reorganization of the spinal Nature Medicine,2000,6:521-528.
[97]Vasudeva Raghavendra,Joyce A.DeLeo.The role of astrocytes and microglia inpersistent pain.Advances in Molecular and Cell Biology,2004,31：951–966.
[98]Wieseler-Frank J，Maier SF，Watkins LR，et al.Glial activation andpathological pain.Neurochemistry International，2004，45：389–395.
[99]Zhang RX,Liu B,Wang L,et al.Spinal glial activation in a new rat model ofbone cancer pain produced by prostate cancer cell inoculation of thetibia.Pain,2005,118(1-2)：125-136.
[100]Watkins LR,Maier SF.Gliaia novel drug discovery target for clinicalpain.Nat Rev Drug Discov,2003,2：973-985.
[101]陈敏，项红兵.胶质细胞参与疼痛中枢敏感化的机制[J].中国临床康复，2005，9（45）：127-129.
[102]Schwei MJ,Honore P,Rogers SD,et al.Neurochemical and cellularreorganization of the spinal cord in a murine model of bone cancer pain.JNeurosci，1999,19：10886-10897.
[103]Honore P.，Rogers SD，Sehwei MJ，et al. Murine models of inflammatory，neuropathie and cancer Pain eaeh generates a unique se of neurochemieal changesin the spinal cord and sensory neurons. Neuroscience,2000,98:585-598.
[104]Zhang RX，Liu B，Wang LB，et al. Spinal glial activation in a new rat modelof bone cancer pain produced by prostate cancer cell inoculation of the tibia.Pain,2005,118:125-136.
[105]Watkins RW, Milligan ED, Maier SF.Spinal cord glial new player in pain.Pain,2001,93(3):201-205.
[106]Watkins LR, Milligan ED, Maier SF.Glia activation:a driving force forpathological pain.Trends Neurosci,2001,24(8):450-455.
[107]Song P,Zhao ZQ.The involvement of glial cells in the development of morphinetolerance.Neurosci Res,2001,39(3):281-286.
[108]Hauser KF,Stiene MA,Mattson MP,et al.mu-Opioid receptor–induced Ca2+mobilization and astroglial development morphine inhibits DNA synthesis andstimulates cellular hypertrophy through a Ca2+-dependent mechanism. BrainRes.1996,720(1-2):191-203.
[109]张博爱，马兴荣，贾延劼，等.尼美舒利对大鼠的脑保护作用[J].郑州大学学报（医学版），2006，41（3）：483-486.
[110]WakitaH, TomimotoH, Akiguchi I, et al. Protective effect of cyclosporineAon whiter matter change in the rat brain after chronic cerebral hypoperfusion.
Stroke,1995,26(8):1415