新生期体表炎性刺激诱发成年大鼠内脏痛过敏的表观调控机制研究

发布时间：2018-03-16 22:43

本文选题：肠易激综合症　切入点：不良刺激　出处：《苏州大学》2014年博士论文　论文类型：学位论文

【摘要】：研究背景：肠易激综合症(Irritable Bowel Syndrome, IBS)是临床常见的功能性胃肠疾病(Functional Gastrointestinal Disorders, FGIDs)之一,患者的主要症状表现为肠道运动功能紊乱和慢性腹痛,疼痛症状随排便过程而加重,但无明显肠道炎症或病理、生化指标的异常。目前,对于IBS的发病机理仍然不清楚,临床治疗手段短缺,现有的治疗方法和疗效均不佳,因此,对IBS腹痛等症状形成与发展的生物学机制进行研究具有重要意义。研究目的： (1)阐述新生期体表炎性刺激(Neonatal Somatic Inflammation, NSI)诱发内脏痛觉的特性,验证“新生期体表炎性刺激可以增加成年个体慢性内脏痛敏感性”的研究假设。 (2)探讨硫化氢(Hydrogen Sulfide, H2S)是否参与新生期体表炎性刺激引起成年大鼠结肠特异性背根节(DRG)神经元(T13-L2)兴奋性改变以及痛相关分子辣椒素受体或野香草受体1(Transient Receptor Potential Vanilloid1, TRPV1)的可塑性变化。 (3)研究表观调控机制参与新生期体表炎性刺激诱发H2S合酶胱硫醚-β-合成酶(Cystathionine β-synthase, CBS)基因变化的机制。 (4)初步结合临床标本进行CBS和TRPV1的分子生物学检测,以期为临床IBS诊断和治疗提供新靶点。研究方法： (1)在个体发育不同时间点(大鼠出生后10d、3w、6w),给予不同强度的炎性刺激(完全弗氏佐剂CFA、卡拉胶/角叉菜胶Carrageenan和福尔马林Formalin),通过结直肠扩张(Colorectal Distension, CRD)实验进行腹壁撤回反射(Abdominal Withdrawal Reflex, AWR)评分以检测成年大鼠的内脏痛反应。 (2)采用膜片钳方法记录新生期体表炎性刺激对成年SD大鼠结肠特异性DRG神经元(T13-L2)兴奋性的影响；同时,观察硫化氢是否参与新生期体表炎性刺激诱发的神经元兴奋性的改变。 (3)通过Real-time PCR和Western blotting方法分别检测新生期体表炎性刺激诱发的大鼠结肠DRG(T13-L2)以及肠道粘膜/肌肉组织中硫化氢合酶CBS和TRPV1分子在mRNA和蛋白质水平的表达情况。 (4)应用免疫荧光染色方法观察CBS与TRPV1在结肠特异性DRG神经元上的共表达情况。 (5)体外原代培养DRG神经元,给予硫化氢供体硫氢化钠(NaHS)孵育神经元,观察TRPV1的表达情况。 (6)通过甲基化特异性PCR(MSP)和亚硫酸氢盐处理测序法(BSP)分析比较新生期体表炎性刺激引起SD大鼠结肠DRG神经元内CBS基因的甲基化程度的差异。同时,采用Real-time PCR方法检测DNA甲基转移酶基因Dnmts (Dnmt3a和Dnmt3b)、甲基CpG结合域蛋白基因MBDs (MBD2和MBD4)和DNA修复酶-胸腺嘧啶DNA糖基化酶基因(TDG)在rnRNA水平的表达情况。 (7)在临床受试者知情同意并符合伦理学情况下,肠镜采集IBS患者和对照组肠粘膜组织,分析比较IBS患者和对照组的肠粘膜内CBS和TRPV1分子的表达差异。免疫组织化学方法检测IBS患者和对照组的肠粘膜组织内CBS和TRPV1的分布情况。实验结果： (1)通过CRD实验进行AWR评分发现：给予出生第10天SD大鼠体表炎性刺激即完全弗氏佐剂CFA(CFA:NS=2:1)处理,可以诱发成年大鼠的AWR评分显著升高,即表现为内脏痛觉过敏,且这种作用可以从第6周持续到第12周。而在出生后第21天给予CFA处理,却不能诱发成年大鼠的内脏痛觉过敏。同样,在出生后第42天给予CFA处理,也不能诱发大鼠内脏痛觉过敏。新生期给予不同强度的炎性刺激,如角叉菜胶(2%)、福尔马林(5%)及低浓度CFA(CFA:NS=1:10),均不能诱发成年大鼠的内脏痛觉过敏。 (2)应用膜片钳记录荧光染料DiI标记的结肠特异性DRG神经元(T13-L2)的兴奋性。结果发现：与对照组相比,实验组的静息膜电位(RP)绝对值显著降低,即静息膜电位显著去极化；同时,刺激的基强度显著降低,两倍和三倍基强度刺激下的放电频率显著增加。以上结果表明,与对照组相比,新生期第10天CFA炎性处理后肠特异性DRG神经元的兴奋性增加。此外,CBS抑制齐AOAA可翻转新生期体表炎性刺激诱发的结肠特异性DRG神经元的兴奋性增加,即与对照组相比,静息膜电位绝对值和基强度均升高,两倍和三倍基强度下的放电频率显著降低。 (3)通过Western blotting和Real-time PCR方法发现：新生期体表炎性刺激可以诱发SD大鼠结肠DRG T13-L2内CBS蛋白表达水平升高,持续时间可以从处理后8小时、3周、6周均存在显著性差异,而12周时无显著性差异。在转录水平,选取3周和6周龄SD大鼠结肠DRG进行检测,发现：与对照组相比,新生期体表炎性刺激可诱发3周龄和6周龄SD大鼠DRG T13-L2内CBS基因mRNA水平表达升高,具有显著性差异。 (4)通过Western blotting方法发现：新生期体表炎性刺激可引起6周龄SD大鼠结肠DRG神经元内TRPV1蛋白表达水平上调。给予SD大鼠连续7天腹腔注射CBS的抑制剂AOAA(10mg/kgi.p.)能够翻转NSI诱发的内脏痛觉过敏,即与同时间点下的对照组相比,AOAA明显提高NSI大鼠的痛阈,最大抑制效应产生在注射后30min,镇痛作用均持续60min。然而给予健康正常SD大鼠腹腔注射10mg/kg的AOAA对其痛阈无明显影响。同时CBS抑制剂AOAA可以翻转NSI诱发的大鼠结肠DRG神经元内TRPV1的表达水平。 (5)免疫荧光染色发现：CBS与TRPV1在结肠特异性DRG神经元上存在共表达。 (6)体外原代培养急性分离的DRG神经元细胞,给予硫氢化钠(100gm)处理,模拟体内H2S产生增多,收集细胞检测TRPV1分子在蛋白水平的表达情况。发现：与对照组相比,实验组的TRPV1表达水平显著上调。 (7)应用MSP和BSP方法发现：新生期体表炎性刺激可以诱发3周龄和6周龄SD大鼠结肠DRG神经元T13-L2内CBS基因的启动子调控区CpG岛2的低甲基化。同时,NSI诱发6周龄SD雄性大鼠DRG T13-L2内Dnmt3a和Dnmt3b基因的mRNA表达水平下调,而MBD2、MBD4和TDG基因的mRNA表达水平无显著性差异。 (8)新生期第10天完全弗氏佐剂处理后6周龄SD大鼠肠粘膜和肌层内CBS和TRPV1的蛋白均呈上调表达,具有显著性差异。 (9)与对照组相比,IBS患者肠粘膜内的CBS和TRPV1蛋白表达同样出现了显著升高。同时免疫组织化学染色结果显示：IBS患者肠粘膜组织内CBS和TRPV1分子的表达分布的阳性信号均高于对照组。结论： (1)新生期体表炎性刺激可增加成年个体慢性内脏痛敏感性,且推测这种作用具有时间窗口的依赖性、刺激强度的依赖性和作用部位的非依赖性等特性。 (2) CBS-H2S信号系统的增强在新生期体表炎性刺激诱发的内脏痛觉过敏中发挥重要作用,即这种增强可能是通过兴奋肠特异性DRG神经元,导致外周敏化,产生内脏痛觉过敏。 (3)CBS基因的表观调控特征：结肠特异性神经元内的硫化氢合酶基因CBS的低甲基化状态,可引起CBS基因表达的上调,即硫化氢合成增加,参与内脏痛过敏。 (4)新生期体表炎性刺激可以诱发成年大鼠肠相关组织内的CBS和TRPV1蛋白表达上调,同时临床IBS患者肠粘膜组织内的CBS和TRPV1表达水平也出现显著升高,提示疼痛相关基因可能作为肠易激综合症疼痛诊断和治疗的新靶点。
[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.

【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R574.4

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