本文選題：腸易激綜合癥　切入點(diǎn)：不良刺激　出處：《蘇州大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：研究背景： 腸易激綜合癥(Irritable Bowel Syndrome, IBS)是臨床常見的功能性胃腸疾病(Functional Gastrointestinal Disorders, FGIDs)之一,患者的主要癥狀表現(xiàn)為腸道運(yùn)動功能紊亂和慢性腹痛,疼痛癥狀隨排便過程而加重,但無明顯腸道炎癥或病理、生化指標(biāo)的異常。目前,對于IBS的發(fā)病機(jī)理仍然不清楚,臨床治療手段短缺,現(xiàn)有的治療方法和療效均不佳,因此,對IBS腹痛等癥狀形成與發(fā)展的生物學(xué)機(jī)制進(jìn)行研究具有重要意義。 研究目的： (1)闡述新生期體表炎性刺激(Neonatal Somatic Inflammation, NSI)誘發(fā)內(nèi)臟痛覺的特性,驗(yàn)證“新生期體表炎性刺激可以增加成年個體慢性內(nèi)臟痛敏感性”的研究假設(shè)。 (2)探討硫化氫(Hydrogen Sulfide, H2S)是否參與新生期體表炎性刺激引起成年大鼠結(jié)腸特異性背根節(jié)(DRG)神經(jīng)元(T13-L2)興奮性改變以及痛相關(guān)分子辣椒素受體或野香草受體1(Transient Receptor Potential Vanilloid1, TRPV1)的可塑性變化。 (3)研究表觀調(diào)控機(jī)制參與新生期體表炎性刺激誘發(fā)H2S合酶胱硫醚-β-合成酶(Cystathionine β-synthase, CBS)基因變化的機(jī)制。 (4)初步結(jié)合臨床標(biāo)本進(jìn)行CBS和TRPV1的分子生物學(xué)檢測,以期為臨床IBS診斷和治療提供新靶點(diǎn)。 研究方法： (1)在個體發(fā)育不同時間點(diǎn)(大鼠出生后10d、3w、6w),給予不同強(qiáng)度的炎性刺激(完全弗氏佐劑CFA、卡拉膠/角叉菜膠Carrageenan和福爾馬林Formalin),通過結(jié)直腸擴(kuò)張(Colorectal Distension, CRD)實(shí)驗(yàn)進(jìn)行腹壁撤回反射(Abdominal Withdrawal Reflex, AWR)評分以檢測成年大鼠的內(nèi)臟痛反應(yīng)。 (2)采用膜片鉗方法記錄新生期體表炎性刺激對成年SD大鼠結(jié)腸特異性DRG神經(jīng)元(T13-L2)興奮性的影響；同時,觀察硫化氫是否參與新生期體表炎性刺激誘發(fā)的神經(jīng)元興奮性的改變。 (3)通過Real-time PCR和Western blotting方法分別檢測新生期體表炎性刺激誘發(fā)的大鼠結(jié)腸DRG(T13-L2)以及腸道粘膜/肌肉組織中硫化氫合酶CBS和TRPV1分子在mRNA和蛋白質(zhì)水平的表達(dá)情況。 (4)應(yīng)用免疫熒光染色方法觀察CBS與TRPV1在結(jié)腸特異性DRG神經(jīng)元上的共表達(dá)情況。 (5)體外原代培養(yǎng)DRG神經(jīng)元,給予硫化氫供體硫氫化鈉(NaHS)孵育神經(jīng)元,觀察TRPV1的表達(dá)情況。 (6)通過甲基化特異性PCR(MSP)和亞硫酸氫鹽處理測序法(BSP)分析比較新生期體表炎性刺激引起SD大鼠結(jié)腸DRG神經(jīng)元內(nèi)CBS基因的甲基化程度的差異。同時,采用Real-time PCR方法檢測DNA甲基轉(zhuǎn)移酶基因Dnmts (Dnmt3a和Dnmt3b)、甲基CpG結(jié)合域蛋白基因MBDs (MBD2和MBD4)和DNA修復(fù)酶-胸腺嘧啶DNA糖基化酶基因(TDG)在rnRNA水平的表達(dá)情況。 (7)在臨床受試者知情同意并符合倫理學(xué)情況下,腸鏡采集IBS患者和對照組腸粘膜組織,分析比較IBS患者和對照組的腸粘膜內(nèi)CBS和TRPV1分子的表達(dá)差異。免疫組織化學(xué)方法檢測IBS患者和對照組的腸粘膜組織內(nèi)CBS和TRPV1的分布情況。 實(shí)驗(yàn)結(jié)果： (1)通過CRD實(shí)驗(yàn)進(jìn)行AWR評分發(fā)現(xiàn)：給予出生第10天SD大鼠體表炎性刺激即完全弗氏佐劑CFA(CFA:NS=2:1)處理,可以誘發(fā)成年大鼠的AWR評分顯著升高,即表現(xiàn)為內(nèi)臟痛覺過敏,且這種作用可以從第6周持續(xù)到第12周。而在出生后第21天給予CFA處理,卻不能誘發(fā)成年大鼠的內(nèi)臟痛覺過敏。同樣,在出生后第42天給予CFA處理,也不能誘發(fā)大鼠內(nèi)臟痛覺過敏。新生期給予不同強(qiáng)度的炎性刺激,如角叉菜膠(2%)、福爾馬林(5%)及低濃度CFA(CFA:NS=1:10),均不能誘發(fā)成年大鼠的內(nèi)臟痛覺過敏。 (2)應(yīng)用膜片鉗記錄熒光染料DiI標(biāo)記的結(jié)腸特異性DRG神經(jīng)元(T13-L2)的興奮性。結(jié)果發(fā)現(xiàn)：與對照組相比,實(shí)驗(yàn)組的靜息膜電位(RP)絕對值顯著降低,即靜息膜電位顯著去極化；同時,刺激的基強(qiáng)度顯著降低,兩倍和三倍基強(qiáng)度刺激下的放電頻率顯著增加。以上結(jié)果表明,與對照組相比,新生期第10天CFA炎性處理后腸特異性DRG神經(jīng)元的興奮性增加。此外,CBS抑制齊AOAA可翻轉(zhuǎn)新生期體表炎性刺激誘發(fā)的結(jié)腸特異性DRG神經(jīng)元的興奮性增加,即與對照組相比,靜息膜電位絕對值和基強(qiáng)度均升高,兩倍和三倍基強(qiáng)度下的放電頻率顯著降低。 (3)通過Western blotting和Real-time PCR方法發(fā)現(xiàn)：新生期體表炎性刺激可以誘發(fā)SD大鼠結(jié)腸DRG T13-L2內(nèi)CBS蛋白表達(dá)水平升高,持續(xù)時間可以從處理后8小時、3周、6周均存在顯著性差異,而12周時無顯著性差異。在轉(zhuǎn)錄水平,選取3周和6周齡SD大鼠結(jié)腸DRG進(jìn)行檢測,發(fā)現(xiàn)：與對照組相比,新生期體表炎性刺激可誘發(fā)3周齡和6周齡SD大鼠DRG T13-L2內(nèi)CBS基因mRNA水平表達(dá)升高,具有顯著性差異。 (4)通過Western blotting方法發(fā)現(xiàn)：新生期體表炎性刺激可引起6周齡SD大鼠結(jié)腸DRG神經(jīng)元內(nèi)TRPV1蛋白表達(dá)水平上調(diào)。給予SD大鼠連續(xù)7天腹腔注射CBS的抑制劑AOAA(10mg/kgi.p.)能夠翻轉(zhuǎn)NSI誘發(fā)的內(nèi)臟痛覺過敏,即與同時間點(diǎn)下的對照組相比,AOAA明顯提高NSI大鼠的痛閾,最大抑制效應(yīng)產(chǎn)生在注射后30min,鎮(zhèn)痛作用均持續(xù)60min。然而給予健康正常SD大鼠腹腔注射10mg/kg的AOAA對其痛閾無明顯影響。同時CBS抑制劑AOAA可以翻轉(zhuǎn)NSI誘發(fā)的大鼠結(jié)腸DRG神經(jīng)元內(nèi)TRPV1的表達(dá)水平。 (5)免疫熒光染色發(fā)現(xiàn)：CBS與TRPV1在結(jié)腸特異性DRG神經(jīng)元上存在共表達(dá)。 (6)體外原代培養(yǎng)急性分離的DRG神經(jīng)元細(xì)胞,給予硫氫化鈉(100gm)處理,模擬體內(nèi)H2S產(chǎn)生增多,收集細(xì)胞檢測TRPV1分子在蛋白水平的表達(dá)情況。發(fā)現(xiàn)：與對照組相比,實(shí)驗(yàn)組的TRPV1表達(dá)水平顯著上調(diào)。 (7)應(yīng)用MSP和BSP方法發(fā)現(xiàn)：新生期體表炎性刺激可以誘發(fā)3周齡和6周齡SD大鼠結(jié)腸DRG神經(jīng)元T13-L2內(nèi)CBS基因的啟動子調(diào)控區(qū)CpG島2的低甲基化。同時,NSI誘發(fā)6周齡SD雄性大鼠DRG T13-L2內(nèi)Dnmt3a和Dnmt3b基因的mRNA表達(dá)水平下調(diào),而MBD2、MBD4和TDG基因的mRNA表達(dá)水平無顯著性差異。 (8)新生期第10天完全弗氏佐劑處理后6周齡SD大鼠腸粘膜和肌層內(nèi)CBS和TRPV1的蛋白均呈上調(diào)表達(dá),具有顯著性差異。 (9)與對照組相比,IBS患者腸粘膜內(nèi)的CBS和TRPV1蛋白表達(dá)同樣出現(xiàn)了顯著升高。同時免疫組織化學(xué)染色結(jié)果顯示：IBS患者腸粘膜組織內(nèi)CBS和TRPV1分子的表達(dá)分布的陽性信號均高于對照組。 結(jié)論： (1)新生期體表炎性刺激可增加成年個體慢性內(nèi)臟痛敏感性,且推測這種作用具有時間窗口的依賴性、刺激強(qiáng)度的依賴性和作用部位的非依賴性等特性。 (2) CBS-H2S信號系統(tǒng)的增強(qiáng)在新生期體表炎性刺激誘發(fā)的內(nèi)臟痛覺過敏中發(fā)揮重要作用,即這種增強(qiáng)可能是通過興奮腸特異性DRG神經(jīng)元,導(dǎo)致外周敏化,產(chǎn)生內(nèi)臟痛覺過敏。 (3)CBS基因的表觀調(diào)控特征：結(jié)腸特異性神經(jīng)元內(nèi)的硫化氫合酶基因CBS的低甲基化狀態(tài),可引起CBS基因表達(dá)的上調(diào),即硫化氫合成增加,參與內(nèi)臟痛過敏。 (4)新生期體表炎性刺激可以誘發(fā)成年大鼠腸相關(guān)組織內(nèi)的CBS和TRPV1蛋白表達(dá)上調(diào),同時臨床IBS患者腸粘膜組織內(nèi)的CBS和TRPV1表達(dá)水平也出現(xiàn)顯著升高,提示疼痛相關(guān)基因可能作為腸易激綜合癥疼痛診斷和治療的新靶點(diǎn)。
[Abstract]:Research background:
Irritable bowel syndrome (Irritable Bowel, Syndrome, IBS) is a common functional gastrointestinal disease (Functional Gastrointestinal, Disorders, FGIDs) is one of the main symptoms of the patients showed intestinal motility disorder and chronic abdominal pain, pain symptoms increase with the defecation process, but no obvious intestinal inflammation or abnormal pathological, biochemical indexes at present. And for the pathogenesis of IBS remains unclear, the clinical treatment shortage, treatment methods and curative effect of existing were poor, therefore, it is very important to study the biological mechanisms of the formation and development of IBS, abdominal pain and other symptoms.
The purpose of the study is:
(1) to describe the characteristics of visceral pain evoked by Neonatal Somatic Inflammation (NSI) in the neonatal period, and to verify that the hypothesis of "neonatal inflammatory stimulation on the body surface can increase the sensitivity of chronic visceral pain in adults".
(2) of hydrogen sulfide (Hydrogen Sulfide H2S) is involved in the neonatal skin inflammatory stimulation caused by adult rat colon specific dorsal root ganglion (DRG) neurons (T13-L2) excitability change and pain related molecular capsaicin receptor receptor 1 (Transient or vanillon Receptor Potential Vanilloid1, TRPV1) of plasticity.
(3) the mechanism of epigenetic regulation is involved in the changes of H2S synthase Cystathionine beta -synthase (CBS) gene induced by inflammatory stimulation on the neonatal surface.
(4) the molecular biological detection of CBS and TRPV1 is preliminarily combined with clinical specimens, so as to provide new targets for the diagnosis and treatment of clinical IBS.
Research methods:
(1) in the ontogeny of different time points (postnatal rats 10d, 3W, 6W), inflammatory stimulation of different intensity (complete Freund's adjuvant CFA / carrageenan, carrageenan and formalin Formalin, Carrageenan (Colorectal) by colorectal distension Distension, CRD) the experiment of Abdominal withdrawal reflex (Abdominal Withdrawal Reflex, AWR) reaction to detect visceral pain score in adult rats.
(2) patch clamp method was used to record the effect of neonatal inflammatory stimulation on the excitability of colon specific DRG neurons (T13-L2) in adult SD rats, and to observe whether hydrogen sulfide is involved in the change of neuronal excitability induced by inflammatory stimulation on the neonatal stage.
(3) Real-time PCR and Western blotting methods were used to detect colon DRG (T13-L2) induced by inflammatory stimulation in neonatal rats, and the expression of CBS and TRPV1 molecules in mRNA and protein levels in intestinal mucosa / muscle tissues.
(4) the co expression of CBS and TRPV1 on colonic specific DRG neurons was observed by immunofluorescence staining.
(5) the DRG neurons were cultured in vitro, and the hydrogen sulfide donor sodium hydrogen sulfide (NaHS) was incubated with neurons, and the expression of TRPV1 was observed.
(6) by methylation specific PCR (MSP) and bisulfite sequencing analysis processing (BSP) caused by differences in the degree of methylation of CBS gene SD in rat colon DRG neuronal stimulation compared to neonatal body inflammation. At the same time, using the Real-time PCR method to detect DNA methyltransferase gene (Dnmt3a and Dnmts Dnmt3b), methyl CpG binding domain protein gene MBDs (MBD2 and MBD4) and DNA repair enzyme thymine DNA glycosylase (TDG) gene expression in rnRNA level.
(7) in clinical subjects informed consent and ethical case, colonoscopy acquisition IBS patients and control group of intestinal mucosa, differential expression analysis between IBS patients and controls in the intestinal mucosa of CBS and TRPV1 molecules. The distribution of intestinal mucosa was detected by immunohistochemistry in patients with IBS and control group in CBS and TRPV1.
Experimental results:
(1) through the CRD experiment AWR were found: give birth to tenth days on the inflammatory stimulation of SD rats with complete Freund's adjuvant (CFA:NS=2:1 CFA), can induce adult rat AWR score increased significantly, which shows the visceral hyperalgesia, and this effect can last from sixth weeks to twelfth weeks. In twenty-first days after birth to CFA treatment, but did not induce adult rat visceral hyperalgesia. Similarly, CFA treated at forty-second days after birth, nor in rats induced by visceral hyperalgesia. Inflammatory stimulation in neonatal period with different strength, such as carrageenan (2%), Faure Marin (5%) and low concentration of CFA (CFA:NS=1:10), were unable to induce adult rat visceral hyperalgesia.
(2) the application of patch clamp DiI fluorescent dye labeled colon specific DRG (T13-L2) neuron excitability. Results: compared with the control group, the resting membrane potential (RP) of the experimental group significantly reduced the absolute value, namely the resting membrane potential was depolarized; at the same time, medium intensity stimulation significantly reduced the discharge frequency two times and three times the base strength under stimulation increased significantly. The above results show that, compared with the control group, the new period of tenth days of CFA treatment of inflammatory intestinal specific excitability of DRG neurons increased. In addition, CBS AOAA can inhibit Qi excitatory flip neonatal inflammatory stimulation induced surface colon specific DRG neurons that increase, compared with the control group, the absolute value of the resting membrane potential and strength were increased, the discharge frequency of two times and three times the base strength decreased significantly.
(3) by Western blotting and Real-time PCR found that neonatal skin inflammatory stimulation can increase in the expression level of CBS protein induced by SD in rat colon DRG T13-L2, duration from 8 hours after treatment, 3 weeks and 6 weeks were significantly different at 12 weeks there was no significant difference in transcription. The level of selection of colon DRG 3 week and 6 week old SD rats were detected and found that: compared with the control group, neonatal skin inflammatory stimulation can induce CBS gene mRNA levels of 3 and 6 weeks old SD rats of DRG T13-L2 expression increased, with significant difference.
(4) by Western blotting method showed that neonatal skin inflammatory stimulation can induce the expression of TRPV1 protein up-regulated in colon DRG neurons of SD rats aged 6 weeks. SD rats were given 7 consecutive days intraperitoneal injection of CBS inhibitor AOAA (10mg/kgi.p.) can turn NSI induced visceral hyperalgesia, compared to control group with the same at the time of the AOAA was significantly increased in NSI rats after injection pain threshold, 30min had the greatest inhibitory effect, analgesic effect was 60min. but 10mg/kg was given by intraperitoneal injection of rat normal SD AOAA had no obvious effect on the pain threshold. At the same time CBS inhibitor AOAA expression level of NSI induced colon flip DRG neurons in rats TRPV1.
(5) immunofluorescence staining showed that there was co expression of CBS and TRPV1 on colonic specific DRG neurons.
(6) in vitro primary culture of acute DRG neurons, which were treated with sodium hydrogen sulfide (100gm), increased the number of H2S in vivo and detected the expression of TRPV1 at protein level. It was found that the expression level of TRPV1 in the experimental group was significantly higher than that in the control group.
(7) found that the application of MSP and BSP methods: the promoter region CpG island of neonatal skin inflammation can be induced in 3 week old and 6 week old SD rat colon DRG neurons in T13-L2 CBS 2 gene hypomethylation. At the same time, NSI induced 6 week old male SD rats in Dnmt3a and DRG T13-L2 Dnmt3b gene mRNA expression, while MBD2, MBD4 and TDG gene mRNA expression levels had no significant difference.
(8) the expression of CBS and TRPV1 in the intestinal mucosa and muscularis of SD rats at 6 weeks after tenth days of complete Freund's adjuvant treatment in the neonatal period was up to up expression, and there was a significant difference.
(9) compared with the control group, the expression of CBS and TRPV1 protein in the intestinal mucosa of IBS patients also increased significantly. Meanwhile, immunohistochemical staining showed that the positive signals of the expression and distribution of CBS and TRPV1 molecules in the intestinal mucosa tissues of IBS patients were higher than those of the control group.
Conclusion:
(1) neonatal inflammatory stimulation can increase the sensitivity of chronic visceral pain in adults. It is speculated that this effect is dependent on time window, dependence of stimulus intensity and independence of location.
(2) enhancement of CBS-H2S signal system plays an important role in visceral hyperalgesia induced by inflammatory stimulation in the neonatal period, which is probably due to peripheral sensitization and visceral hyperalgesia induced by excitability of intestinal specific DRG neurons.
(3) the apparent regulation characteristics of CBS gene: the hypo methylation state of CBS in colon specific neurons can increase the expression of CBS gene, that is, the synthesis of hydrogen sulfide increases, and is involved in visceral hypersensitivity.
(4) neonatal skin inflammatory stimulation can induce rat intestinal tissue in CBS and TRPV1 protein expression in adult patients with IBS at the same time, the clinical intestinal mucosa in CBS and TRPV1 expression levels also increased significantly, indicating that pain related genes may be irritable bowel syndrome pain diagnosis and treatment of the new target.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R574.4

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 Piero Portincasa;Antonio Moschetta;Giuseppe Baldassarre;Donato F. Altomare;Giuseppe Palasciano;;Pan-enteric dysmotility,impaired quality of life and alexithymia in a large group of patients meeting ROMEⅡ criteria for irritable bowel syndrome[J];World Journal of Gastroenterology;2003年10期

2 Tauseef Ali;James Choe;Ahmed Awab;Theodore L Wagener;William C Orr;;Sleep,immunity and inflammation in gastrointestinal disorders[J];World Journal of Gastroenterology;2013年48期

，

本文編號：1621981