【作者】 周娟；

【导师】 李为民；

【作者基本信息】 复旦大学 ， 中西医结合基础， 2008， 硕士

【摘要】 内脏高敏感性(visceral hyperalgesia)是指内脏对正常生理性或伤害性刺激的感觉阈值降低,反应增强,其发生机制非常复杂。以往研究多局限于胃肠道局部病变,如肠嗜铬细胞和肥大细胞的活化、肠壁内脑肠肽及其受体的表达异常等,即外周致敏机制。但内脏感觉的形成是一个复杂的过程,内脏感觉中枢传导通路和大脑高级中枢的异常,包括神经递质及其受体的表达和活性,均可影响对内脏刺激的感知。肠易激综合征(irritable bowel syndrome,IBS)是临床上一种常见病,其主要临床表现之一是腹痛和腹部不适,属于一种慢性内脏痛敏。IBS的局部肠道虽然没有明显器质性病变,但研究表明IBS病人对阈下肠道刺激感觉过敏。近年来对IBS发病机制的研究显示,脊髓中枢对内脏感觉传入投射区域的敏感性升高是形成IBS慢性内脏痛敏的重要机制。脊髓背角和侧角的内脏感觉投射神经元和中间神经元的兴奋性异常升高是形成内脏中枢敏化的机制之一,其中一些神经递质或调质如五羟色胺递质受体系统参与发挥了作用。针刺镇痛是祖国医学的瑰宝之一,具有疗效确切,毒副作用少,经济简单等优点。国内外大量临床和基础研究表明,针刺治疗各类疼痛包括内脏痛具有显著镇痛作用。AI-Chaer于2001年报道了一种能够模拟临床IBS的大鼠模型。我们在成功复制了该IBS模型大鼠后,观察了电针对肠易激综合症的治疗作用,结果发现电针能明显缓解模型大鼠慢性内脏痛敏。然而目前对于上述针刺治疗IBS模型大鼠慢性内脏痛敏作用的机制研究还有待于进一步的开展。鉴于慢性内脏痛的形成和发展与脊髓的谷氨酸递质受体系统有着密切的联系,本研究选择了脊髓NR1受体为研究对象,观察和分析脊髓NR1受体在IBS慢性内脏痛敏形成中及针刺对其疗效中的作用。具体内容报告如下:一.研究内容:1.建立IBS大鼠模型参照AI-Chaer等报道的大鼠IBS模型,将雄性SD新生鼠,自出生后8天起,每天给予结直肠扩张刺激(在这个过程中改进造模方法,根据老鼠的状态给予不同量的结直肠刺激),连续两周,使其在成年后形成慢性内脏痛敏状态。通过测定模型大鼠体重和腹泻情况、腹部撤回反射(abdominal withdrawal reflex,AWR),对模型进行综合评估。2.运用神经药理学方法,观察脊髓中枢NMDA受体在IBS大鼠慢性痛敏维持中的作用鞘内埋管,给予NMDA受体拮抗剂MK-801,观察给药前后IBS大鼠内脏痛敏行为的变化。3.运用分子生物学方法,观察脊髓中枢部位NMDA受体在IBS大鼠慢性痛敏形成过程中的表达规律1)运用Western Blot方法,从蛋白水平观察NR1受体的表达变化;2)运用RT-PCR方法,从mRNA水平观察NR1受体的表达变化。4.运用分子生物学方法,观察电针过程中脊髓中枢NMDA受体表达变化规律1)运用Western Blot方法,从蛋白水平观察电针前后NR1受体的表达改变;2)运用RT-PCR方法,从mRNA水平观察电针前后NR1受体的表达改变。二.研究结果:1.大鼠IBS慢性内脏痛敏模型的建立和评价(1)IBS大鼠模型的建立参照Al-Chaer等报道的大鼠IBS模型,并对报道的造模方法进行改进,制造成功的IBS大鼠内脏痛敏表现明显,腹泻体征表现突出,内脏痛持续时间长。通过造模方法的改进,大大提高了模型的存活率和稳定性。(2)成年IBS大鼠AWR评分异常升高成年IBS大鼠在清醒的状态下,对不同强度的结直肠扩张刺激(CRD)所产生的腹部撤回反射(AWR)进行半定量行为评分。结果表明:与正常成年大鼠相比,在各个诱发刺激强度下,IBS大鼠的AWR评分显著升高。(3)成年IBS大鼠与正常大鼠腹泻体征比较比较IBS大鼠(6周龄时)和同龄正常大鼠的腹泻体征的差异。结果表明,IBS大鼠伴有腹泻体征发生率与同龄正常大鼠比较具有显著性差异。该结果提示IBS大鼠具有明显的腹泻体征。(4)成年IBS大鼠与正常大鼠体重比较比较IBS大鼠(各个时间阶段)和同时间段正常大鼠的体重差异。结果表明,IBS大鼠(各个时间阶段)与同时间段正常大鼠体重比较无明显差异。提示造模本身对IBS大鼠体重无明显影响。2.脊髓兴奋性氨基酸递质受体系统在IBS大鼠慢性内脏痛敏中的作用2.1鞘内注射MK-801对IBS大鼠AWR评分的影响本实验观察了鞘内给予不同剂量的NMDA受体特异性拮抗剂MK-801对IBS大鼠AWR评分的影响。大鼠鞘内埋管3天后进行埋管位置鉴定,4天后进行实验。给药前进行AWR评分基础值记录。实验分为两组:正常对照组(Normal)和IBS模型组(Model),两组分别给予低、中和高剂量的MK-801(分别为0.01μg、0.1μg和1μg的MK-801)。结果表明:正常对照组大鼠鞘内注射各种剂量MK-801后AWR评分与注射前相比没有明显变化;IBS模型组鞘内注射低剂量MK-801后AWR评分呈现降低的趋势,但无统计学差异;IBS模型组鞘内注射中剂量和高剂量MK-801后AWR评分与注射前相比显著降低。该结果提示,i.t.MK-801可剂量相关性地降低IBS大鼠AWR评分。2.2 IBS模型大鼠脊髓NR1受体mRNA的表达显著升高运用RT-PCR方法检测IBS模型大鼠脊髓NR1受体mRNA的表达变化。实验分为正常组(Normal)和IBS模型组(Model)。Trizol一步法提取总mRNA。RT-PCR结果显示,与正常组相比,模型组脊髓腰膨大NR1受体mRNA表达明显升高。2.3 IBS模型大鼠脊髓NR1受体蛋白的表达显著升高运用western blot方法检测IBS模型大鼠脊髓NR1受体蛋白的表达变化。实验分为:正常组(Normal)和IBS模型组(Model)。western blot结果显示,与正常组相比,模型组脊髓腰膨大NR1受体蛋白表达明显升高。3.电针缓解IBS大鼠内脏痛敏本实验观察了单次电针在短时间内(停针后20min)对IBS大鼠AWR的影响。实验分为四组:正常对照组(Normal),模型组(Model),模型加电针组(Model+EA)和模型加假电针组(Model+Sham EA)。结果表明:在不同强度(20,40,60,80mmHg)的内脏痛诱发刺激下,与模型组相比,模型加电针组AWR评分明显降低,而假电针组无明显改变。提示单次电针在短时间内可以明显地缓解内脏痛敏。4.脊髓兴奋性氨基酸递质受体系统在电针缓解IBS慢性内脏痛中的作用4.1电针降低IBS大鼠脊髓NR1受体mRNA的表达运用RT-PCR方法检测电针对IBS模型大鼠脊髓NR1受体mRNA表达的影响。实验分为:正常组(Normal)、IBS模型组(Model)、模型加电针组(Model+EA)和模型加假电针组(Model+Sham EA)。RT-PCR结果显示,与模型组相比,模型加电针组脊髓腰膨大NR1受体mRNA表达明显降低;而模型加假电针组无明显变化。4.2电针降低IBS大鼠脊髓NR1受体蛋白的表达运用western blot方法检测电针对IBS模型大鼠脊髓NR1受体蛋白表达的影响。实验分为:正常组(Normal)、IBS模型组(Model)、模型加电针组(Model+EA)和模型加假电针组(Model+Sham EA)。Western blot结果显示,与模型组相比,模型加电针组脊髓腰膨大NR1受体蛋白表达明显降低,而模型加假电针组无明显变化。三、研究结论:1.制作成功的IBS大鼠模型能较好地模拟了临床IBS的表现:腹痛表现明显,腹泻体征表现突出,是一个比较稳定的慢性内脏痛敏动物模型。通过造模方法的改进,大大提高了模型的存活率和稳定性。2.IBS大鼠脊髓中枢NMDA R1受体表达明显增加,提示了脊髓中枢NMDA R1受体可能参与了功能性慢性内脏痛敏的维持。3.单次电针在短时间内(停针后20min)可以明显缓解慢性内脏痛敏。4.电针可以明显抑制脊髓背角NMDA R1受体的表达,这可能是针刺缓解慢性内脏痛敏的机制之一。更多还原

【Abstract】 Internal organs are sensitive to physiological or nocuous stimulus,which is called visceral hyperalgesia.And its mechanism is complex.Previous study was confined to gastrointestinal local affection such as activated enterochromaffin cells、mast cells and so on.However,the formation of visceral sensation is a complex process.The abnormality of viceral sensorius path and cerebral sophisticated center can affect visceral sensation, including some neurotransmitters and their receptors.Irritable bowel syndrome(IBS) is clinically a common disease characterized most commonly by abdominal pain or discomfort,which belongs to chronic visceral pain. Alouthgh no conspicuous structural affection on local intestines was found in IBS patients,some research indicated that people who suffered from IBS had a colon,or large intestine,that was particularly sensitive,which was also the base of chronic viseral hypersensitivity.Recent studies of IBS demonstrated that increased levels of sensitivity of visceral afferent projection to spinal cord play a role in IBS’s formation.Numerous studies indicated abnormal excitement of visceral sense projection neuron and endaxoneuron in spinal dorsal horn and lateral horn is one of central sensitization mechanism,with some neurotransmitters or neuromodulators such as glutamate involved.Many sufferers of IBS seek treatment using acupuncture,one of the important parts of Traditional Chinese Medicine.Evidences have already indicated that acupuncture is effective for treating various pains including viscral pain.Al-Chaer reported a model of chronic visceral hypersensitivity in adult rats,which can also simulate clinical IBS symptoms.In our previous study,we examined the effect of electro-acupuncture(EA) on chronic visceral pain in rats using an IBS model(a chronic visceral hypersensitivity model) and found that EA could significantly alleviate visceral hyperalgesia.However, how dose acupuncture work that still needs to be further explained. In the study,spinal N-methyl-D-aspartate receptors-1(NMDAR1,an NMDA subtype receptor of glutamata) was investigated to obtain the evidences of possible involvement of the receptors in acupuncture’s effect on chronic visceral hyperalgesia in rats.Ⅰ.Content of the study1.Animal modelTo produce IBS model rats,daily mechanical colon distention which was repeated twice a day at a 30 min interval was performed on male Sprague-Dawley neonatal rats beginning 8-21 days after their birth according to the scale of Al-Chaer et al.The model was assessed by observing both the behavioral responses such as body weight,diarrhea, abdominal withdrawal reflex(AWR).2.Experiments were performed to observe pain threshold of IBS rats before & after intrathecal injection(i.t.) of MK-801(a non-competitive antagonist of the NMDA receptor).3.The expression of NMDA R1 in the spinal cord was investigated by western blot analysis,RT-PCR in IBS rats.1) Western blotting was used to detect the expression of NMDA R1 at protein level.2) RT-PCR was used to detect the expression of NMDA R1 at mRNA level.4.The expression of NMDA R1 in the spinal cord was investigated by western blot analysis and RT-PCR in IBS rats under EA treatment.1) Western blotting was used to detect the expression of NMDA R1 before & after single EA stimulus.2) RT-PCR was used to detect the expression of NMDA R1 before & after single EA stimulus.Ⅱ.Results of the study1.Assessment of IBS model 1.1 production of IBS modelAccording to Al-Chaer’s report,we produced the IBS model rats successfully. Further,we improved the method of model production and made the IBS model more stable with long term lasting visceral pain and more apparent sign of diarrhea.1.2 Behavioral test for assessing visceral pain sensationBehavioral responses to graded strengths ofcolorectal distention(CRD)(20,40,60, and 80 mmHg) were assessed by observing the abdominal withdrawal reflex(AWR). Semi-quantitative scores were used for judgment of the responses to CRD stimulus.AWR scores were assigned according to the scale of Al-Chaer et al.And the averaged responsive value was taken for analysis.There is a significantly enhanced response to CRD stimulation in IBS rats,but no such effects in normal rats stimulated by graded CRD stimulation at above strengths.1.3 Comparison of diarrhea signs between IBS rats and normal onesThere is a significant increase on incidence of diarrhea in IBS rats compared with normal rats.1.4 Comparison of body weight between IBS rats and normal onesThere were no significant differences on body weight among the above group of rats at the same time.2.NMDA receptor plays a role in chronic visceral pain of IBS rat2.1 The effect intrathecal injection of MK-801 on AWR scores in IBS ratThe rats were treated with intrathecal injection of MK-801,3 days after which the location was assessed and then experiment was conducted one day later.Before injection of MK-801 in each group,AWR scores were detected to obtain the base value.Rats were divided into 2 groups(n=8 in each group):normal group(normal rats) and IBS model group(IBS rats).Both groups were treated with MK-801 at doses of 0.01μg,0.1μg and 1μg.The IBS rats which were treated with MK-801 at dose of 0.1μg and 1μg produced a remarkable reduction of the enhanced responses to CRD stimulation,but not in 0.01μg group.That indicates intrathecal injection of MK-801 could depress pain threshold in a dose-dependent manner.2.2 The expression of spinal NMDA R1 mRNA in IBS ratRT-PCR was used to detect the expression of NR1 at mRNA level in the spinal cord. Rats were divided into 2 groups(n = 8 in each group):normal group(normal rats) and IBS model group(IBS rats).In contrast to normal rats,there was significant increase of NR1 mRNA expression in model rats.2.3 The expression of spinal NMDA R1 protein in IBS ratWestern blotting was used to detect the expression of NR1 at protein level in the spinal cord.Rats were divided into 2 groups(n = 8 in each group):normal group(normal rats) and IBS model group(IBS rats).In contrast to normal rats,there was significant increase of NR1 expression in model rats.3.Relief of visceral pain in IBS rats by EA treatmentAWR assessment was performed during 20-90 min after EA treatment.Rats were divided into 4 groups(n = 8 in each group):normal group(normal rats),IBS model group (IBS rats),EA group(IBS rats treated with EA) and sham EA group(IBS rats treated with EA).In the EA group,EA produced a partial but significant reduction of the enhanced responses to CRD stimulation,but no analgesic effects by sham EA on AWR scores induced by graded CRD stimulation at above strengths.It shows that single EA treatment could alleviate visceral pain within a short time period.4.NMDA receptor plays a role in EA relieving visceral pain in IBS rat4.1 The expression of spinal NMDA R1 mRNA in IBS rat treated with single EART-PCR was used to detect the expression of NR1 at mRNA level in the spinal cord. Rats were divided into 4 groups(n = 8 in each group):normal group(normal rats),IBS model group(IBS rats),EA group(IBS rats treated with single EA) and sham EA group (IBS rats treated with sham EA).There was significant reduction of NR1 mRNA expression in EA group rats but not in other groups.4.2 The expression of spinal NMDA R1 protein in IBS rat treated with single EAWestern blotting was used to detect the expression of NR1 at protein level in the spinal cord.Rats were divided into 4 groups(n = 8 in each group):normal group(normal rats),IBS model group(IBS rats),EA group(IBS rats treated with single EA) and sham EA group(IBS rats treated with sham EA).There was significant reduction of NR1 expression in EA group rats but not in other groups.Ⅲ.Conclusion of the study1、The IBS model rats in this study can simulate clinical IBS symptoms such as conspicuous abdominal pain and diarrhea in humanbeings.It is an experimental model of chronic visceral hypersensitivity.We improved the method of IBS model production,made the model more stable and increased the survival rate of model animals.2、The increased expression of NMDA R1 receptors in the spinal cord of IBS model rats indicates that the excitatory amino acid system may be involved in IBS chronic visceral pain.3、EA treatment can alleviate chronic visceral hypersensitivity effectively in the IBS model rats.4、The above effect of EA treatment may be induced by a decrease in the expression of NMDA receptors in the spinal cord of IBS model rats.更多还原