去甲基化酶TET1参与术后切口痛的表观遗传调控机制

发布时间：2018-09-10 05:58

【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。
[Abstract]:Background Chronic post-surgical pain syndrome (CPSP) is a common clinical problem and its mechanism is unclear. Surveys show that about 10-50% of patients suffer from postoperative knife-edge pain, and up to 10% of them recover from the primary disease after surgery, but still suffer from persistent chronic pain that is difficult to cure. CPSP has become a challenging research topic and hotspot in the medical field because of its unclear pathogenesis and untargeted clinical treatment. In the relevant clinical reports, it is often shown that patients in elective surgery often have anxiety, tension and noisy environment. Insufficient sleep leads to CPSP. These factors may affect the endocrine disorder of the central nervous system and the changes of the internal and external environment of the nervous system. However, the specific molecular mechanism is unclear, especially the epigenetic mechanism of the central and peripheral neurogenesis. In the animal model of stomachache, the expression of TET1 protein in the dorsal root ganglion and spinal cord of rats with chronic postoperative pain induced by perioperative stress was found to be down-regulated for the first time. This project will elucidate the behavioral characteristics of postoperative pain prolongation caused by mild and severe perioperative sleep deprivation, reveal the molecular basis of increased excitatory activity of pain-related neurons induced by mild perioperative sleep deprivation, and explore the role of epigenetic regulation mechanism induced by mild perioperative sleep deprivation in postoperative pain prolongation. Methods (1) Animal models were made and successfully divided into 4 groups (n < 5 / group) N group (sham); IN group: rat model of foot incision pain in left hind paw (left foot incision 1 cm and blunt tendon separation); S group: rats were wrapped and bound with soft wire mesh. The rats in the IN+S group were treated with left plantar incision and perioperative physical and psychological stress for 6 hours, and the models were established by detecting the changes of mechanical pain, hot pain, cold pain and recovery time, Western-blot and immunofluorescence. (2) Changes of methylase and demethylase in ganglia and spinal cord of chronic pain model after operation were screened; CDS with down-regulation of Si-TET1 and synthesis of TET1 carried by herpes simplex virus was designed to up-regulate TET1, and to observe the effect of TET1 on pain. The expression of three methylase DNMT1, DNMT3a, DNMT3b and three demethylase TET1, TET2 and TET3 were detected by Western-blot and q-PCR, respectively; the expression of si-TET1 was microinjected into DRG and Spinal cord to observe whether TET1 decreased, and the changes of downstream related proteins after TET1 decreased, and the behavioral changes of rats were observed by HSV-TET1. To investigate whether TET1 is an important and necessary factor involved in the chronic change of incision pain. (3) To detect the regulation of glucocorticoid on TET1, to detect the content of serum glucocorticoid in stress rats; to cultivate primary DRG neurons. The effects of corticosterone or glucocorticoid receptor antagonist Ru486 on TET1 expression in DRG neurons, the effects of intraperitoneal injection of glucocorticoid on postoperative incisional pain, and whether the incisional pain caused by corticosterone was reversed by GR receptor antagonist Ru486 were observed. The binding of glucocorticoid receptor GR to the promoter region of TET1 was detected by luciferase assay, and the regulatory effect of GR on the promoter region of TET1 was investigated. (4) To explore the mechanism of TET1 regulating downstream protein, Western-blot, qPCR was used to detect the expression of MOR, KOR and Kv1.2 in IN + S group compared with other groups. 1 and HSV-TET1 up-regulated TET1 to observe the effect on pain-related genes; 7 pairs of primers were designed to detect the binding between TET1 and MOR promoter by bioinformatics software, and the binding degree of TET1 to MOR promoter was detected by CHIP method; the expression and distribution of TET1 in spinal cord and ganglion were detected by immunofluorescence, and the expression and distribution of TET1 and MOR, TET1 and KO were detected by TEIP method. Results 1. Animal models were successfully made. Compared with IN group, in + S group, the thresholds of mechanical pain, hot pain and cold pain were lower; in IN group, mechanical pain, hot pain and cold pain were lower. On the 9th day after operation, the expression of microglial marker OX42 and astrocyte GFAP were increased in the operation side by immunofluorescence assay. The expression of P ERK1, P ERK2 and GFAP in the spinal cord of IN+S group was higher than that of IN group on the 9th day after operation by Western blot. The expression of methylase DNMT1, DNMT3a and DNMT3b in the spinal cord and DRG of the four groups was not changed. The expression of demethylase TET1 decreased in the operation side of IN+S group, but the expression of TET2 and TET3 remained unchanged. The expression of TET1 decreased in the operation side of DRG and S Pinal cord after microinjection of SITET1 respectively. At the same time, the mechanical pain threshold, hot pain threshold and cold pain threshold of the operation side were decreased; HSV-TET1 was used to inject DRG and S Pinal cord of the operation side of IN+S group, respectively, to observe and find that it can reverse the increased pain and shorten the postoperative pain duration of IN+S group. 3.4 groups of rats immediately after stress, orbital blood was taken to detect the content of serum glucocorticoid hormones in stress rats, the results were obvious. Compared with non-stress group, the levels of serum glucocorticoids in S group and IN+S group increased as much as 1000 times, the preference of S group and IN+S group decreased, the stress state of forced swimming prolonged, the expression of TET1 decreased after primary cultured DRG neurons were given glucocorticoid CORT, and the expression of TET1 increased after intraperitoneal injection of glucocorticoid receptor blocker Ru486. Glucocorticoid prolonged postoperative incision pain after three consecutive days and shortened the pain time after intrathecal administration of Ru486. Four pairs of primers were designed for the promoter region of TET1. GR antibody pulldown chromatin immunoprecipitation (CHIP) results showed that GR binds directly to the promoter region of TET1, and luciferase reporter gene (luciferase) showed glucocorticoid receptor. The expression of TET1 was negatively regulated by somatic GR. Western-blot and qPCR results showed that the expression of MOR and KOR decreased but the expression of Kv1.2 remained unchanged. Conditional site preference test showed that the function of MOR and KOR decreased in IN+S group. After injection of Si-TET1, the expression of MOR and KOR decreased; after transfection of HSV-TET1 into primary cultured DRG cells, the expression of MOR and KOR was up-regulated by Western-blot, qPCR, and the expression of MOR and KOR was reversed by microinjection of IN+S or HSV-TET1. Chromosomal immunoprecipitation (CHIP) showed that TET1 binds directly to the promoter region of MOR. CHIP and double luciferase reporter genes showed that TET1 regulates the expression of MOR. DNA-blot showed that the total methylation level remained unchanged, but high-throughput sequencing showed that the methylation of MOR promoter region in IN+S group was increased compared with that in IN group. Activation of glucocorticoid receptors inhibits the expression of TET1. TET1 results in persistent postoperative incisional pain by affecting the balance of KOR, 5 MC and 5 MHC in the promoter regions of MOR. Therefore, TET1 is an important and necessary molecule involved in the occurrence and development of chronic postoperative pain. It provides a theoretical basis for opening up new ideas for the treatment of CPSP, and may be a potential target for prevention and treatment of CPSP.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R402

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