發(fā)布時(shí)間：2018-09-10 05:58

【摘要】：研究背景慢性術(shù)后疼痛(chronic post-surgical pain syndrome,CPSP)是臨床常見(jiàn)的難題且機(jī)制不明。調(diào)查顯示大約10-50%的患者遭遇術(shù)后刀口痛,其中高達(dá)10%的患者術(shù)后原發(fā)疾病痊愈,但仍然遭受難以治愈的持續(xù)慢性疼痛即慢性術(shù)后疼痛�，F(xiàn)CPSP發(fā)病率有逐年上升趨勢(shì),且因其發(fā)病分子機(jī)制不清,臨床處理無(wú)針對(duì)性,目前尚無(wú)好治療方法、療效不佳,因此CPSP成為醫(yī)學(xué)領(lǐng)域內(nèi)挑戰(zhàn)性的研究課題和熱點(diǎn)。在相關(guān)的臨床報(bào)導(dǎo)中,多顯示出擇期手術(shù)期患者往往出現(xiàn)焦慮、緊張、環(huán)境的嘈雜導(dǎo)致患者睡眠不足,導(dǎo)致CPSP。這些因素都會(huì)影響到患者的中樞神經(jīng)系統(tǒng)內(nèi)分泌的紊亂,使神經(jīng)系統(tǒng)內(nèi)外環(huán)境的變化,然而具體的分子機(jī)制不明,尤其是中樞和周?chē)纳窠?jīng)元發(fā)生的相關(guān)表觀遺傳機(jī)制不清。本實(shí)驗(yàn)利用圍手術(shù)期應(yīng)激延遲術(shù)后切口痛動(dòng)物模型,首次發(fā)現(xiàn)圍手術(shù)期應(yīng)激造成的慢性術(shù)后痛大鼠脊背根神經(jīng)節(jié)及脊髓內(nèi)TET1蛋白表達(dá)下調(diào),因此本研究聚焦于TET1參與圍手術(shù)期應(yīng)激造成的慢性術(shù)后痛的發(fā)生發(fā)展及相關(guān)機(jī)制,為神經(jīng)病理性疼痛的治療提供新思路。研究目的本項(xiàng)目將闡明圍術(shù)期輕度與重度睡眠不足引起術(shù)后痛延長(zhǎng)的行為學(xué)特征;揭示圍術(shù)期輕度睡眠不足引起與痛覺(jué)傳遞相關(guān)神經(jīng)元興奮性活動(dòng)增加的分子基礎(chǔ);探討圍術(shù)期輕度睡眠不足誘導(dǎo)的表觀調(diào)控機(jī)制在術(shù)后痛延長(zhǎng)中的作用。以期從分子-細(xì)胞-整體行為角度揭示CPSP發(fā)生和維持的機(jī)制。研究方法(1)制作動(dòng)物模型,檢測(cè)模型的成功將動(dòng)物分為4組(n≥5/組)N組(sham);IN組:大鼠左后爪足底切口痛模型(左足底切口1cm并鈍性分離肌腱);S組:將大鼠用軟鐵絲網(wǎng)包裹并捆綁每天6h并禁食水(或睡眠剝奪每天6h并禁食水),連續(xù)3d;制作IN+S組(并給予左足底切口并圍手術(shù)期身體及心理應(yīng)激6h);通過(guò)檢測(cè)各組機(jī)械痛,熱痛,冷痛的時(shí)程變化,恢復(fù)時(shí)間的變化,確定模型成功;Western-blot及免疫熒光檢測(cè)神經(jīng)元、小膠質(zhì)細(xì)胞、星形膠質(zhì)細(xì)胞是否活化;電生理檢測(cè)IN+S組與IN組脊神經(jīng)節(jié)神經(jīng)元興奮性的變化。(2)篩查術(shù)后慢性疼痛模型的神經(jīng)節(jié)和脊髓內(nèi)甲基化酶與去甲基化酶的改變;設(shè)計(jì)Si-TET1下調(diào)及合成單純皰疹病毒攜帶的TET1的CDS上調(diào)TET1,觀察TET1對(duì)疼痛的影響。利用Western-blot方法,qPCR方法檢測(cè)3種甲基化酶DNMT1、DNMT3a、DNMT3b和3種去甲基化酶TET1、TET2、TET3的表達(dá)量;在DRG和Spinal cord分別顯微注射si-TET1,觀察TET1是否下降,以及TET1下降后后對(duì)下游相關(guān)蛋白的變化、對(duì)大鼠行為學(xué)的變化;利用HSV-TET1在DRG和Spinal cord分別顯微注射,觀察對(duì)于下游蛋白的影響和疼痛行為的影響;探索TET1是否是參與切口痛的慢性變的重要且必要因素。(3)檢測(cè)糖皮質(zhì)激素對(duì)TET1的調(diào)控檢測(cè)應(yīng)激大鼠的血清糖皮質(zhì)激素皮質(zhì)酮的含量;培養(yǎng)原代DRG神經(jīng)元,分別給予皮質(zhì)酮或糖皮質(zhì)激素受體阻滯劑Ru486,檢測(cè)在DRG神經(jīng)元對(duì)TET1表達(dá)的影響。腹腔注射糖皮質(zhì)激素檢測(cè)對(duì)術(shù)后切口痛的影響;觀察皮質(zhì)酮引起的術(shù)后切口痛是否被GR受體阻滯劑Ru486翻轉(zhuǎn)。利用染色質(zhì)免疫共沉淀(CHIP)、雙熒光素酶報(bào)告基因(luciferase)等方法檢測(cè)糖皮質(zhì)激素受體GR與TET1啟動(dòng)子區(qū)的結(jié)合及GR對(duì)其的調(diào)控作用。(4)探索TET1對(duì)下游蛋白調(diào)控的機(jī)制Western-blot,qPCR檢測(cè)IN+S組與其它組相比MOR、KOR、Kv1.2的表達(dá)變化;在體外原代細(xì)胞培養(yǎng)、在體顯微注射利用si-TET1下調(diào)TET1及HSV-TET1上調(diào)TET1,觀察對(duì)疼痛相關(guān)基因的影響;通過(guò)生物信息軟件,以MOR的啟動(dòng)子為研究對(duì)象,設(shè)計(jì)7對(duì)引物,CHIP法檢測(cè)TET1與MOR啟動(dòng)子是否結(jié)合及應(yīng)激狀態(tài)下結(jié)合程度的變化;免疫熒光檢測(cè)TET1在脊髓和神經(jīng)節(jié)的表達(dá)分布,TET1和MOR、TET1和KOR的免疫雙標(biāo);檢測(cè)動(dòng)物模型IN+S組與IN組相比MOR,KOR、Kv1.2是否變化;給予MOR、KOR激動(dòng)劑或阻斷劑觀察行為學(xué)改變。研究結(jié)果1.制作動(dòng)物模型成功。與IN組相比,IN+S組于第7d出現(xiàn)行為學(xué)差異,機(jī)械痛、熱痛、冷痛閾值均降低;IN組機(jī)械痛、熱痛、冷痛在術(shù)后第9d恢復(fù)正常,IN+S延長(zhǎng)致13d以后。免疫熒光檢測(cè)于術(shù)后第9d術(shù)側(cè)小膠質(zhì)細(xì)胞標(biāo)記物OX42、星形膠質(zhì)細(xì)胞物GFAP均表達(dá)增加;Western-blot結(jié)果顯示與IN組相比,IN+S組于術(shù)后9d脊髓p ERK1、p ERK2、GFAP表達(dá)增加;電生理檢測(cè)IN+S組比IN組脊神經(jīng)節(jié)神經(jīng)元興奮性增加。2.Western-blot,qPCR結(jié)果顯示4組模型大鼠脊髓及DRG中甲基化酶DNMT1、DNMT3a、DNMT3b表達(dá)并無(wú)變化;去甲基化酶TET1于IN+S組術(shù)側(cè)表達(dá)下降,但TET2、TET3的表達(dá)量并無(wú)改變;正常大鼠DRG和Spinal cord分別顯微注射si TET1后術(shù)側(cè)TET1表達(dá)下降的同時(shí)術(shù)側(cè)機(jī)械痛閾、熱痛閾、冷痛閾均下降;利用HSV-TET1分別顯微注射IN+S組的術(shù)側(cè)DRG和Spinal cord,觀察發(fā)現(xiàn)可以逆轉(zhuǎn)IN+S組增強(qiáng)的疼痛和縮短術(shù)后疼痛時(shí)長(zhǎng)。3.4組大鼠于應(yīng)激后,立即眼眶取血檢測(cè)應(yīng)激大鼠的血清糖皮質(zhì)激素的含量,結(jié)果顯示與未應(yīng)激組相比,S組及IN+S組血清糖皮質(zhì)激素的含量上升高達(dá)上千倍;S組及IN+S糖水偏愛(ài)減少;被迫游泳應(yīng)激狀態(tài)時(shí)間延長(zhǎng);于原代培養(yǎng)DRG神經(jīng)元,給予糖皮質(zhì)激素CORT后TET1表達(dá)下降;而糖皮質(zhì)激素受體阻滯劑Ru486后TET1表達(dá)回升;腹腔注射糖皮質(zhì)激素連續(xù)三天后延長(zhǎng)術(shù)后切口疼痛,鞘內(nèi)給予Ru486后疼痛時(shí)間縮短;針對(duì)TET1的啟動(dòng)子區(qū)設(shè)計(jì)4對(duì)引物,GR抗體pulldown染色質(zhì)免疫共沉淀(CHIP)結(jié)果顯示GR與TET1的啟動(dòng)子區(qū)直接結(jié)合,雙熒光素酶報(bào)告基因(luciferase)顯示糖皮質(zhì)激素受體GR負(fù)調(diào)控TET1的表達(dá)。4.4組大鼠DRG及脊髓的Western-blot,qPCR結(jié)果顯示,MOR、KOR表達(dá)下降但Kv1.2表達(dá)不變;條件位置偏愛(ài)實(shí)驗(yàn)顯示IN+S組MOR、KOR功能下降。Si-TET1轉(zhuǎn)染原代培養(yǎng)DRG細(xì)胞后Western-blot,qPCR結(jié)果顯示MOR、KOR表達(dá)下降但Kv1.2表達(dá)不變,DRG顯微注射Si-TET1后MOR、KOR表達(dá)下降;HSV-TET1轉(zhuǎn)染原代培養(yǎng)DRG細(xì)胞后Western-blot,qPCR結(jié)果顯示MOR、KOR表達(dá)上調(diào),,DRG顯微注射IN+S組術(shù)側(cè)DRG或脊髓HSV-TET1后縮短疼痛時(shí)長(zhǎng)的同時(shí)翻轉(zhuǎn)MOR、KOR表達(dá)下降的現(xiàn)象;針對(duì)MOR的啟動(dòng)子區(qū)設(shè)計(jì)6對(duì)引物,TET1抗體pulldown染色質(zhì)免疫共沉淀(CHIP)結(jié)果顯示TET1與MOR的啟動(dòng)子區(qū)直接結(jié)合,CHIP和雙熒光素酶報(bào)告基因顯示TET1正調(diào)控MOR的表達(dá),DNA-blot顯示總甲基化水平不變,但高通量測(cè)序結(jié)果顯示IN+S組較IN組術(shù)側(cè)MOR啟動(dòng)子區(qū)甲基化增加。研究結(jié)論圍手術(shù)期的應(yīng)激造成糖皮質(zhì)激素受體激活抑制TET1的表達(dá),TET1通過(guò)影響KOR、MOR的啟動(dòng)子區(qū)5mc和5mhc的平衡導(dǎo)致KOR、MOR表達(dá)和功能,產(chǎn)生持續(xù)術(shù)后切口痛。因此TET1是參與術(shù)后慢性疼痛的發(fā)生發(fā)展的重要和必要分子,研究TET1對(duì)更全面闡明表觀遺傳參與疼痛機(jī)制提供理論依據(jù),為治療CPSP開(kāi)辟新的思路,可能是預(yù)防和治療CPSP的潛在靶點(diǎn)。
[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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R402

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