儿童功能性便秘的流行病学调查、功能分类及部分类型的病理生理基础研究
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摘要
前言
儿童便秘为常见的临床症状,而不是一种独立的疾病。据Thowa大学和Heston大学医院儿科门诊统计,儿童便秘的发生率为3%—8%,其中90%—95%是功能性便秘。按照我国13亿人口统计,约有3亿儿童,其中有二千万左右的儿童患有便秘。便秘对儿童个体生长发育危害巨大,病因目前尚不清楚,治疗上存在泻药滥用问题;如何判定儿童便秘病因,并制订有针对性的措施进行治疗,正是本研究所要解决的问题。
由于便秘在医学中地位及重要性的迅速提高,国内外相继对便秘及其病因等进行了众多的流行病学调查研究,尤其在成人便秘方面投入了大量的人力物力。迄今为止,国内己有北京、广东广州、成都、重庆、杭州、天津、上海、西安等地区对成人便秘及排便习惯进行了流行病学调查,国家更将老年人的便秘调查与多因素分析列为国家“九五”攻关项目(科研编号96-906-05-09),拨巨资支持。但是由于正常人群的排便习惯受饮食习惯、精神心理因素、环境和种族、文化背景等诸多因素的影响,儿童处于生理发育期,胃肠系统尚不成熟,因此儿童FC的患病情况相差较大,尤其是国内,此类研究相对少见,本研究从儿童最常见而且尚未解决的问题入手,首先对中国北方具有代表性的城市儿童进行大范围的流行病学调查,探讨北方儿童便秘的患病率,并在调查的基础上提出我国儿童便秘的诊断标准。
功能性便秘是一个复杂的病理生理过程,其确切的发病机制目前尚不清楚,结直肠动力、盆底肌群的功能以及肠道菌群紊乱都可能与功能性便秘有关。在众多的研究中,结肠动力低下被认为是功能性便秘发生的重要因素之一,已有动物实验表明,内源性麻酚类物质可以抑制胃肠动力,主要是通过分布于神经末梢的受体CB1起作用。脂肪酰胺水解酶(Fatty acid amide hydrolase,FAAH)在体内能催化内源性麻酚类物质的水解,因此在理论上可以通过调节FAAH的活性来控制内源性麻酚类物质,以达到调节胃肠动力的目的。到目前为止,关于FAAH是否在人类的胃肠道中的作用还未见报道,因此本研究着眼于此,力图通过免疫组化、蛋白印迹和定量PCR技术从不同水平观察FAAH的表达情况,探讨其在便秘的发生过程中的作用。另外,随着分子生物学技术在微生物领域应用的不断进步和微生态学的不断完善,越来越多的肠道菌群被克隆和分离出来,细菌在胃肠道中的作用也逐步得到确认,人们对肠道菌群的认识已不仅仅停留在以往的消化和吸收方面,越来越多的作用被开发出来,研究显示,肠道菌群可以通过其代谢产物调节胃肠动力,尤其是短链脂肪酸,可以通过多种不同的机制影响胃肠动力功能,因此本研究应用PCR-SSCP和BOX-PCR技术对正常肠道菌群进行DNA指纹图谱分析,观察肠道菌群在正常和便秘儿童肠道中的分布比例关系并结合应用气相色谱技术观察细菌代谢产物短链脂肪酸在正常和便秘儿童肠道中的分布比例关系,探讨肠道菌群及其代谢产物短链脂肪酸在儿童功能性便秘发病中的作用。
材料和方法
一、流行病学调查
应用分层、整群随机抽样方法对中国北方5市(包括北京、天津、沈阳、吉林和哈尔滨)常住儿童中2～14岁的儿童进行问卷调查,调查问卷由专门从事流行病学调查的专家协助设计,首先进行预调查,然后进行普查,共发放问卷20000份,其中北京6000份,天津4000份,沈阳4500份,哈尔滨3000份,长春2500份,根据普查结果了解按儿童罗马Ⅱ标准判断的FC的检出率,再对筛出的部分人在医院进行精查(结肠传输试验,钡灌肠及排便造影,消化道测压)以排除结肠器质性疾病,计算我国北方城市儿童儿童功能性便秘的患病率。详细记录与儿童功能性便秘有关的各种危险因素以及与排便有关的各种相关症状,对各种危险因素与儿童功能性便秘的关系进行逐步logistic回归分析,分析其对于儿童功能性便秘的贡献;按儿童罗马Ⅱ标准筛选确定便秘组,分析各相关症状在便秘组和非便秘组发生的频率,用卡方对单因素进行比较,再对有意义的症状用logistic回归模型作拟合优度检测,探讨儿童功能性便秘的危险因素,初步制定适合我国儿童的便秘标准。
二、排便功能检测
研究对象分为对照组和便秘组。对照组33例,平均年龄5岁;便秘组96例,平均年龄6.5岁,均符合儿童功能性便秘罗马Ⅱ诊断标准;联合应用结肠传输试验,X线排便造影和肛门直肠测压对对照组和便秘组儿童进行全面检查,分析其影像学及功能学表现的差异,探讨这些异常在儿童功能性便秘的病理生理及其对便秘的诊断和分型的意义;
三、慢传输性便秘儿童结肠壁内FAAH的表达及意义
研究对象分为便秘组和对照组,对照组16例,其中男10例,女6例,平均年龄7.5岁。便秘组男15例,女7例,平均年龄7岁,均符合STC的标准,分别取上述病例结肠标本,按部位分为升、降和乙状结肠,其中对照组升结肠5例,降结肠3例,乙状结肠8例;便秘组升结肠4例,降结肠18例,乙状结肠22例;应用免疫组化,Western blotting,Real Time PCR对便秘组和对照组儿童结肠壁内FAAH的表达分析,探讨FAAH在正常结肠壁内的定位和定量表达及其意义,并联合应用免疫荧光双重染色技术观察FAAH与CB1受体的位置与分布,探讨FAAH在生理条件下对结肠功能进行调控的作用机制;同时还进一步联合应用高压液相色谱技术对便秘组儿童结肠壁内FAAH的生物活性进行定量检测和分析,探讨其在儿童功能性便秘发病中的作用。
四、功能性便秘儿童肠道菌群DNA指纹图谱的建立及意义
研究对象分为对照组和便秘组,采用儿童功能性便秘罗马Ⅱ标准确立便秘组,便秘组15例,男9例,女6例,平均年龄6.5岁;对照组35例,男24例,女11例,平均年龄6岁。取对照组和便秘组所有儿童粪便标本进行分析。应用粪便细菌基因组DNA提取试剂盒提取粪便细菌基因组DNA,分别应用细菌16S rDNA的V3区引物和BOX基因组重复序列的引物BOX A1R进行扩增,然后进行电泳和图像分析,分别建立基于16SrDNA和BOX基因组重复序列的粪便细菌DNA指纹图谱,探讨PCR-SSCP和BOX-PCR对于粪便细菌的分离效果及其对粪便细菌菌种鉴定的意义;将扩增产物进行克隆测序,测序后对序列的同源性在GenBank数据库中使用BLAST工具进行比较,初步确定细菌的种属和分类,探讨肠道菌群在功能性便秘儿童的变化趋势及其对便秘产生的意义。
五、功能性便秘儿童粪便短链脂肪酸的检测与分析
应用气相色谱技术对便秘组和对照组儿童粪便中短链脂肪酸的含量进行定量检测,分析短链脂肪酸在功能性便秘儿童肠道内的变化趋势,进一步探讨肠道微生态菌落及其代谢产物短链脂肪酸在儿童功能性便秘发病中的意义。
结果
一、流行病学调查
本次调查共发放问卷20000份,其中北京6000份,天津4000份,沈阳4500份,哈尔滨3000份,长春2500份。回收19638份,其中北京5452份,天津3788份,沈阳4066份,哈尔滨2713份,长春2267份,回收率98.19%;有效问卷19286份,有效率96.43%;有913名符合儿童罗马Ⅱ诊断标准,占被调查人数的4.73%;儿童FC的患病率具有地域差异性和年龄差异性特点,从地区上看,北京的患病率最高,为5.02%,长春最低,约4.27%,统计学差异显著(P＜0.001);从年龄上看,婴幼儿期(2-3岁)的患病率最高,学龄期(7-11岁)最低,统计学差异显著(P＜0.001)。性别分布无明显差异。逐步logistic回归证实人工喂养、添加辅食过早、饮食纤维低、挑食偏食、排便恐惧、抑郁、焦虑、体重、遗传、高经济收入及为FC的危险因素。
对便秘的相关症状逐个进行单因素卡方分析比较,结果显示包括排便次数＜3次/周,大便干硬成团块状,排便费力,腹痛,恶心呕吐等在内的22种症状虽可在非FC人群中不同程度存在,但FC组发生频率比非FC组明显为高,统计学差异显著,多因素逐步logistic回归分析,结果显示排便次数＜3次/周,大便干硬成团块状,排便费力,需要泻药协助排便等症状在FC发生率更高,对诊断FC最有意义,减少的其他症状对FC诊断无影响,并以国际通用罗马Ⅱ标准为黄金标准,对拟定儿童FC症状学标准诊断试验的灵敏度,特异度和诊断准确率进行评价,结果显示,具备2条者的特异度和灵敏度均较适中,但诊断准确率最高。
二、排便功能检测
结肠传输时间测定结果显示,便秘组的TGITT、LCTT和RSTT较对照组明显延长,RCTT在两组无显著性差异。儿童FC结肠传输时间延长具有以下几种形式:①RCTT、LCTT和RSTT都延长;②单纯RSTT延长;③RCTT、LCTT和RSTT都延长,但RSTT延长更显著;④TGITT正常。X线排便造影显示耻骨直肠肌痉挛综合症14例:排便状态下直肠肛管角不开大,直肠后壁耻骨直肠肌压迹加深;肛门外括约肌痉挛4例:直肠肛管角正常开大,但会阴下降不明显,肛管前壁出现指状压迹;肠疝(包括小肠疝和结肠疝)3例:小肠或结肠下端疝入直肠膀胱陷凹,压迫直肠前壁;直肠粘膜脱垂和套叠3例:排便时肛管上方出现直肠粘膜影像;直肠前突2例:排便时直肠前壁向前方突出深度超过2cm;会阴下降综合症1例:盆膈摆动幅度大,下降时超过2cm;盆底痉挛综合症2例:表现为“鹅征”。肛门直肠测量结果显示,便秘组的肛管最大收缩压升高,对称指数明显降低而向量容积虽有增加,但不显著。
三、慢传输性便秘儿童结肠壁内FAAH的表达及意义
应用免疫组化,Western blotting,Real Time PCR对便秘组和对照组儿童结肠壁内FAAH的表达进行分析发现,正常结肠无论是升结肠、降结肠还是乙状结肠其壁内蛋白水平和mRNA水平均存在FAAH的表达,各段表达无显著差异,并且免疫染色显示FAAH主要定位于肠壁肌间神经元和粘膜的吸收细胞和肠腺体;在正常结肠,结合免疫荧光双重染色和共聚焦显微镜发现肌间神经元中FAAH的分布在位置上与CB1受体呈对应趋势,而在吸收细胞和肠腺体中未见此改变;在便秘儿童结肠,FAAH的表达显著减弱,结合高压液相色谱技术发现,FAAH的水解活性也显著降低,差异具有统计学意义。
四、功能性便秘儿童肠道菌群DNA指纹图谱的建立及意义
对照组和便秘组的所有样品分别进行3次以上的PCR-SSCP和BOX-PCR指纹图谱分析,得到了稳定可重复的图谱,无论便秘组还是对照组个体,图谱中均可见较多的电泳条带,从条带位置看,在对照组与便秘组,对照组之间以及便秘组之间既存在个体一致性条带,同时也存在个体特异性条带,且各条带亮度不鞠嗤?与PCR-SSCP图谱相比BOX-PCR图谱的泳带条数更多,各泳带之间界限清晰。测序结果显示,便秘组和对照组肠道内即存在相同的细菌菌种,也存在各自不一的菌种。在便秘组,菌种的种类较对照组明显增多,统计学差异显著;结合BOX-PCR指纹图谱的条带位置,对照组优势菌主要为拟杆菌属,埃希氏菌属,双歧杆菌属,乳杆菌属;便秘组的优势菌主要为脆弱拟杆菌属,埃希氏菌属,真杆菌属和梭菌属。
五、功能性便秘儿童粪便短链脂肪酸的检测与分析
气相色谱分析结果显示功能性便秘儿童粪便中每克干粪中短链脂肪酸含量较对照组减少,统计学差异显著,但便秘儿童粪便中含水量减少,短链脂肪酸显著升高,明显高于对照组,统计学差异显著,且各短链脂肪酸比例失调。
结论
(1)儿童FC是儿童期的常见病多发病,好发于2～3岁儿童;人工喂养、添加辅食过早、饮食纤维低、挑食偏食、排便恐惧、抑郁、焦虑、体重、遗传、高经济收入可能是FC的危险因素,应该对民众进行广泛的宣教做针对性预防和治疗。
(2)我国儿童FC的症状学诊断标准为:①排便次数＜3次/周;②大便干硬成团块状;③排便费力;④需要泻药协助排便。具备上述的4个诊断标尺中2条者,症状持续至少2月即可诊断便秘。
(3)儿童功能性便秘可以分为以下几种类型:①慢传输型便秘,近端结肠各节段传输功能异常。②出口梗阻型便秘:结肠出口处肛门直肠区域存在动力异常。③混合型便秘:同时存在结肠传输障碍和出口动力异常;联合应用结肠传输时间,X线排便造影和肛门直肠测压对儿童功能性便秘的病因诊断和临床分型具有重要意义。
(4)人类正常结肠壁内无论是蛋白还是核酸水平均存在FAAH的表达,FAAH主要表达在结肠肌间神经丛的神经元内和肠粘膜表面的吸收细胞和腺体内,在肌间神经丛内FAAH的分布与CB1受体呈对应趋势。
(5)慢传输性便秘儿童结肠壁内FAAH的表达减低,水解活性减弱,FAAH可能参与了慢传输性便秘的发生。
(6)功能性便秘儿童肠道菌群的菌属多样性增加,肠道内存在与正常儿童相似的常驻菌,但优势菌种不同,各肠道菌属间存在比例失调。
(7)功能性便秘儿童粪便中的短链脂肪酸含量减少,比例失调,功能性便秘的产生可能与肠道内短链脂肪酸吸收过度以及局部的短链脂肪酸浓度升高有关。
Introduction
Constipation is a common complaint in childhood; it is a series of symptoms, but not an independent disease. The incidence reported by Thowa and Heston university hospital was 3%-8% among which 90%-95% was the functional constipation. On the base of this data, there were 20 million children involved in our country with the total amount of children was up to 300 million. Constipation does harm to the physical and mental health in children, and further body development. Till now the pathophysiological ground of the functional constipation in childhood is unclear, and fortunately, drug abuse remained the main problem in treatment. The aim of this investigation was just to work on these fields so as to resolve these problems mentioned above.
With attention to the importance of constipation in medical science, there were numerous surveys carried out in the epidemiology of the functional constipation in the world, especially in adulthood. Till now, there have been many epidemiologic investigations of adult constipation and the normal stooling pattern reported in Beijing, Guangzhou, Chengdu, Chongqing, Hangzhou, Tianjin, Shanghai, Xi'an, and other cities. The government has listed the constipation survey of aged people into the "ninth five-year" project (the number is 96-906-05-09) and put lots of attention in it. The normal stooling pattern could be influenced by many factors, such as the dietary habit, the psychological factor, the circumstance, the race, the culture and so on. The children are in physiological development stage and the gastrointestinal system is still immature. So there are many differences between children and adult functional constipation. The epidemiologic investigation of the functional constipation in childhood is relatively rare, especially in China. At the beginning of these unsolved problems, the study was carried out on the cluster epidemiologic investigation of the children in representative cities in the north of China. The aim was to acquire the incidence of the functional constipation in childhood and to found the criteria of diagnosis, and further to explore the normal stooling pattern as well.
Functional constipation in childhood is a complicated pathophysiologic process. The exact pathogenesis is not clear till now. Colorectal motility, function of pelvic floor muscle and disorder of intestinal flora might be either involved. In the past years, there have been numerous published issues reported that colon inertia was considered to be an important factor lead to the functional constipation. Animal experiments have demonstrated that endogenous cannabinoids could inhibit the gastrointestinal motility, mainly by the receptor CB1 distributed in the terminal nerve. Fatty acid amide hydrolase (FAAH) is a novel membrane enzyme could catalyze the hydrolysis of endogenous cannabinoids in vivo. Thus the mount of endogenous cannabinoids could theoretically be controlled by the activity and the amount of FAAH expression both in vivo and in vitro to achieve the purpose of gastrointestinal motility regulation. Till now not any reports have been published reflecting whether FAAH was expressed or acted in human gastrointestinal tract. Another aim of this study was to try hard to observe the expression of FAAH in human colon by means of the immunohistochemistry, the Western blotting and the quantitative PCR technique both on the protein and the mRNA level, and to explore its roles in the development of slow transit constipation in childhood. In addition, with the rapidly development of molecular ecology, the molecular biological technique was widely used in identification and classification of the microorganism, more and more intestinal flora have been isolated and identified, the function of these floras in gastrointestinal tract was also gradually confirmed that beyond the traditional digestion and absorption. More and further functions such as nutrition and immunity and motility charge have been explored. As revealed that gastrointestinal motility could be regulated by metabolic product of intestinal flora, especially the short-chain fatty acid, which can influence the human gastrointestinal motility through many different mechanisms. So the DNA finger prints of the normal and abnormal intestinal flora was established by PCR-SSCP and BOX-PCR method, and clone sequencing based on BOX-PCR was carried out to observe the variation of the proportion of the intestinal flora in normal and functional constipation children. Meanwhile, the gas chromatographic technique was also combined used so that the amount and proportion of the short chain fatty in the normal and functional constipation children could also be observed. From above, it is promising that the role of the intestinal flora and its metabolic product-short chain fatty acid in the pathogenesis of the functional constipation in childhood could be approached.
Materials and Methods
1、Epidemiology
A screening program for FC in childhood was carried out in 5 northern cities (Beijing, Tianjin, Shenyang, Changchun, and Harbin) of China according to symptoms using Rome II criteria. Random clustered sampling of the inhabitants was carried out under stratification of city, zone and school or school nursery. Sample size of each area was in proportion to the population of the area. The range of age was 2-14. All subjects studied were requested to fill in a questionnaire distributed by the teacher who was trained prior to survey. A preliminary program was firstly performed before the screening program, after that the formal screening program was performed, according to the result of the screening, a small number of patients who fulfilled the criteria were further selected to undergo detail clinical examination in the hospital including laboratory examination, colonic transit time, defecography or/and barium enema, electromyologram and anorectal manometry to exclude organic disease of the colon. Prevalence of FC of the population was then adjusted by the rate of correct diagnosis during detailed study. Potential risk factors and the relative symptoms were explored by comparing frequencies between FC group and non-FC group using X~2 and logistic analysis so that the symptomatic criteria for diagnosis could be approached.
2、Physical examination
All the subjects involved in this study were divided into 2 groups. One group was control, and 33 healthy volunteers (21 males and 12 females) aged 2-13 years (mean 5 years) were involved in this group. The other was constipation group, in which 25 patients (15 males and 10 females) aged 3-14 years (mean 7 years) referred to constipation according to the Rome II criteria were involved. In this study the simplified method of radio opaque markers was used to determine the total gastrointestinal transit time and segmental colonic transit time of the normal and constipated children, and simultaneously X ray defecography and anorectal manometry were also combined used to explore the etiology and classification of the functional constipation in childhood.
3、Expression of FAAH in human colon and its mode of action inthe slow-transit constipation in children.
The investigate objects were divided into functional constipation (FC) group and control group. 16 children were involved in the control group including 10 boys and 6 girls, with a mean age of 7.5 years, and 22 children in the FC group, including 15 boys and 7 girls, the mean age was 7 years. All of the subjects met the standard of STC. All of the colon samples were classified into ascending colon, descending colon and sigmoid colon basing on the position. Among the total, there were 5 ascending colon, 3 descending colon and 8 sigmoid colon samples in control group and 4 ascending colon samples, 18 descending colon samples and 22 sigmoid colon samples in constipation group obtained. Immunohistochemistry, Western blotting and Real Time PCR were performed to detect the expression of FAAH both in human colon and in FC patients. By these means, the localization and quantitative expression of FAAH and its roles in normal colon were approached. Furthermore, the immonofluorescence double-staining was used to observe the location and distribution of FAAH and CB1 receptors. Actions of FAAH on colonic function in physiological condition were further to explored. Meanwhile, quantitive detection of the activity of FAAH in human colon was done by high pressure liquid chromatography and its function in pathogenesis in children functional constipation was approached.
4、Foundation of the DNA finger-print of the intestinal flora infunctional constipation in children.
Investigation objects were divided into control group and functional constipation (FC) group. A Rome II criterion in childhood was used to establish the FC group. There were 15 children in FC group, including 9 boys and 6 girls, with a mean age of 6.5-year-old. There were 35 children in control group, including 24 boys and 11 girls, with a mean age of 6-year-old. Stool samples of FC group and control group were analyzed. We extracted stool bacterial genome DNA utilizing stool bacterial genome DNA extracting kit and carried out amplification of BOX AIR (primer of BOX genome repetitive sequence) and bacterial 16S rDNA V3 region primer. Then, electrophoresis and image analysis establishing on stool bacterial DNA finger print of 16S rDNA and BOX genome's repetitive sequence was carried out. The significance of PCR-SSCP and BOX-PCR on stool bacteria abstraction effect and stool bacterial flora identification was approached. Amplification products were clone-sequencing and the homology of arrays was compared utilizing BLAST instrument in GenBank database. We initially definited the genus and classification of bacteria and approached the changing tendency of intestinal flora in children FC and the significance of intestinal flora on constipation genesis.
5、Variations of the short chain fatty acid in functionalconstipation in children and their probable roles of action.
Quantitive detection of short-chain fatty acid content in children of control group and FC group was carried out combining gas chromatographic technique and analysis of short-chain fatty acid's changing tendency in children FC was done. Advanced, the significances of intestinal microecology bacteria and its metabolic product-short-chain fatty acid in morbility of FC in childhood were approached.
Results
1、Epidemiology
In this investigation, 20,000 questionnaires were released, including 6,000 pieces in Beijing, 4000 pieces in Tianjin, 4500 pieces in Shenyang, 3000 pieces in Harbin and 2500 pieces in Changchun. Among the total, 19638 pieces were regained, including Beijing's 5452 pieces, Tianjin's 3788 pieces, Shenyang's 4066 pieces, Harbin's 2713 pieces and Changchun's 2267 pieces. The recovery rate was 98.19%. Among these, there were 19286 effective questionnaires, with an effective power of 96.43%. 913 children met children Rome II diagnostic criteria, accounting 4.73% of investigated children. The morbility rate of children FC had area and age variability. Seeing from area, Beijing had the highest morbidity of 5.02% and Changchun had the lowest of approximately 4.27 % (P<0.001). Seeing from age, infantile children (2-3 years old) hadthe highest morbidity and school age children (7-11 years old) had the lowest(p<0.001). There were no obvious differences in sex distribution. Stepwise logistic regression demonstrated that artificial feeding, adding assistant-food too early, low dietary fiber, dietary bias, coprophobia, depression, anxiety, overweight, heredity and high economic income were risk factors of FC.
Constipation related symptoms were compared one by one using single factor chi-square analysis. The result revealed: the 22 symptoms (defecating less than 3 times per week, dry hard bolus-shape stool, difficult defecation, abdominal pain, nausea and vomiting and so on) could exist in control group, but FC group had a higher frequency. The statistic difference was significant. Multiple factor stepwise logistic regression analysis showed defecating less than 3 times per week, dry hard bolus-shape feces, difficult defecation, defecation needing assist of drug and so on had a higher incidence rate in FC and they had much significance on diagnosis of FC. The absence of other symptoms had no influence on diagnosis of FC. International common used Rome II criteria was established as the golden criteria to evaluate the sensitivity, specificity and diagnostic accurate rate of symptomatic standard diagnostic test in FC children. The result showed: children have 2 items above had the highest diagnostic accurate rate but medium sensitivity and specificity.
2、Physical examination
In the constipated children,the TGITT, LCTT and RSTT were significantly longer than those in controls, while the RCTT had no significant difference. Radiological presentation of the colonic transit time could be present as three forms: all the colonic segment were abnormal, only the outlet area was abnormal and/or both of them; X ray defecography demonstrated that the puborectal muscle syndrome was noted in 21 children(21/96, 21.88%), the spastic external sphincter was in 9(9/96, 9.38%), the sastic pelvic floor syndrome was in 4(4/96, 4.17%), and the rectal prolapse or intussusception was in 8(8/96, 8.33%), the perineal floor hernia was in 10(10/96, 10.42%), and the rectocele and the perineal descent syndrome were both noted in 3(3/96, 3.13%) respectively. Each syndrome has its special radiological presentations on the X ray defecography film, which could be easily distinguished from each other. The MaxP(S) of in FC group was higher and the VSI was lower than those of the control, while V had no difference.
3、Expression of FAAH in human colon and its action in theslow-transit constipation in children.
Immunohistochemistry, Western blotting, Real Time PCR were utilized in analysis of FAAH expression in colon wall of FC group and control group. The result showed: FAAH expressed at both protein and mRNA level in normal colon wall, regardless of ascending colon, descending colon or sigmoid colon. There was no significant deviation in different segment. Immunostaining showed FAAH mainly localized in intermuscular neuron of bowel wall, absorptive cell and intestinal gland of mucosa. In normal colon, there was a tendency that FAAH's expression location in intermuscular neurons corresponded to CBI receptor. It was found combining immunofluorescence double staining and confocal microscopy. But such alteration was not detected in absorptive cells or intestinal gland. In colon of FC children, the expression of FAAH significantly decreased. Combining high pressure liquid chromatography technique, we found that the hydrolytic activity of FAAH also significantly decreased. The difference had statistical significance.
4、Foundation of the DNA finger-print of the intestinal flora infunctional constipation in children.
All samples of control group and FC group were analyzed more than 3 times utilizing PCR-SSCP and BOX-PCR finger print. We got stable and repeatable pictures. There were many electrophoresis strips in pictures of objects in control group and FC group. From the location of strips, there were consistent strips and individual specific strips exist between control group and FC group. The brightness of the strips were different. Comparing with PCR-SSCP pictures, BOX-PCR pictures had more strips with distinct margins. The result of sequencing revealed: there were identical and distinct bacterial strains exist in FC group and control group. In FC group, there were obviously more strain types with significant statistic difference. Combining the strip locations of BOX-PCR finger print, predominant bacteria of control group were mainly Bacteroides, Escherichia, Bifidobacterium and Acidobacterium; predominant bacteria of FC group were mainly Bacteroides fragilis, Escherichia, Eubacterium and Clostridia.
5、Variations of the short chain fatty acid in functional constipationin children and their probable roles of action.
The results of gas chromatographic analysis revealed: short-chain fatty acid content in per gram dry stool decreased in children troubled by FC. Statistic difference was significant. But water content of FC group decreased with notably elevated short-chain fatty acid concentration, which was obviously higher than control group, and the short-chain fatty acids were disproportioned.
Conclusions
(1) FC is a common disorder in childhood in the North of China which deserves greater care. Higher prevalence is noted in age 2-3. The feeding patterns, earlier feed adding, dietary bias, fiber intake, and fear of defecation, heredity, exposure to anxiety and depression, and income might be the most important risk factors. Therefore the popular knowledge shoule be announced to the public so as to prevent FC from happening.
(2) The symptomatic criterion for FC diagnosis in children in our country is:①defecation frequency is less than 3 times per week;②dry, hard, bolus-shape stool is noted in most defecation;③hard defecation is noted in most defecation;④defecation assisted by drugs or fingers. Those owing 2 of the above 4 and the symptoms last at least 2 months could be committed a diagnosis of FC.
(3) FC in childhood could be classified into several subtypes as follow:①Slow-transit constipation: disorder of the transit function was found in any of the colonic segment.②Outlet-obstruction: dynamic abnormality was only found in the anorectal region, in other words, the outlet area.③Mixed type: both the colonic transit and the outlet dynamic disorder exist. Combined use of colonic transit time, X-ray defecography, and anorectal manomatry has important significance on etiological diagnosis and classification of FC in children.
(4) Expression of FAAH on protein and nucleic acid levels was both found in the normal colon in human. It mainly expresses in the myenteric plexus in colon, absorptive cells and glands on the surface of intestinal mucosa. In the myenteric plexus, FAAH have a tendency of comparative distribution with CB1 receptor.
(5) In the slow transit constipation, the expression and activity of FAAH decrease. FAAH may take part in pathogenesis of the slow transit constipation in children.
(6) The category of the intestinal flora increase in FC in children. The resident bacteria are similar to the normal children, but the predominant bacteria are different and disproportion exists among the intestinal flora.
(7) Short-chain fatty acid content decrease with error ratio in stools of FC children. Pathogenesis of FC may relate to excessive absorption of short-chain fatty chain in bowel and increasing of local concentration of the short-chain fatty acid.
儿童便秘为常见的临床症状,而不是一种独立的疾病。据Thowa大学和Heston大学医院儿科门诊统计,儿童便秘的发生率为3%—8%,其中90%—95%是功能性便秘。按照我国13亿人口统计,约有3亿儿童,其中有二千万左右的儿童患有便秘。便秘对儿童个体生长发育危害巨大,病因目前尚不清楚,治疗上存在泻药滥用问题;如何判定儿童便秘病因,并制订有针对性的措施进行治疗,正是本研究所要解决的问题。
由于便秘在医学中地位及重要性的迅速提高,国内外相继对便秘及其病因等进行了众多的流行病学调查研究,尤其在成人便秘方面投入了大量的人力物力。迄今为止,国内己有北京、广东广州、成都、重庆、杭州、天津、上海、西安等地区对成人便秘及排便习惯进行了流行病学调查,国家更将老年人的便秘调查与多因素分析列为国家“九五”攻关项目(科研编号96-906-05-09),拨巨资支持。但是由于正常人群的排便习惯受饮食习惯、精神心理因素、环境和种族、文化背景等诸多因素的影响,儿童处于生理发育期,胃肠系统尚不成熟,因此儿童FC的患病情况相差较大,尤其是国内,此类研究相对少见,本研究从儿童最常见而且尚未解决的问题入手,首先对中国北方具有代表性的城市儿童进行大范围的流行病学调查,探讨北方儿童便秘的患病率,并在调查的基础上提出我国儿童便秘的诊断标准。
功能性便秘是一个复杂的病理生理过程,其确切的发病机制目前尚不清楚,结直肠动力、盆底肌群的功能以及肠道菌群紊乱都可能与功能性便秘有关。在众多的研究中,结肠动力低下被认为是功能性便秘发生的重要因素之一,已有动物实验表明,内源性麻酚类物质可以抑制胃肠动力,主要是通过分布于神经末梢的受体CB1起作用。脂肪酰胺水解酶(Fatty acid amide hydrolase,FAAH)在体内能催化内源性麻酚类物质的水解,因此在理论上可以通过调节FAAH的活性来控制内源性麻酚类物质,以达到调节胃肠动力的目的。到目前为止,关于FAAH是否在人类的胃肠道中的作用还未见报道,因此本研究着眼于此,力图通过免疫组化、蛋白印迹和定量PCR技术从不同水平观察FAAH的表达情况,探讨其在便秘的发生过程中的作用。另外,随着分子生物学技术在微生物领域应用的不断进步和微生态学的不断完善,越来越多的肠道菌群被克隆和分离出来,细菌在胃肠道中的作用也逐步得到确认,人们对肠道菌群的认识已不仅仅停留在以往的消化和吸收方面,越来越多的作用被开发出来,研究显示,肠道菌群可以通过其代谢产物调节胃肠动力,尤其是短链脂肪酸,可以通过多种不同的机制影响胃肠动力功能,因此本研究应用PCR-SSCP和BOX-PCR技术对正常肠道菌群进行DNA指纹图谱分析,观察肠道菌群在正常和便秘儿童肠道中的分布比例关系并结合应用气相色谱技术观察细菌代谢产物短链脂肪酸在正常和便秘儿童肠道中的分布比例关系,探讨肠道菌群及其代谢产物短链脂肪酸在儿童功能性便秘发病中的作用。
材料和方法
一、流行病学调查
应用分层、整群随机抽样方法对中国北方5市(包括北京、天津、沈阳、吉林和哈尔滨)常住儿童中2～14岁的儿童进行问卷调查,调查问卷由专门从事流行病学调查的专家协助设计,首先进行预调查,然后进行普查,共发放问卷20000份,其中北京6000份,天津4000份,沈阳4500份,哈尔滨3000份,长春2500份,根据普查结果了解按儿童罗马Ⅱ标准判断的FC的检出率,再对筛出的部分人在医院进行精查(结肠传输试验,钡灌肠及排便造影,消化道测压)以排除结肠器质性疾病,计算我国北方城市儿童儿童功能性便秘的患病率。详细记录与儿童功能性便秘有关的各种危险因素以及与排便有关的各种相关症状,对各种危险因素与儿童功能性便秘的关系进行逐步logistic回归分析,分析其对于儿童功能性便秘的贡献;按儿童罗马Ⅱ标准筛选确定便秘组,分析各相关症状在便秘组和非便秘组发生的频率,用卡方对单因素进行比较,再对有意义的症状用logistic回归模型作拟合优度检测,探讨儿童功能性便秘的危险因素,初步制定适合我国儿童的便秘标准。
二、排便功能检测
研究对象分为对照组和便秘组。对照组33例,平均年龄5岁;便秘组96例,平均年龄6.5岁,均符合儿童功能性便秘罗马Ⅱ诊断标准;联合应用结肠传输试验,X线排便造影和肛门直肠测压对对照组和便秘组儿童进行全面检查,分析其影像学及功能学表现的差异,探讨这些异常在儿童功能性便秘的病理生理及其对便秘的诊断和分型的意义;
三、慢传输性便秘儿童结肠壁内FAAH的表达及意义
研究对象分为便秘组和对照组,对照组16例,其中男10例,女6例,平均年龄7.5岁。便秘组男15例,女7例,平均年龄7岁,均符合STC的标准,分别取上述病例结肠标本,按部位分为升、降和乙状结肠,其中对照组升结肠5例,降结肠3例,乙状结肠8例;便秘组升结肠4例,降结肠18例,乙状结肠22例;应用免疫组化,Western blotting,Real Time PCR对便秘组和对照组儿童结肠壁内FAAH的表达分析,探讨FAAH在正常结肠壁内的定位和定量表达及其意义,并联合应用免疫荧光双重染色技术观察FAAH与CB1受体的位置与分布,探讨FAAH在生理条件下对结肠功能进行调控的作用机制;同时还进一步联合应用高压液相色谱技术对便秘组儿童结肠壁内FAAH的生物活性进行定量检测和分析,探讨其在儿童功能性便秘发病中的作用。
四、功能性便秘儿童肠道菌群DNA指纹图谱的建立及意义
研究对象分为对照组和便秘组,采用儿童功能性便秘罗马Ⅱ标准确立便秘组,便秘组15例,男9例,女6例,平均年龄6.5岁;对照组35例,男24例,女11例,平均年龄6岁。取对照组和便秘组所有儿童粪便标本进行分析。应用粪便细菌基因组DNA提取试剂盒提取粪便细菌基因组DNA,分别应用细菌16S rDNA的V3区引物和BOX基因组重复序列的引物BOX A1R进行扩增,然后进行电泳和图像分析,分别建立基于16SrDNA和BOX基因组重复序列的粪便细菌DNA指纹图谱,探讨PCR-SSCP和BOX-PCR对于粪便细菌的分离效果及其对粪便细菌菌种鉴定的意义;将扩增产物进行克隆测序,测序后对序列的同源性在GenBank数据库中使用BLAST工具进行比较,初步确定细菌的种属和分类,探讨肠道菌群在功能性便秘儿童的变化趋势及其对便秘产生的意义。
五、功能性便秘儿童粪便短链脂肪酸的检测与分析
应用气相色谱技术对便秘组和对照组儿童粪便中短链脂肪酸的含量进行定量检测,分析短链脂肪酸在功能性便秘儿童肠道内的变化趋势,进一步探讨肠道微生态菌落及其代谢产物短链脂肪酸在儿童功能性便秘发病中的意义。
结果
一、流行病学调查
本次调查共发放问卷20000份,其中北京6000份,天津4000份,沈阳4500份,哈尔滨3000份,长春2500份。回收19638份,其中北京5452份,天津3788份,沈阳4066份,哈尔滨2713份,长春2267份,回收率98.19%;有效问卷19286份,有效率96.43%;有913名符合儿童罗马Ⅱ诊断标准,占被调查人数的4.73%;儿童FC的患病率具有地域差异性和年龄差异性特点,从地区上看,北京的患病率最高,为5.02%,长春最低,约4.27%,统计学差异显著(P＜0.001);从年龄上看,婴幼儿期(2-3岁)的患病率最高,学龄期(7-11岁)最低,统计学差异显著(P＜0.001)。性别分布无明显差异。逐步logistic回归证实人工喂养、添加辅食过早、饮食纤维低、挑食偏食、排便恐惧、抑郁、焦虑、体重、遗传、高经济收入及为FC的危险因素。
对便秘的相关症状逐个进行单因素卡方分析比较,结果显示包括排便次数＜3次/周,大便干硬成团块状,排便费力,腹痛,恶心呕吐等在内的22种症状虽可在非FC人群中不同程度存在,但FC组发生频率比非FC组明显为高,统计学差异显著,多因素逐步logistic回归分析,结果显示排便次数＜3次/周,大便干硬成团块状,排便费力,需要泻药协助排便等症状在FC发生率更高,对诊断FC最有意义,减少的其他症状对FC诊断无影响,并以国际通用罗马Ⅱ标准为黄金标准,对拟定儿童FC症状学标准诊断试验的灵敏度,特异度和诊断准确率进行评价,结果显示,具备2条者的特异度和灵敏度均较适中,但诊断准确率最高。
二、排便功能检测
结肠传输时间测定结果显示,便秘组的TGITT、LCTT和RSTT较对照组明显延长,RCTT在两组无显著性差异。儿童FC结肠传输时间延长具有以下几种形式:①RCTT、LCTT和RSTT都延长;②单纯RSTT延长;③RCTT、LCTT和RSTT都延长,但RSTT延长更显著;④TGITT正常。X线排便造影显示耻骨直肠肌痉挛综合症14例:排便状态下直肠肛管角不开大,直肠后壁耻骨直肠肌压迹加深;肛门外括约肌痉挛4例:直肠肛管角正常开大,但会阴下降不明显,肛管前壁出现指状压迹;肠疝(包括小肠疝和结肠疝)3例:小肠或结肠下端疝入直肠膀胱陷凹,压迫直肠前壁;直肠粘膜脱垂和套叠3例:排便时肛管上方出现直肠粘膜影像;直肠前突2例:排便时直肠前壁向前方突出深度超过2cm;会阴下降综合症1例:盆膈摆动幅度大,下降时超过2cm;盆底痉挛综合症2例:表现为“鹅征”。肛门直肠测量结果显示,便秘组的肛管最大收缩压升高,对称指数明显降低而向量容积虽有增加,但不显著。
三、慢传输性便秘儿童结肠壁内FAAH的表达及意义
应用免疫组化,Western blotting,Real Time PCR对便秘组和对照组儿童结肠壁内FAAH的表达进行分析发现,正常结肠无论是升结肠、降结肠还是乙状结肠其壁内蛋白水平和mRNA水平均存在FAAH的表达,各段表达无显著差异,并且免疫染色显示FAAH主要定位于肠壁肌间神经元和粘膜的吸收细胞和肠腺体;在正常结肠,结合免疫荧光双重染色和共聚焦显微镜发现肌间神经元中FAAH的分布在位置上与CB1受体呈对应趋势,而在吸收细胞和肠腺体中未见此改变;在便秘儿童结肠,FAAH的表达显著减弱,结合高压液相色谱技术发现,FAAH的水解活性也显著降低,差异具有统计学意义。
四、功能性便秘儿童肠道菌群DNA指纹图谱的建立及意义
对照组和便秘组的所有样品分别进行3次以上的PCR-SSCP和BOX-PCR指纹图谱分析,得到了稳定可重复的图谱,无论便秘组还是对照组个体,图谱中均可见较多的电泳条带,从条带位置看,在对照组与便秘组,对照组之间以及便秘组之间既存在个体一致性条带,同时也存在个体特异性条带,且各条带亮度不鞠嗤?与PCR-SSCP图谱相比BOX-PCR图谱的泳带条数更多,各泳带之间界限清晰。测序结果显示,便秘组和对照组肠道内即存在相同的细菌菌种,也存在各自不一的菌种。在便秘组,菌种的种类较对照组明显增多,统计学差异显著;结合BOX-PCR指纹图谱的条带位置,对照组优势菌主要为拟杆菌属,埃希氏菌属,双歧杆菌属,乳杆菌属;便秘组的优势菌主要为脆弱拟杆菌属,埃希氏菌属,真杆菌属和梭菌属。
五、功能性便秘儿童粪便短链脂肪酸的检测与分析
气相色谱分析结果显示功能性便秘儿童粪便中每克干粪中短链脂肪酸含量较对照组减少,统计学差异显著,但便秘儿童粪便中含水量减少,短链脂肪酸显著升高,明显高于对照组,统计学差异显著,且各短链脂肪酸比例失调。
结论
(1)儿童FC是儿童期的常见病多发病,好发于2～3岁儿童;人工喂养、添加辅食过早、饮食纤维低、挑食偏食、排便恐惧、抑郁、焦虑、体重、遗传、高经济收入可能是FC的危险因素,应该对民众进行广泛的宣教做针对性预防和治疗。
(2)我国儿童FC的症状学诊断标准为:①排便次数＜3次/周;②大便干硬成团块状;③排便费力;④需要泻药协助排便。具备上述的4个诊断标尺中2条者,症状持续至少2月即可诊断便秘。
(3)儿童功能性便秘可以分为以下几种类型:①慢传输型便秘,近端结肠各节段传输功能异常。②出口梗阻型便秘:结肠出口处肛门直肠区域存在动力异常。③混合型便秘:同时存在结肠传输障碍和出口动力异常;联合应用结肠传输时间,X线排便造影和肛门直肠测压对儿童功能性便秘的病因诊断和临床分型具有重要意义。
(4)人类正常结肠壁内无论是蛋白还是核酸水平均存在FAAH的表达,FAAH主要表达在结肠肌间神经丛的神经元内和肠粘膜表面的吸收细胞和腺体内,在肌间神经丛内FAAH的分布与CB1受体呈对应趋势。
(5)慢传输性便秘儿童结肠壁内FAAH的表达减低,水解活性减弱,FAAH可能参与了慢传输性便秘的发生。
(6)功能性便秘儿童肠道菌群的菌属多样性增加,肠道内存在与正常儿童相似的常驻菌,但优势菌种不同,各肠道菌属间存在比例失调。
(7)功能性便秘儿童粪便中的短链脂肪酸含量减少,比例失调,功能性便秘的产生可能与肠道内短链脂肪酸吸收过度以及局部的短链脂肪酸浓度升高有关。
Introduction
Constipation is a common complaint in childhood; it is a series of symptoms, but not an independent disease. The incidence reported by Thowa and Heston university hospital was 3%-8% among which 90%-95% was the functional constipation. On the base of this data, there were 20 million children involved in our country with the total amount of children was up to 300 million. Constipation does harm to the physical and mental health in children, and further body development. Till now the pathophysiological ground of the functional constipation in childhood is unclear, and fortunately, drug abuse remained the main problem in treatment. The aim of this investigation was just to work on these fields so as to resolve these problems mentioned above.
With attention to the importance of constipation in medical science, there were numerous surveys carried out in the epidemiology of the functional constipation in the world, especially in adulthood. Till now, there have been many epidemiologic investigations of adult constipation and the normal stooling pattern reported in Beijing, Guangzhou, Chengdu, Chongqing, Hangzhou, Tianjin, Shanghai, Xi'an, and other cities. The government has listed the constipation survey of aged people into the "ninth five-year" project (the number is 96-906-05-09) and put lots of attention in it. The normal stooling pattern could be influenced by many factors, such as the dietary habit, the psychological factor, the circumstance, the race, the culture and so on. The children are in physiological development stage and the gastrointestinal system is still immature. So there are many differences between children and adult functional constipation. The epidemiologic investigation of the functional constipation in childhood is relatively rare, especially in China. At the beginning of these unsolved problems, the study was carried out on the cluster epidemiologic investigation of the children in representative cities in the north of China. The aim was to acquire the incidence of the functional constipation in childhood and to found the criteria of diagnosis, and further to explore the normal stooling pattern as well.
Functional constipation in childhood is a complicated pathophysiologic process. The exact pathogenesis is not clear till now. Colorectal motility, function of pelvic floor muscle and disorder of intestinal flora might be either involved. In the past years, there have been numerous published issues reported that colon inertia was considered to be an important factor lead to the functional constipation. Animal experiments have demonstrated that endogenous cannabinoids could inhibit the gastrointestinal motility, mainly by the receptor CB1 distributed in the terminal nerve. Fatty acid amide hydrolase (FAAH) is a novel membrane enzyme could catalyze the hydrolysis of endogenous cannabinoids in vivo. Thus the mount of endogenous cannabinoids could theoretically be controlled by the activity and the amount of FAAH expression both in vivo and in vitro to achieve the purpose of gastrointestinal motility regulation. Till now not any reports have been published reflecting whether FAAH was expressed or acted in human gastrointestinal tract. Another aim of this study was to try hard to observe the expression of FAAH in human colon by means of the immunohistochemistry, the Western blotting and the quantitative PCR technique both on the protein and the mRNA level, and to explore its roles in the development of slow transit constipation in childhood. In addition, with the rapidly development of molecular ecology, the molecular biological technique was widely used in identification and classification of the microorganism, more and more intestinal flora have been isolated and identified, the function of these floras in gastrointestinal tract was also gradually confirmed that beyond the traditional digestion and absorption. More and further functions such as nutrition and immunity and motility charge have been explored. As revealed that gastrointestinal motility could be regulated by metabolic product of intestinal flora, especially the short-chain fatty acid, which can influence the human gastrointestinal motility through many different mechanisms. So the DNA finger prints of the normal and abnormal intestinal flora was established by PCR-SSCP and BOX-PCR method, and clone sequencing based on BOX-PCR was carried out to observe the variation of the proportion of the intestinal flora in normal and functional constipation children. Meanwhile, the gas chromatographic technique was also combined used so that the amount and proportion of the short chain fatty in the normal and functional constipation children could also be observed. From above, it is promising that the role of the intestinal flora and its metabolic product-short chain fatty acid in the pathogenesis of the functional constipation in childhood could be approached.
Materials and Methods
1、Epidemiology
A screening program for FC in childhood was carried out in 5 northern cities (Beijing, Tianjin, Shenyang, Changchun, and Harbin) of China according to symptoms using Rome II criteria. Random clustered sampling of the inhabitants was carried out under stratification of city, zone and school or school nursery. Sample size of each area was in proportion to the population of the area. The range of age was 2-14. All subjects studied were requested to fill in a questionnaire distributed by the teacher who was trained prior to survey. A preliminary program was firstly performed before the screening program, after that the formal screening program was performed, according to the result of the screening, a small number of patients who fulfilled the criteria were further selected to undergo detail clinical examination in the hospital including laboratory examination, colonic transit time, defecography or/and barium enema, electromyologram and anorectal manometry to exclude organic disease of the colon. Prevalence of FC of the population was then adjusted by the rate of correct diagnosis during detailed study. Potential risk factors and the relative symptoms were explored by comparing frequencies between FC group and non-FC group using X~2 and logistic analysis so that the symptomatic criteria for diagnosis could be approached.
2、Physical examination
All the subjects involved in this study were divided into 2 groups. One group was control, and 33 healthy volunteers (21 males and 12 females) aged 2-13 years (mean 5 years) were involved in this group. The other was constipation group, in which 25 patients (15 males and 10 females) aged 3-14 years (mean 7 years) referred to constipation according to the Rome II criteria were involved. In this study the simplified method of radio opaque markers was used to determine the total gastrointestinal transit time and segmental colonic transit time of the normal and constipated children, and simultaneously X ray defecography and anorectal manometry were also combined used to explore the etiology and classification of the functional constipation in childhood.
3、Expression of FAAH in human colon and its mode of action inthe slow-transit constipation in children.
The investigate objects were divided into functional constipation (FC) group and control group. 16 children were involved in the control group including 10 boys and 6 girls, with a mean age of 7.5 years, and 22 children in the FC group, including 15 boys and 7 girls, the mean age was 7 years. All of the subjects met the standard of STC. All of the colon samples were classified into ascending colon, descending colon and sigmoid colon basing on the position. Among the total, there were 5 ascending colon, 3 descending colon and 8 sigmoid colon samples in control group and 4 ascending colon samples, 18 descending colon samples and 22 sigmoid colon samples in constipation group obtained. Immunohistochemistry, Western blotting and Real Time PCR were performed to detect the expression of FAAH both in human colon and in FC patients. By these means, the localization and quantitative expression of FAAH and its roles in normal colon were approached. Furthermore, the immonofluorescence double-staining was used to observe the location and distribution of FAAH and CB1 receptors. Actions of FAAH on colonic function in physiological condition were further to explored. Meanwhile, quantitive detection of the activity of FAAH in human colon was done by high pressure liquid chromatography and its function in pathogenesis in children functional constipation was approached.
4、Foundation of the DNA finger-print of the intestinal flora infunctional constipation in children.
Investigation objects were divided into control group and functional constipation (FC) group. A Rome II criterion in childhood was used to establish the FC group. There were 15 children in FC group, including 9 boys and 6 girls, with a mean age of 6.5-year-old. There were 35 children in control group, including 24 boys and 11 girls, with a mean age of 6-year-old. Stool samples of FC group and control group were analyzed. We extracted stool bacterial genome DNA utilizing stool bacterial genome DNA extracting kit and carried out amplification of BOX AIR (primer of BOX genome repetitive sequence) and bacterial 16S rDNA V3 region primer. Then, electrophoresis and image analysis establishing on stool bacterial DNA finger print of 16S rDNA and BOX genome's repetitive sequence was carried out. The significance of PCR-SSCP and BOX-PCR on stool bacteria abstraction effect and stool bacterial flora identification was approached. Amplification products were clone-sequencing and the homology of arrays was compared utilizing BLAST instrument in GenBank database. We initially definited the genus and classification of bacteria and approached the changing tendency of intestinal flora in children FC and the significance of intestinal flora on constipation genesis.
5、Variations of the short chain fatty acid in functionalconstipation in children and their probable roles of action.
Quantitive detection of short-chain fatty acid content in children of control group and FC group was carried out combining gas chromatographic technique and analysis of short-chain fatty acid's changing tendency in children FC was done. Advanced, the significances of intestinal microecology bacteria and its metabolic product-short-chain fatty acid in morbility of FC in childhood were approached.
Results
1、Epidemiology
In this investigation, 20,000 questionnaires were released, including 6,000 pieces in Beijing, 4000 pieces in Tianjin, 4500 pieces in Shenyang, 3000 pieces in Harbin and 2500 pieces in Changchun. Among the total, 19638 pieces were regained, including Beijing's 5452 pieces, Tianjin's 3788 pieces, Shenyang's 4066 pieces, Harbin's 2713 pieces and Changchun's 2267 pieces. The recovery rate was 98.19%. Among these, there were 19286 effective questionnaires, with an effective power of 96.43%. 913 children met children Rome II diagnostic criteria, accounting 4.73% of investigated children. The morbility rate of children FC had area and age variability. Seeing from area, Beijing had the highest morbidity of 5.02% and Changchun had the lowest of approximately 4.27 % (P<0.001). Seeing from age, infantile children (2-3 years old) hadthe highest morbidity and school age children (7-11 years old) had the lowest(p<0.001). There were no obvious differences in sex distribution. Stepwise logistic regression demonstrated that artificial feeding, adding assistant-food too early, low dietary fiber, dietary bias, coprophobia, depression, anxiety, overweight, heredity and high economic income were risk factors of FC.
Constipation related symptoms were compared one by one using single factor chi-square analysis. The result revealed: the 22 symptoms (defecating less than 3 times per week, dry hard bolus-shape stool, difficult defecation, abdominal pain, nausea and vomiting and so on) could exist in control group, but FC group had a higher frequency. The statistic difference was significant. Multiple factor stepwise logistic regression analysis showed defecating less than 3 times per week, dry hard bolus-shape feces, difficult defecation, defecation needing assist of drug and so on had a higher incidence rate in FC and they had much significance on diagnosis of FC. The absence of other symptoms had no influence on diagnosis of FC. International common used Rome II criteria was established as the golden criteria to evaluate the sensitivity, specificity and diagnostic accurate rate of symptomatic standard diagnostic test in FC children. The result showed: children have 2 items above had the highest diagnostic accurate rate but medium sensitivity and specificity.
2、Physical examination
In the constipated children,the TGITT, LCTT and RSTT were significantly longer than those in controls, while the RCTT had no significant difference. Radiological presentation of the colonic transit time could be present as three forms: all the colonic segment were abnormal, only the outlet area was abnormal and/or both of them; X ray defecography demonstrated that the puborectal muscle syndrome was noted in 21 children(21/96, 21.88%), the spastic external sphincter was in 9(9/96, 9.38%), the sastic pelvic floor syndrome was in 4(4/96, 4.17%), and the rectal prolapse or intussusception was in 8(8/96, 8.33%), the perineal floor hernia was in 10(10/96, 10.42%), and the rectocele and the perineal descent syndrome were both noted in 3(3/96, 3.13%) respectively. Each syndrome has its special radiological presentations on the X ray defecography film, which could be easily distinguished from each other. The MaxP(S) of in FC group was higher and the VSI was lower than those of the control, while V had no difference.
3、Expression of FAAH in human colon and its action in theslow-transit constipation in children.
Immunohistochemistry, Western blotting, Real Time PCR were utilized in analysis of FAAH expression in colon wall of FC group and control group. The result showed: FAAH expressed at both protein and mRNA level in normal colon wall, regardless of ascending colon, descending colon or sigmoid colon. There was no significant deviation in different segment. Immunostaining showed FAAH mainly localized in intermuscular neuron of bowel wall, absorptive cell and intestinal gland of mucosa. In normal colon, there was a tendency that FAAH's expression location in intermuscular neurons corresponded to CBI receptor. It was found combining immunofluorescence double staining and confocal microscopy. But such alteration was not detected in absorptive cells or intestinal gland. In colon of FC children, the expression of FAAH significantly decreased. Combining high pressure liquid chromatography technique, we found that the hydrolytic activity of FAAH also significantly decreased. The difference had statistical significance.
4、Foundation of the DNA finger-print of the intestinal flora infunctional constipation in children.
All samples of control group and FC group were analyzed more than 3 times utilizing PCR-SSCP and BOX-PCR finger print. We got stable and repeatable pictures. There were many electrophoresis strips in pictures of objects in control group and FC group. From the location of strips, there were consistent strips and individual specific strips exist between control group and FC group. The brightness of the strips were different. Comparing with PCR-SSCP pictures, BOX-PCR pictures had more strips with distinct margins. The result of sequencing revealed: there were identical and distinct bacterial strains exist in FC group and control group. In FC group, there were obviously more strain types with significant statistic difference. Combining the strip locations of BOX-PCR finger print, predominant bacteria of control group were mainly Bacteroides, Escherichia, Bifidobacterium and Acidobacterium; predominant bacteria of FC group were mainly Bacteroides fragilis, Escherichia, Eubacterium and Clostridia.
5、Variations of the short chain fatty acid in functional constipationin children and their probable roles of action.
The results of gas chromatographic analysis revealed: short-chain fatty acid content in per gram dry stool decreased in children troubled by FC. Statistic difference was significant. But water content of FC group decreased with notably elevated short-chain fatty acid concentration, which was obviously higher than control group, and the short-chain fatty acids were disproportioned.
Conclusions
(1) FC is a common disorder in childhood in the North of China which deserves greater care. Higher prevalence is noted in age 2-3. The feeding patterns, earlier feed adding, dietary bias, fiber intake, and fear of defecation, heredity, exposure to anxiety and depression, and income might be the most important risk factors. Therefore the popular knowledge shoule be announced to the public so as to prevent FC from happening.
(2) The symptomatic criterion for FC diagnosis in children in our country is:①defecation frequency is less than 3 times per week;②dry, hard, bolus-shape stool is noted in most defecation;③hard defecation is noted in most defecation;④defecation assisted by drugs or fingers. Those owing 2 of the above 4 and the symptoms last at least 2 months could be committed a diagnosis of FC.
(3) FC in childhood could be classified into several subtypes as follow:①Slow-transit constipation: disorder of the transit function was found in any of the colonic segment.②Outlet-obstruction: dynamic abnormality was only found in the anorectal region, in other words, the outlet area.③Mixed type: both the colonic transit and the outlet dynamic disorder exist. Combined use of colonic transit time, X-ray defecography, and anorectal manomatry has important significance on etiological diagnosis and classification of FC in children.
(4) Expression of FAAH on protein and nucleic acid levels was both found in the normal colon in human. It mainly expresses in the myenteric plexus in colon, absorptive cells and glands on the surface of intestinal mucosa. In the myenteric plexus, FAAH have a tendency of comparative distribution with CB1 receptor.
(5) In the slow transit constipation, the expression and activity of FAAH decrease. FAAH may take part in pathogenesis of the slow transit constipation in children.
(6) The category of the intestinal flora increase in FC in children. The resident bacteria are similar to the normal children, but the predominant bacteria are different and disproportion exists among the intestinal flora.
(7) Short-chain fatty acid content decrease with error ratio in stools of FC children. Pathogenesis of FC may relate to excessive absorption of short-chain fatty chain in bowel and increasing of local concentration of the short-chain fatty acid.
引文
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