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上传于:2015-06-17

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靶向抑制延髓下行易化作用在大鼠骨癌痛中的镇痛效应及其机制北行研究

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内容提示: 分分分分 类类类类 号号号号学号学号学号学号 D200978413D200978413D200978413D200978413学校代码学校代码学校代码学校代码10487104871048710487密级密级密级密级博士学位论文靶向抑制延髓下行易化作用在大鼠骨癌痛靶向抑制延髓下行易化作用在大鼠骨癌痛靶向抑制延髓下行易化作用在大鼠骨癌痛靶向抑制延髓下行易化作用在大鼠骨癌痛中的镇痛效应及其机制研究中的镇痛效应及其机制研究中的镇痛效应及其机制研究中的镇痛效应及其机制研究学 位 申 请 人 :学 位 申 请 人 :学 位 申 请 人 :学 位 申 请 人 : 刘希江刘希江刘希江刘希江学 科 专 业 :学 科 专 业...

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分分分分 类类类类 号号号号学号学号学号学号 D200978413D200978413D200978413D200978413学校代码学校代码学校代码学校代码10487104871048710487密级密级密级密级博士学位论文靶向抑制延髓下行易化作用在大鼠骨癌痛靶向抑制延髓下行易化作用在大鼠骨癌痛靶向抑制延髓下行易化作用在大鼠骨癌痛靶向抑制延髓下行易化作用在大鼠骨癌痛中的镇痛效应及其机制研究中的镇痛效应及其机制研究中的镇痛效应及其机制研究中的镇痛效应及其机制研究学 位 申 请 人 :学 位 申 请 人 :学 位 申 请 人 :学 位 申 请 人 : 刘希江刘希江刘希江刘希江学 科 专 业 :学 科 专 业 :学 科 专 业 :学 科 专 业 : 麻醉学麻醉学麻醉学麻醉学指 导 教 师 :指 导 教 师 :指 导 教 师 :指 导 教 师 : 田玉科田玉科田玉科田玉科教授教授教授教授答 辩 日 期 :答 辩 日 期 :答 辩 日 期 :答 辩 日 期 :2012201220122012 年年年年 4 4 4 4 月月月月 A A A A dissertationdissertationdissertationdissertation submittedScienceScienceScienceScience andsubmittedsubmittedsubmitted to to to to Huazhongandandand TechnologyTechnologyTechnologyTechnology forHuazhongHuazhongHuazhong Universityforforfor thethethethe DegreeDegreeDegreeDegree of of of ofUniversityUniversityUniversity of of of ofDoctorDoctorDoctorDoctor of of of of MedicineMedicineMedicineMedicineMechanismsMechanismsMechanismsMechanisms of of of of analgesicdescendingdescendingdescendingdescending facilitationfacilitationfacilitationfacilitation fromratratratrat modelmodelmodelmodel of of of of canceranalgesicanalgesicanalgesic effectfromfromfrom thecancercancercancerinducedeffecteffecteffect of of of of targetedthethethe medullamedullamedullamedulla oblongatainducedinducedinduced bonebonebonebone paintargetedtargetedtargeted inhibitioninhibitioninhibitioninhibition of of of ofoblongataoblongataoblongata in in in in a a a apainpainpainPhPhPhPhD D D D. . . . CandidateCandidateCandidateCandidate: : : : LiuLiuLiuLiu XijiangXijiangXijiangXijiangMajorMajorMajorMajor: : : :AnesthesiologyAnesthesiologyAnesthesiologyAnesthesiologySupervisorSupervisorSupervisorSupervisor: : : : Prof.Prof.Prof.Prof. Dr.Dr.Dr.Dr. TianTianTianTianYukeYukeYukeYukeH H H Huazhonguazhonguazhonguazhong UniversityUniversityUniversityUniversity of of of of ScienceScienceScienceScience & & & & TechnologyTechnologyTechnologyTechnologyWuhanWuhanWuhanWuhan 430074,430074,430074,430074, P. P. P. P. R.R.R.R. ChinaChinaChinaChinaAprilAprilAprilApril, , , , 2020202012121212 独创性声明独创性声明独创性声明独创性声明本人郑重声明, 本学位论文是本人在导师指导下进行的研究工作及取得的研究成果的总结。尽我所知,除文中已经标明引用的内容外,本论文不包含任何其他个人或集体已经发表或撰写过的研究成果。对本文的研究做出贡献的个人和集体,均已在文中以明确方式标明。本人完全意识到本人将承担本声明引起的一切法律后果。学位论文作者签名:日期:年月日学位论文版权使用授权书学位论文版权使用授权书学位论文版权使用授权书学位论文版权使用授权书本学位论文作者完全了解学校有关保留、使用学位论文的规定,即:学校有权保留并向国家有关部门或机构送交论文的复印件和电子版,允许论文被查阅和借阅。 本人授权华中科技大学可以将本学位论文的全部或部分内容编入有关数据库进行检索,可以采用影印、缩印或扫描等复制手段保存和汇编本学位论文。保密□ ,在_____年解密后适用本授权书。本论文属于不保密□。(请在以上方框内打“√” )学位论文作者签名:指导教师签名:日期:年月日日期:年月日 华华华华 中中中中 科科科科 技技技技 大大大大 学学学学 博博博博 士士士士 学学学学 位位位位 论论论论 文文文文0目目目目录录录录主要缩略词表主要缩略词表主要缩略词表主要缩略词表················································································································································································································································································································································································································1中文摘要中文摘要中文摘要中文摘要················································································································································································································································································································································································································································3英文摘要英文摘要英文摘要英文摘要················································································································································································································································································································································································································································9正文正文正文正文········································································································································································································································································································································································································································································17第一部分第一部分第一部分第一部分 骨癌痛大鼠脊髓背角骨癌痛大鼠脊髓背角骨癌痛大鼠脊髓背角骨癌痛大鼠脊髓背角5- 5- 5- 5-羟色胺含量的变化及意义羟色胺含量的变化及意义羟色胺含量的变化及意义羟色胺含量的变化及意义················································································17前言····································································································································································································································································································································································································································17材料与方法····································································································································································································································································································································································································18结果····································································································································································································································································································································································································································24讨论····································································································································································································································································································································································································································31第二部分第二部分第二部分第二部分 靶向抑制靶向抑制靶向抑制靶向抑制RVMRVMRVMRVM区区区区5- 5- 5- 5-羟色胺能下行易化作用在大鼠骨癌痛的镇羟色胺能下行易化作用在大鼠骨癌痛的镇羟色胺能下行易化作用在大鼠骨癌痛的镇羟色胺能下行易化作用在大鼠骨癌痛的镇痛效应评价痛效应评价痛效应评价痛效应评价················································································································································································································································································································································35前言····································································································································································································································································································································································································································35材料与方法····································································································································································································································································································································································································36结果····································································································································································································································································································································································································································41讨论····································································································································································································································································································································································································································46第三部分第三部分第三部分第三部分 RVMRVMRVMRVM区区区区5- 5- 5- 5-羟色胺能下行易化作用参与大鼠骨癌痛的机制研究羟色胺能下行易化作用参与大鼠骨癌痛的机制研究羟色胺能下行易化作用参与大鼠骨癌痛的机制研究羟色胺能下行易化作用参与大鼠骨癌痛的机制研究································································································································································································································································································································································································································50前言····································································································································································································································································································································································································································50材料与方法····································································································································································································································································································································································································50结果····································································································································································································································································································································································································································52讨论····································································································································································································································································································································································································································59第四部分第四部分第四部分第四部分 RVMRVMRVMRVM区胶质细胞活化参与大鼠骨癌痛的机制研究区胶质细胞活化参与大鼠骨癌痛的机制研究区胶质细胞活化参与大鼠骨癌痛的机制研究区胶质细胞活化参与大鼠骨癌痛的机制研究········································································63 华华华华 中中中中 科科科科 技技技技 大大大大 学学学学 博博博博 士士士士 学学学学 位位位位 论论论论 文文文文1前言····································································································································································································································································································································································································································63材料与方法····································································································································································································································································································································································································64结果····································································································································································································································································································································································································································67讨论····································································································································································································································································································································································································································73综述综述综述综述········································································································································································································································································································································································································································································80综述综述综述综述1 1 1 1 骨癌痛机制与治疗研究进展骨癌痛机制与治疗研究进展骨癌痛机制与治疗研究进展骨癌痛机制与治疗研究进展························································································································································································································································80综述综述综述综述2 2 2 2 下行痛觉调控系统与丝裂原活化蛋白激酶研究进展下行痛觉调控系统与丝裂原活化蛋白激酶研究进展下行痛觉调控系统与丝裂原活化蛋白激酶研究进展下行痛觉调控系统与丝裂原活化蛋白激酶研究进展····················································································92致谢致谢致谢致谢································································································································································································································································································································································································································································101附录附录附录附录 攻读攻读攻读攻读学位学位学位学位期间发表期间发表期间发表期间发表论文目录论文目录论文目录论文目录························································································································································································································································102 华华华华 中中中中 科科科科 技技技技 大大大大 学学学学 博博博博 士士士士 学学学学 位位位位 论论论论 文文文文1主主主主 要要要要 缩缩缩缩 略略略略 词词词词 表表表表索引索引英文缩写英文缩写英文全称英文全称中文名称中文名称BBDNFBrain-derived neurotrophic factor脑源性神经营养因子CCIBPCancer induced bone pain癌症诱导的骨痛DDMSODimethyl sulfoxide二甲基亚砜DLFDorsolateral funiculus背外侧索GGFAPGlial fibrillary acidicprotein胶质纤维酸性蛋白GiANucleus reticularis gigantocellularis parsalpha网状巨细胞旁核α部GiVentral nucleus reticularisgigantocellularis网状巨细胞腹侧核HHRPHorseradish peroxidase辣根过氧化物酶HPLCHigh performance liquidchromatography高效液相色谱IIL-1βInterleukin-1β白介素-1βIL-6Interleukin-6白介素-6MMWDMechanical withdrawal duration机械缩爪持续时间NNRMNucleus raphe magnus中缝大核PPCPApara-chlorophenylalanine对氯苯基丙氨酸PWTMechanical paw withdrawal threshold机械缩爪阈值p38MAPKp38 mitogen-activated protein kinasep38 丝裂原活化蛋白激酶 华华华华 中中中中 科科科科 技技技技 大大大大 学学学学 博博博博 士士士士 学学学学 位位位位 论论论论 文文文文2RRVMRostral ventromedial medulla延髓头端腹内侧区SSNLSpinal nerve ligation脊神经结扎TTNF-αTumor necrosis factor α肿瘤坏死因子-αTPHTryptophan hydroxylase色氨酸羟化酶5-HT5-hydroxytryptamine5-羟色胺5,7-DHT5,7-di-hydroxytryptamine5,7-双羟色胺 华华华华 中中中中 科科科科 技技技技 大大大大 学学学学 博博博博 士士士士 学学学学 位位位位 论论论论 文文文文3中文摘要中文摘要中文摘要中文摘要靶向抑制延髓下行易化作用在大鼠骨癌痛中的镇痛效应及其机靶向抑制延髓下行易化作用在大鼠骨癌痛中的镇痛效应及其机靶向抑制延髓下行易化作用在大鼠骨癌痛中的镇痛效应及其机靶向抑制延髓下行易化作用在大鼠骨癌痛中的镇痛效应及其机制研究制研究制研究制研究博士研究生博士生导师刘希江田玉科 教授华中科技大学同济医学院附属同济医院麻醉学教研室研究背景研究背景研究背景研究背景癌症可表现出生理和心理的多种症状,但是癌症引起的疼痛往往是关键症状之一。未经治疗的疼痛可严重影响患者的生活质量,并能影响癌症患者对治疗的耐受程度从而影响癌症患者的预后。 随着癌症诊疗技术的进步, 癌症患者生存时间明显延长,缓解癌症患者的疼痛,提高其生活质量成为突出问题。据估计癌症痛的患病率在新诊断出的癌症患者中为 25%, 在积极治疗的癌症患者中为 33%, 而在晚期癌症患者中癌症痛的患病率超过 75%。 由于现有临床治疗癌症痛措施的局限性, 约 45%的癌症患者的疼痛未得到有效控制。 因而, 对癌痛机制和镇痛手段的全面深入研究显得尤为重要。癌症导致的骨痛(简称骨癌痛)是癌症患者最常见的一种疼痛,也是肿瘤发生骨转移时最常见的症状之一。大约三分之一的晚期癌症患者会发生骨转移,而在乳腺癌和前列腺癌晚期患者中超过 70%的患者发生骨转移,骨转移也是癌症痛最常见的原因。骨癌痛是癌症痛的典型代表,其主要表现是:持续的进行性的背景痛( Ongoingpain) ,爆发痛(Breakthrough pain)、痛觉过敏(Hyperalgesia)和痛觉超敏(Allodynia) 。目前,临床上尚缺乏有效且副作用小的骨癌痛治疗措施,未合理控制的疼痛不但严重影响患者生活质量而且显著缩短生存期,这与对骨癌痛的发病机制缺乏了解有关。 骨癌痛发生机制复杂,局部浸润的肿瘤细胞、炎症细胞及破/成骨细胞等产生的各种生物学活性物质,诱导外周和中枢神经系统的敏化,共同参与了骨癌痛的形成。并且,不同类型和不同阶段的肿瘤以及不同的肿瘤生长部位,均可影响骨癌痛的发生发展。近来研究表明,中枢神经下行易化系统激活对慢性疼痛的维持起重要作用。延髓头端腹内侧区(rostral ventromedial medulla,RVM)是下行痛觉调控系统的关键结构。 华华华华 中中中中 科科科科 技技技技 大大大大 学学学学 博博博博 士士士士 学学学学 位位位位 论论论论 文文文文4RVM 主要由中缝大核、网状巨细胞核α部和腹侧网状巨细胞核等神经核团组成,是整合脊髓上中枢痛觉调控信息并下行传递至脊髓的中继站。RVM 内 50%左右的神经元为 5-羟色胺(5-hydroxytryptamine, 5-HT)能神经元,其中含有 5-HT 生物合成的限速酶--色氨酸羟化酶(Tryptophan hydroxylase, TPH) ,其产生的 5-HT 可下行投射到脊髓背角,是下行调控系统参与脊髓疼痛处理的主要神经递质,来自 RVM 区的下行 5-HT对脊髓的痛觉信息处理起重要作用。同时,在神经病理性痛等慢性疼痛当中,RVM区胶质细胞活化并参与慢性疼痛的形成。但是,在骨癌痛中来自 RVM 区并下行投射到脊髓背角的 5-HT 对脊髓痛觉信息处理的具体作用以及骨癌痛中 RVM 区胶质细胞是否参与下行痛觉调控均尚不清楚。以 RVM 为治疗靶点研究癌痛治疗策略可不受肿瘤生长、转移部位的限制,还可有效控制癌症治疗引起的多发性神经病理性痛。基于此, 本研究拟通过探讨从 RVM 区投射到脊髓的 5-HT 能下行通路以及 RVM 区胶质细胞在骨癌痛大鼠疼痛机制中的作用,以期寻找癌痛治疗的新靶点和新的治疗策略。研究方法与结果研究方法与结果研究方法与结果研究方法与结果1 1 1 1.骨癌痛大鼠脊髓背角.骨癌痛大鼠脊髓背角 5- 5- 5- 5-羟色胺含量的变化及意义羟色胺含量的变化及意义方法:方法:实验一(骨癌痛大鼠脊髓背角 5-HT 含量与其痛阈的相关性分析) :雌性SD 大鼠 57 只,随机分为 3 组,每组 19 只。1)正常对照组(Naïve) :不作任何处理;2)假手术组(Sham) :于右侧胫骨上段骨髓腔注射 D-hank’s 液 10 μl ;3)骨癌痛组(CIBP) :于右侧胫骨上段骨髓腔接种 10 μl(约 3×104)Walker256 大鼠乳腺癌细胞悬液制备胫骨癌痛模型。实验二(鞘内注射 5-羟色胺能选择性神经毒素 5,7-DHT对骨癌痛大鼠痛阈的影响) :雌性 SD 大鼠 56 只,随机分为 4 组,每组 14 只。1)Sham-Saline 组:假手术后第 10 天鞘内置管,第 14 天鞘内给生理盐水(20μl) ;2)Sham-5,7-DHT 组:假手术后第 10 天鞘内置管,第 14 天鞘内给 5,7-DHT(60μg) ;3)CIBP-Saline 组:造模后第 10 天鞘内置管,第 14 天鞘内给生理盐水(20μl) ;4)CIBP-5,7-DHT 组:造模后第 10 天鞘内置管,第 14 天鞘内给 5,7-DHT(60μg) 。采用痛觉行为学和组织病理学方法鉴定模型构建成功。分别于术后第 7、14 和 21 天取各组大鼠脊髓背角组织,采用高效液相色谱法检测脊髓背角组织中 5-HT 含量,并与相 华华华华 中中中中 科科科科 技技技技 大大大大 学学学学 博博博博 士士士士 学学学学 位位位位 论论论论 文文文文5应时点的机械缩爪阈值和缩爪持续时间做双变量相关分析。 检测鞘内给予 5,7-DHT 对大鼠痛觉行为学的影响。 采用高效液相色谱分析和免疫荧光染色检测 5,7-DHT 对脊髓背角 5-羟色胺能神经的毁损效应。结果结果::肿瘤细胞接种后 7 ~21 d 大鼠机械缩爪阈值明显降低,缩爪持续时间延长,移动诱发痛评分进行性升高 (P<0.05) 。 光镜下肿瘤注射部位可见大量肿瘤细胞浸润,骨组织结构严重破坏, 表明模型制备成功。 与对照组相比, 骨癌痛组大鼠接种后 7 ~21d 时机械痛阈降低,接种后 7、14 和 21 d 时脊髓背角 5-HT 含量升高,且 5-HT 含量与机械痛阈呈显著相关 (P<0.05) 。 鞘内注射 5,7-DHT 后可显著降低脊髓背角中 5-HT含量,并能显著缓解骨癌痛大鼠的疼痛行为。2 2 2 2.靶向抑制.靶向抑制 RVMRVMRVMRVM 区区 5- 5- 5- 5-羟色胺能下行易化作用在骨癌痛大鼠的镇痛效应评价羟色胺能下行易化作用在骨癌痛大鼠的镇痛效应评价方法:方法:雌性 SD 大鼠 108 只,随机分为 4 组,每组 27 只。1)Sham-Saline 组: 假手术后第 14 天 RVM 区给生理盐水(0.5μl) ;2)Sham-PCPA 组:假手术后第 14 天RVM区给PCPA (50μg) ; 3) CIBP-Saline组: 造模后第14天RVM区给生理盐水 (0.5μl) ;4)CIBP-PCPA 组:造模后第 14 天 RVM 区给 PCPA(50μg) 。检测各组大鼠不同时点的机械缩爪阈值、缩爪持续时间和移动诱发痛评分。免疫印迹法检测 RVM 区 TPH 的表达。高效液相色谱法检测 RVM 给药后不同时点脊髓背...

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