【作者】 曹靖；

【导师】 高晓群；

【作者基本信息】 郑州大学 ， 人体解剖学与组织胚胎学， 2017， 博士

【摘要】 研究背景慢性术后疼痛(chronic post-surgical pain syndrome,CPSP)是临床常见的难题且机制不明。调查显示大约10-50%的患者遭遇术后刀口痛,其中高达10%的患者术后原发疾病痊愈,但仍然遭受难以治愈的持续慢性疼痛即慢性术后疼痛。现CPSP发病率有逐年上升趋势,且因其发病分子机制不清,临床处理无针对性,目前尚无好治疗方法、疗效不佳,因此CPSP成为医学领域内挑战性的研究课题和热点。在相关的临床报导中,多显示出择期手术期患者往往出现焦虑、紧张、环境的嘈杂导致患者睡眠不足,导致CPSP。这些因素都会影响到患者的中枢神经系统内分泌的紊乱,使神经系统内外环境的变化,然而具体的分子机制不明,尤其是中枢和周围的神经元发生的相关表观遗传机制不清。本实验利用围手术期应激延迟术后切口痛动物模型,首次发现围手术期应激造成的慢性术后痛大鼠脊背根神经节及脊髓内TET1蛋白表达下调,因此本研究聚焦于TET1参与围手术期应激造成的慢性术后痛的发生发展及相关机制,为神经病理性疼痛的治疗提供新思路。研究目的本项目将阐明围术期轻度与重度睡眠不足引起术后痛延长的行为学特征;揭示围术期轻度睡眠不足引起与痛觉传递相关神经元兴奋性活动增加的分子基础;探讨围术期轻度睡眠不足诱导的表观调控机制在术后痛延长中的作用。以期从分子-细胞-整体行为角度揭示CPSP发生和维持的机制。研究方法(1)制作动物模型,检测模型的成功将动物分为4组(n≥5/组)N组(sham);IN组:大鼠左后爪足底切口痛模型(左足底切口1cm并钝性分离肌腱);S组:将大鼠用软铁丝网包裹并捆绑每天6h并禁食水(或睡眠剥夺每天6h并禁食水),连续3d;制作IN+S组(并给予左足底切口并围手术期身体及心理应激6h);通过检测各组机械痛,热痛,冷痛的时程变化,恢复时间的变化,确定模型成功;Western-blot及免疫荧光检测神经元、小胶质细胞、星形胶质细胞是否活化;电生理检测IN+S组与IN组脊神经节神经元兴奋性的变化。(2)筛查术后慢性疼痛模型的神经节和脊髓内甲基化酶与去甲基化酶的改变;设计Si-TET1下调及合成单纯疱疹病毒携带的TET1的CDS上调TET1,观察TET1对疼痛的影响。利用Western-blot方法,qPCR方法检测3种甲基化酶DNMT1、DNMT3a、DNMT3b和3种去甲基化酶TET1、TET2、TET3的表达量;在DRG和Spinal cord分别显微注射si-TET1,观察TET1是否下降,以及TET1下降后后对下游相关蛋白的变化、对大鼠行为学的变化;利用HSV-TET1在DRG和Spinal cord分别显微注射,观察对于下游蛋白的影响和疼痛行为的影响;探索TET1是否是参与切口痛的慢性变的重要且必要因素。(3)检测糖皮质激素对TET1的调控检测应激大鼠的血清糖皮质激素皮质酮的含量;培养原代DRG神经元,分别给予皮质酮或糖皮质激素受体阻滞剂Ru486,检测在DRG神经元对TET1表达的影响。腹腔注射糖皮质激素检测对术后切口痛的影响;观察皮质酮引起的术后切口痛是否被GR受体阻滞剂Ru486翻转。利用染色质免疫共沉淀(CHIP)、双荧光素酶报告基因(luciferase)等方法检测糖皮质激素受体GR与TET1启动子区的结合及GR对其的调控作用。(4)探索TET1对下游蛋白调控的机制Western-blot,qPCR检测IN+S组与其它组相比MOR、KOR、Kv1.2的表达变化;在体外原代细胞培养、在体显微注射利用si-TET1下调TET1及HSV-TET1上调TET1,观察对疼痛相关基因的影响;通过生物信息软件,以MOR的启动子为研究对象,设计7对引物,CHIP法检测TET1与MOR启动子是否结合及应激状态下结合程度的变化;免疫荧光检测TET1在脊髓和神经节的表达分布,TET1和MOR、TET1和KOR的免疫双标;检测动物模型IN+S组与IN组相比MOR,KOR、Kv1.2是否变化;给予MOR、KOR激动剂或阻断剂观察行为学改变。研究结果1.制作动物模型成功。与IN组相比,IN+S组于第7d出现行为学差异,机械痛、热痛、冷痛阈值均降低;IN组机械痛、热痛、冷痛在术后第9d恢复正常,IN+S延长致13d以后。免疫荧光检测于术后第9d术侧小胶质细胞标记物OX42、星形胶质细胞物GFAP均表达增加;Western-blot结果显示与IN组相比,IN+S组于术后9d脊髓p ERK1、p ERK2、GFAP表达增加;电生理检测IN+S组比IN组脊神经节神经元兴奋性增加。2.Western-blot,qPCR结果显示4组模型大鼠脊髓及DRG中甲基化酶DNMT1、DNMT3a、DNMT3b表达并无变化;去甲基化酶TET1于IN+S组术侧表达下降,但TET2、TET3的表达量并无改变;正常大鼠DRG和Spinal cord分别显微注射si TET1后术侧TET1表达下降的同时术侧机械痛阈、热痛阈、冷痛阈均下降;利用HSV-TET1分别显微注射IN+S组的术侧DRG和Spinal cord,观察发现可以逆转IN+S组增强的疼痛和缩短术后疼痛时长。3.4组大鼠于应激后,立即眼眶取血检测应激大鼠的血清糖皮质激素的含量,结果显示与未应激组相比,S组及IN+S组血清糖皮质激素的含量上升高达上千倍;S组及IN+S糖水偏爱减少;被迫游泳应激状态时间延长;于原代培养DRG神经元,给予糖皮质激素CORT后TET1表达下降;而糖皮质激素受体阻滞剂Ru486后TET1表达回升;腹腔注射糖皮质激素连续三天后延长术后切口疼痛,鞘内给予Ru486后疼痛时间缩短;针对TET1的启动子区设计4对引物,GR抗体pulldown染色质免疫共沉淀(CHIP)结果显示GR与TET1的启动子区直接结合,双荧光素酶报告基因(luciferase)显示糖皮质激素受体GR负调控TET1的表达。4.4组大鼠DRG及脊髓的Western-blot,qPCR结果显示,MOR、KOR表达下降但Kv1.2表达不变;条件位置偏爱实验显示IN+S组MOR、KOR功能下降。Si-TET1转染原代培养DRG细胞后Western-blot,qPCR结果显示MOR、KOR表达下降但Kv1.2表达不变,DRG显微注射Si-TET1后MOR、KOR表达下降;HSV-TET1转染原代培养DRG细胞后Western-blot,qPCR结果显示MOR、KOR表达上调,,DRG显微注射IN+S组术侧DRG或脊髓HSV-TET1后缩短疼痛时长的同时翻转MOR、KOR表达下降的现象;针对MOR的启动子区设计6对引物,TET1抗体pulldown染色质免疫共沉淀(CHIP)结果显示TET1与MOR的启动子区直接结合,CHIP和双荧光素酶报告基因显示TET1正调控MOR的表达,DNA-blot显示总甲基化水平不变,但高通量测序结果显示IN+S组较IN组术侧MOR启动子区甲基化增加。研究结论围手术期的应激造成糖皮质激素受体激活抑制TET1的表达,TET1通过影响KOR、MOR的启动子区5mc和5mhc的平衡导致KOR、MOR表达和功能,产生持续术后切口痛。因此TET1是参与术后慢性疼痛的发生发展的重要和必要分子,研究TET1对更全面阐明表观遗传参与疼痛机制提供理论依据,为治疗CPSP开辟新的思路,可能是预防和治疗CPSP的潜在靶点。更多还原

【Abstract】 Background:Chronic postoperative pain(chronic post-surgical pain syndrome,CPSP)is a common clinical problem and it’s mechanisms are unknown.Survey shows that approximately 10%-50% of patients suffered postoperative pain,in which of 10%patients suffered chronic pain badly even the primary disease was healed.Because of the CPSP mechanism is unclear,no suitable treatment for CPSP,so there is a research challenging in this field.In clinical reports,peri-operative patients may be interfere with stress,anxiety,environmental noise on sleep,which can affect the central nervous system of patients that lead to endocrine disorders.The pathological changes may induce CPSP.But the specific molecular mechanism of CPSP is still unknown,especially in epigenetic mechanisms of particular central and peripheral neurons.This research used peri-operative stress animal model,found that peri-operative stress could cause chronic postoperative pain,and DNA demethylase TET1 expression in rat dorsal root ganglion and Spinal Cord was decreasd.So we focused on if or how TET1 participated in CPSP which caused by the peri-operative stress.The aim of this research is to detect the underling mechanisms of TET1 involved in development of CPSP,to provide new ideas to treat CPSP or other neuropathic pain based on this experiment.Research purposesThis project will address behavior characteristics of postoperative pain due to lack of perioperative sleep disturbance;reveal the molecular machanism of increasing neuron excitability induced postoperative pain due to lack of sleep;and explore the epigenetic mechanisms which regulate the development of postoperative pain and extend the postoperative pain due sleep disturbance.This reseach aimed to reveal the underling mechanism of development and maintaince of CPSP through the molecular-cellular-whole body,provide new targets for clinical treatment.Research methods(1)animal modelsRats were divided into 4 groups: N Group(Sham);IN group: Rats were anesthetized with 2% isoflurane delivered via a nose cone.The plantar aspect of left hindpaw was prepared in a sterile manner with a 10% povidone-iodine solution.A1-cm longitudinal incision was made with a number 11 blade,through skin and fascia of the plantar aspect of the foot,starting 0.5 cm from the proximal edge of the heel and extending toward the toes.The plantaris muscle was elevated and incised longitudinally.After hemostasis with gentle pressure,the skin was sutured with 5-0nylon.After surgery,the animals were allowed to recover in their cages;S group: The rats were immobilized with metal mesh packaging and restricting the motion of the head and body or put on the platform aroud the water in a tank,Stress was performed at the same time of the day(between 8 AM and 2 pm)without food and water,animals were subjected to a daily 6-h stress for 3 days;IN+S Group : the rats were suffered perioperative stress before or after incision.Determine model success by detecting behavior changes of mechanical 、 thermal 、 cold threshold,and pain recovery time;Determine activity of neurons,microglia,astrocytes by Western-blot and immunofluorescence;electrophysiology in DRG and spinal cord.(2)Explore the changes of Methyltransferase and Demethyltransferase enzymes after the chronic pain model in DRG and spinal cord by Western-blot;Detected behavior changes through downexpression TET1 or overexpression TET1 by Si-TET1 or herpes simplex virus carry TET1 CDS,to test effects of TET1 on regulating CPSP.We used Western-blot 、 qPCR to test the expression of methylation enzyme DNMT1、DNMT3a、DNMT3b and demethylation enzymes TET1、 TET2、 TET3;micro-injected siTET1 separately in DRG or Spinal cord to detect if it can induce pain behavior,micro-injected HSV-TET1 separately in DRG and Spinal cord to observe if it can reverse CPSP behavior changes of mechanical 、 thermal 、 cold threshold,and pain recovery time;From this experient we could know if TET1 is important and necessary elements involved in CPSP.(3)Regulation of glucocorticoid on TET1In order to find which factor caused the changes of TET1,we first detected theserum glucocorticoid levels after rat stress or incision;then we cultured DRG neurons,detected TET1 expression changes in DRG neurons after given corticosteroid or glucocorticoid receptor antagonist Ru486;we still intraperitoneal injected glucocorticoid to observe if glucocorticoid intraperitoneal injection can induced CPSP,and used the glucocorticoid receptor antagonist Ru486 could reverse the CPSP;finally we used Chromatin immunoprecipitation(CHIP)and the dual-luciferase reporter gene(luciferase)to detect the regulation of glucocorticoid receptor GR on TET1 promoter.(4)Explore the regulatory mechanism of TET1 on downstream proteins.Check the pain related genes from DRG and Spinal cord by Western-blot 、qPCR,to find the difference expression between IN+S group and the other groups,especially check MOR、KOR、Kv1.2 expression changes;Overexpressed TET1 by using HSV-TET1 or down-expressed by using si-TET1 transfection in DRG cell culture culture and in vivo micro-injection in DRG and spinal cord to observe the changes of pain-related genes,especially check MOR、KOR、 Kv1.2 expression changes.Then we focus on MOR,through bioinformatics software,we designed 7primers of MOR promoter,we used TET1 antibody to check if TET1 and OPRM1 promoter is binding together and the binding degree changes under stress;we still used immunofluorescence to detect TET1 expression in spinal cord and ganglion and the coexpression in one cell with MOR and KOR;Gave MOR,KOR agonists or antagonists to observe if IN+S behavioral could change.Results1.Animal models are builded successfully.Compared to IN groups,IN+S groups appeared more sensitive pain behavior from 7d to 9d of mechanical pain,thermal pain,cold pain.Compared to IN group,the incision pain disappeared at postoperative 9d,but in IN+S group pain threshold of machanical 、thermal、cold didn’t back to normal untill postoperative 13 d.Immunofluorescence showed microglia marker Iba1 and astrocytes marker GFAP expression increased in ipsilateral side of L5 spinal cord at postoperative 9d;Western-blot showed pERK1,pERK2,GFAP expression increased in ipsilateral side of L5 spinal cord in IN+S Group;Electrophysiology showed excitability of dorsal root ganglion neurons increased inIN+S Group at 9d compared to the IN Group.2.Western-blot、qPCR results displayed methylase enzyme DNMT1、DNMT3a、and DNMT3 b,demethylase enzyme TET2、TET3 expression in the spinal cord and the DRG had no changes,but demethylase TET1 expression decreased in the spinal cord and the DRG of IN+S group compare to IN group.We microinjected si-TET1 to normal rat DRG and Spinal cord respectively and found the threshold of mechanical、thermal、cold decreased compared to negative microinjection.Though microinjected HSV-TET1 to DRG and spinal cord,We found overexpression TET1 can reversed the decrease of the mechanical threshold 、thermal threshold、cold threshold in IN+S Group and shorten the duration of postoperative pain.3.The cortisone lever of blood were increased more than thousand folds in IN+S group or S group compared to N group or IN group.Sucrose preference results showed that rats of IN+S group and S group intake less sucrouse than N group and IN group;Forced swimming test showed the immobilization time in water extended of IN+S and S group compared to N group and IN group;Western blot and qPCR result all showed TET1 expression decreased in DRG or spinal cord after intraperitoneal injection cortisone continuous 3 days and then incision rats,as well as the same phenomenon in DRG cell culture after cortisones stimulating;this decrease of TET1 stimulated by cortisone could be reverse after GR receptor block agent Ru486 offering no matter in cultured cell or intrathecal Ru486 in IN+S rats.We still found GR antibody could pulldown TET1 promtor by chromatin immune precipitation(CHIP),and activate GR could decreased the luciferase by double fluorescent enzyme report gene carried TET1 promotor after transfected into HEK293 cell.4.Compared to IN group,Western-blot、qPCR result showed that MOR or KOR expression decreased in DRG or spinal cord;the function of MOR and KOR were weaken detected by conditioned place preference experiments of IN+S compared to IN group.Western-blot、qPCR showed MOR,KOR expression decreased in cultured DRG cell after Si-TET1 tranfection;Western-blot,qPCR showed MOR,KOR expression increased after transfection of HSV-TET1 in DRG cells.As well as in vivo experiment,Western-blot、qPCR showed MOR,KOR expression decreased after DRG or spinal cord injection of si-TET1;Western-blot、qPCR showed MOR,KORexpression increased after DRG or spinal cord injection of HSV-TET1 in IN+S group,as well as pain behavior reversed compared to microinjection of negative control in IN+S group.We still found TET1 antibody could pulldown MOR promtor by chromatin immune precipitation(CHIP),and TET1 could increased the luciferase by double fluorescent enzyme report gene carried MOR promoter after transfected into PC12 cell.DNA-blot displayed although total methyl of level didn’t variable,but the high-through sequencing results show that MOR promoter methylation increased in IN+S Group compared to IN group.Research conclusionsPerioperative stress caused glucocorticoid receptor activation that inhibited TET1,the decrease of TET1 expression affected the function and expression of MOR、KOR because of 5mc/5hmc increased.TET1 is the important and necessary factor involved in the development and progression of chronic postoperative pain.It is very important epigenetic molecules of TET1 to fully clarify the mechanisms of CPSP to provide a theoretical basis for opening up new ideas for treatment of CPSP.So TET1 might be a potential targets for the prevention and treatment of CPSP.更多还原