【作者】 朱雪萍；

【导师】 陈卫昌；

【作者基本信息】 苏州大学 ， 消化内科学， 2007， 博士

【摘要】 第一部分苏州市儿童肠易激综合征(IBS)的流行病学危险因素研究目的探讨苏州市区小学生肠易激综合征(IBS)的患病率及危险因素。方法采用流行病学横断面研究方法对苏州市沧浪、平江及金阊辖区内公办小学1～6年级小学生进行问卷调查。采用多阶段、随机整群抽样的方法获取调查样本。在调查对象中,按罗马Ⅱ诊断标准,排除其他器质性疾病后确诊IBS病例,其他非IBS病例为对照。拟合非条件logistic多元回归模型,分析影响IBS发病可能的危险因素,为儿童IBS的预防提供科学的依据。结果共发放问卷8000份,回收有效问卷7472份,回收率93.4%;苏州市区1～6年级小学生有症状符合罗马Ⅱ诊断标准的IBS患病率为10.81%。男、女IBS患病率差异无统计学意义(10.3%vs11.3%)。多因素logistic回归模型分析提示:有食物过敏史(OR=1.53,95%CI:1.13～2.07)、儿童期有肠炎史(OR=1.29,95%CI:1.00～1.63)、喜食油炸食物(OR=1.62,95%CI:1.34～1.96)、心情焦虑(OR=1.49,95%CI:1.16～1.93)、儿童幼年时有意外打击史(OR=1.47,95%CI:1.02～2.20)及父母有便秘史(OR=1.81,95%CI:1.46～2.24)为男女儿童IBS发病最可能的危险因素。儿童年龄越大,IBS发病危险有逐渐下降的趋势(OR=0.94,95%CI:0.89～0.99)结论IBS是苏州市区小学生的常见病。影响苏州市区小学生IBS可能的危险因素是:年龄小、有食物过敏史、儿童期有肠炎史、喜食油炸食物、心情焦虑、儿童幼年时有意外打击史及父母有便秘史。因此,提示临床医师应重视此病,积极防治肠炎,调整儿童饮食,避免多食油炸食品及过敏的食物,以更好地防治本病。第二部分幼鼠肠易激综合征模型的建立及鉴定目的建立适合儿童肠易激综合征(IBS)研究的幼鼠IBS动物模型并鉴定。方法应用20只SD新生大鼠,随机分为IBS模型组和正常对照组,IBS模型组进行母子分离、改良机械束缚、结肠芥子油刺激等综合因素干预幼鼠建立IBS的动物模型;正常组不作干预。用自制的扩张器对幼鼠进行直肠扩张,评估不同压力下腹部收缩反射(AWR)阈值,测定幼鼠腹壁肌电活动;利用血管球囊扩张导管以5ml、10ml分别扩张IBS模型组、对照组幼鼠直肠,分别采集两组鼠脑部的fMRI数据,采用脑功能成像分析软件进行数据处理,应用血氧水平依赖性功能性MRI(BOLD-fMRI)技术,研究直肠球囊扩张刺激下肠易激综合征模型幼鼠脑内脏痛觉中枢兴奋情况及其兴奋区分布情况;取乙状结肠降段结肠用分层刮片技术刮取由外向内取1-4层细胞,丫啶橙荧光染色观察幼老细胞的比值并对肠粘膜上皮细胞进行美兰还原反应时间测定;并行结肠组织病理检测;干预后称两组幼鼠体重,观察粪便性状,查粪常规,并收集粪便称湿重和干重,应用Bristol分型观察各组粪便性状进行评分。结果直肠扩张时,在不同压力下模型组腹壁收缩反射(AWR)评分较对照组阈值显著降低(P值均<0.01);不同扩张压力下,腹壁肌电活动随压力增加明显增加(P<0.01);直肠球囊刺激后可引起IBS模型组幼鼠脑内与内脏相关的脑区(脑岛皮质、额前皮质、丘脑及扣带前皮质)神经元活动增强,表现为高兴奋区,而对照组幼鼠无相应兴奋区表现;结肠黏膜分层刮片后丫啶橙染色见IBS模型组粘膜上皮最外层细胞幼老细胞比值较正常对照组明显减少,美蓝还原时间延长(P<0.05);结肠病理检查两组均未见明显异常;IBS模型组腹泻症状明显较正常对照组增加(P<0.05),模型组粪便多为软的团块或泥浆样,而对照组为柔软的香肠状或团块状,两组粪便湿重存在显著差异(P<0.05),而体重、粪便常规检查两组间无明显差异。两组Bristol分型评分比较有显著性差异(P<0.05)。结论用母子分离、改良机械束缚、结肠芥子油刺激等综合因素干预幼鼠可成功建立适合儿童的幼鼠IBS动物模型,其机制可能为内脏敏感性增高。功能MRI是一种较为直观且准确的观察脑功能活动的脑显像技术,本研究显示IBS幼鼠中枢内脑岛皮质、额前皮质及丘脑可能是参与内脏感觉的主要部位。幼鼠IBS模型结肠粘膜上皮细胞功能处于抑制状态。第三部分ICC细胞和脑肠肽在幼鼠IBS模型中作用机制的实验研究目的旨在通过检测幼鼠结肠、脊髓和丘脑组织中脑肠肽NPY和cGRP的mRNA表达和结肠ICC细胞c-kit阳性细胞的表达,探讨脑肠肽、脑肠轴及ICC细胞在幼鼠IBS模型发病机制中的作用。方法根据简单数字法随机将乳鼠分成IBS模型组和正常对照组二组,各10只。IBS模型组用母子分离、改良机械束缚、结肠芥子油刺激等综合因素干预的方法造模,对照组则不做任何干预。采用半定量逆转录多聚酶链式反应法检测二组幼鼠结肠、脊髓、丘脑组织中NPY和cGRP mRNA的表达。另取结肠组织通过免疫组化检测肌间ICC细胞c-kit阳性表达细胞数。结果IBS模型组中NPY在结肠、脊髓、丘脑组织中mRNA表达明显较正常对照组降低,t值分别为9.66,8.55,12.65,(均P<0.01);而cGRP则在脑肠轴各个层面(结肠、脊髓、丘脑)的表达中,IBS组明显较正常对照组增加,t值分别为14.41,12.85,12.41,(均P<0.01)。二组幼鼠结肠ICC细胞c-kit阳性表达细胞数有差异(t=5.294,P<0.01)。结论ICC细胞、脑肠肽可能在幼鼠IBS模型的发生、发展中起重要作用,本研究为深入研究儿童IBS发病机制提供了实验依据。第四部分马来酸曲美布汀干预幼鼠IBS模型的疗效观察目的探讨马来酸曲美布汀干预幼鼠肠易激综合征(IBS)模型后的治疗疗效。方法用母子分离、改良机械束缚、结肠芥子油刺激等综合的方法建立幼鼠IBS模型共20只,随机选10只在干预同时用马来酸曲美布汀3mg/kg.d灌服治疗为治疗组,另10只为模型组,取同期正常生长幼鼠10只为正常对照组。分别对三组幼鼠行肛门直肠球囊扩张刺激下测腹壁肌电活动,AWR评分;处死后取结肠对其肠粘膜上皮细胞进行分层刮片、荧光染色和还原能力测定,RT-PCR检测结肠、脊髓、丘脑中NPY和cGRP的mRNA表达。结果IBS模型组和正常对照组间不同压力下AWR评分均有显著性差异(P<0.05),而治疗组和正常对照组间差异无显著性(P>0.05);在8mmHg压力时,IBS模型组腹壁收缩次数增加达(6.65±1.04)次/3 min ,肌电图振幅增高,与另二组比较有显著性(P<0.01),而治疗组、正常对照组间腹壁收缩活动和肌电图振幅差异无显著性(p>0.05);在12mmHg、15mmHg扩张压力下,IBS模型组腹壁收缩活动增加,肌电图振幅增高(P<0.05),但正常对照组与治疗组间无显著差异(p>0.05);在28mmHg压力扩张下,三组幼鼠腹壁收缩次数及肌电图振幅增高差异均无显著性(P>0.05);三组结肠粘膜由外向内1-4层取材标本中,IBS模型组的肠粘膜上皮细胞美蓝还原反应时间最长(11.8±1.45)min ;NPY在IBS模型组结肠、脊髓、丘脑三个部位的表达均较另二组低,F值分别为11.29,34.15,27.13(均P<0.05),而治疗组和对照组间差异无显著性;cGRP在模型组三个部位的表达均较正常对照组和治疗组增强,F值分别为98.58,39.12,82.67(均P<0.01),而治疗组和对照组间表达无显著性差异(P>0.05)。结论马来酸曲美布汀治疗幼鼠IBS模型有效,可改善其肠道运动功能及内脏高敏感性。更多还原

【Abstract】 PartⅠEpidemiological Study on Risk Factors for Children’s Irritable Bowel Syndrome (IBS) in SuChow cityObjective To investigate the prevelance rate of primary school students’irritable bowel syndrome(IBS) and explore its risk factors in SuChow city.Methods A cross-sectional study was carried out from Nov, 2006 to Mar, 2007. Standard-structured questionnaire including demographic characteristics,possible risk factors for IBS was used to investigate the screened IBS cases (n=808) according to RomeⅡcriteria,excluding the gastrointestinal pathologic-diseases and non-IBS controls (n=6664). Study subjects were sampled from 1-6 grade primary school students in Canglang, Pingjiang and Jingchang districts, SuChow city, using multi-stage randomized cluster sampling method. Multivariate logistic model was used to analyze the possible risk factors for IBS. Odds ratio (OR) and 95% confidence interval (95%CI) were also estimated. Results We totally sent out 8000 and received 7472 questionnaires with 93.4% response rate. The prevalence rate for IBS among 1-6 grade students in SuChow primary schools was 10.81%. The prevalence rate of IBS decreased significantly with increasing of age the school grade (Cochran-Armitage, P<0.05). Multivariate logistic model analysis showed that history of food allergy (OR=1.53,95%CI:1.13~2.07), gastroenterotitis in childhood (OR=1.29, 95%CI: 1.00~1.63), fond of fried food (OR=1.62, 95%CI: 1.34~1.96), anxiety (OR=1.49, 95%CI: 1.16~1.93), accident attack psychologically in childhood (OR=1.47, 95%CI:1.02~2.20) and parental constipation (OR=1.81, 95%CI:1.46~2.24) were the possible risk factors of the IBS. The increasing ages was associated with the increasing IBS risk (OR=0.94, 95%CI: 0.89~0.99). Conclusions IBS is a common disease among pupils, in SuChow city. To decrease the prevalence rate for IBS in primary school students, we should pay more attention to the IBS severity situation in pupils, and adopt some further measures such as preventing enteritis in childhood, decreasing the irritating food intake for children and avoiding from this kind of disease.PartⅡThe Establishment and Identification of the Infant Rat Irritable Bowel Syndrome Animal ModelObjective This investigation was to establish an infant rat animal model of irritable bowel syndrome with“integrative methods”; and to test it. Methods Twenty SD neonatal rats were divided into model group and control group randomly. The model was established by separated from mother rat about 3 hours daily in the neonatal 2 days, binded up mildly and intrarectal administration of mustard oil daily between 8 and 16 days; the control group didn’t intervene. After the intervenence, let the rats have a rest for 2 weeks. The threshold of abdominal withdrawal reflex (AWR) was evaluated during rectal distension by self-made dilator in different tensions ,and the changes of rectal sensitivity were identified by the abdominal electrical activity measured at postnatal 30 days. Vascular ballon-distention cathether were used by 5ml and 10 ml to dilate the rectal of the experimental group rats and control group rats. Blood resonance imaging (BOLD-fMRI) were performed in both groups and the datas were analyzed by the cerebral function imaging analyze soft. The effect of rectal balloon-distention stimulus by BOLD-fMRI in viseral pain center and the correspond stimuli districts of the irritable bowel syndrome model infant rats was examed. The intestinal epithelial cells outer 1-4 layers in descending colon were collected using slide techniques, fluorescence staining for investigation the cells ratio of young to old and reduction ability determination were performed. At last the colon pathology was measured. At the beginning of intervenence, the two groups’feces were scored according to the Bristol pattern, gathered the feces and measured their wet and dried weights and weighed the rats’weights and tested the feces’routine . Results In contrast to the two groups, the model group showed a significant decrease (P<0.01) in the volume threshold of abdominal withdral reflex during rectal distension; there was a significant increase of abdominal electrical activity responding to different distension pressure compaired with control rats (P<0.01). Rectal distention stimulation increased the activity of the viseral pain center in the brian conclude insular cortex, prefrontal cortex, thalamus and anterior cingulate cortex in most model group rats, while no such manifestation in control group rats. The ratio of young to old cells in outest layers of model group’s colonic mucosa epithelial cells more rapidly decreased than that of control group by MTT staining, and the reduction time of methylene more prolonged (P<0.05), significantly. The colon’s pathology had no significant difference in the two groups. The feces of model group were mostly soft mass or muddy, Bristol Scores was 5.3±0.8; which of control group were delicately sausage or mass, Bristol Scores was 3.6±0.5, the difference was significant in the two groups. The wet and dried weights of the model’s feces were 10.8±1.2、3.2±0.4 respectively,and the control’s were 5.4±0.3、3.6±0.7 respectively, there was significant difference in the two groups’wet weights (p<0.05). The symptom of diarrhea in model group was higher than that in control group (p<0.05),and there was no significant different in weights and the routine of feces. Conclusions The‘integrative’methods (concluded to separation from mother、machinism fetter and chronic rectal irritation with mustard oil) to the infant rats may establish the children’s irritable bowel syndrome model, of which mechanism is visceral hypersensitivity possiblely. fMRI is an objective brian imaging technique to measure the change in regional brian activation exactly. It can reveal the brian viseral hypersensitivity areas in response to painful rectal ballon-distention in IBS model rats. In this study insular cortex, prefrontal cortex and thalamus of the IBS rats are the major positions in the CNS processing of viseral perception. The functions of the intestinal epithelial cells in IBS young rat model were in the inhibitory states.PartⅢThe Mechanism Study on ICC and Neuropeptides in Infant Rat Irritable Bowel Syndrome ModelObjective To clarify the effect of neuropeptides and ICC in the animal model for the infant rat IBS by detecting the mRNA expression of neuropeptides(ect NPY and cGRP) in colon,spinalcord and thalamencephalon and the expression c-kit positive of colon ICC. Methods Twenty SD neonatal rats were divided into model group and control group randomly. The model group was established by integrative methods(concluded to separation from mother、machinism fetter and chronic rectal irritation with mustard oil), and the control group didn’t intervene. The expressions of NPY and cGRP for the two groups’rats colon, spinal cord and thalamencephalon were tested by semi-quantity reverse transcription polymerase chain reaction. Meanwhile the number of c-kit positive ICC in intramyo of colon was counted by immunohistochemistry testing. Results The mRNA expressions of NPY in model group at colon, spinal cord and thalamencephalon were rapidly lower than that in control, t value was 9.66,8.55,12.65, respectively, (P<0.01). Otherwise the expressions of cGRP in model group were higher than that in control group, t value was 14.41,12.85,12.41, respectively, (P<0.01). There were differences between the two groups in the number of c-kit positive ICC, the model group with significant decrease (t=5.294,P P<0.01). Conclusions ICC and neuropeptides play an important role in the occurrence and development of the infant rat IBS, and offers a rational reason for the study of pathogensis to children’s IBS.PartⅣThe Effectiveness Study on Trimebutine Maleate in Intervenence of Irritable Bowel Syndrome of Infant Rat ModelObjective To investigate the effectiveness on trimebutine maleate in the treatment of IBS young rat models. Methods 20 young rats animal model with IBS by intergrative methods were established. 20 IBS rats were randomly divided into the treatment group with trimebutine maleate(3mg/kg.d,po as intervened) and IBS model group, and at the same time the 10 infant rats which grew up regularly were control group. Abdominal wall myoelectric activities and AWR Scores were measured when the three groups of the immature rats’anus and rectum were distended by sacculus vestibularis; the intestinal epithelial cells in different colonic mucosa layers were collected using slide techniques, fluorescence staining and reduction ability determination were performed after death, and the mRNA expressions of NPY and cGRP in colon,spinal cord and thalamencephalon were examined by reverse transcription polymerase chain reaction. Results AWR Scores were quite different between model group and control group under different pressures (P<0.05), having no significant difference between treatment group and control group(P>0.05). Under the pressure of 8mmHg, no distinctive changes of abdominal muscle contractions and abdominal electromyogram amplitudes were in the treatment and control groups, while the abdominal muscle contractions reached ( 6.65±1.04) per/3 min in model group (P<0.01) and abdominal electromyogram amplitudes also rapidly increased in comparison with the control and treatment groups. Under the pressure of 12mmHg, the results almost keeped the same, that was the changes and increase of abdominal muscle contractions and abdominal electromyogram amplitudes were distinctive in model group rather than the other two (P<0.05). Under the pressure of 15mmHg, the increase of abdominal muscle contractions in model group was still obvious(P<0.05), but no distinct difference exited between model group and treatment group this time(p>0.05). When the pressure reached 28mmHg, no distinctive differences were there in these three groups. The ratio of young to old cells in outset layers of model group’s colonic mucosa epithelial cells was the lowest by MTT staining, but it keeped nearly the same in the other two groups. And the reduction time of methylene in the outset layers of model group was longest (11.8±1.45)min. The mRNA expressions of NPY in model group at colon, spinal cord and thalamencephalon were rapidly lower than that in treatment and control groups, F value was 11.29,34.15,27.13, respectively, (P<0.05), having no significant difference between treatment and control groups. Otherwise the expressions of cGRP in model group were higher than that in treatment and control groups, F value was 98.58,39.12,82.67, respectively, (P<0.01), and there was no rapid difference between treatment group and control group. Conclusions It is effective for intevene the infant rats model with trimebutine maleate without side effect. Trimebutine maleate can improve the function of gastrointestinal motion and restrain the visceral hypersensitivity, and thus it is quite worthy to popularize it in clinic treatment.更多还原