幼鼠腸易激綜合征模型建立及其發(fā)病機制的實驗研究
發(fā)布時間：2018-08-28 14:59
【摘要】： 第一部分蘇州市兒童腸易激綜合征(IBS)的流行病學危險因素研究 目的探討蘇州市區(qū)小學生腸易激綜合征(IBS)的患病率及危險因素。方法采用流行病學橫斷面研究方法對蘇州市滄浪、平江及金閶轄區(qū)內(nèi)公辦小學1～6年級小學生進行問卷調(diào)查。采用多階段、隨機整群抽樣的方法獲取調(diào)查樣本。在調(diào)查對象中,按羅馬Ⅱ診斷標準,排除其他器質(zhì)性疾病后確診IBS病例,其他非IBS病例為對照。擬合非條件logistic多元回歸模型,分析影響IBS發(fā)病可能的危險因素,為兒童IBS的預防提供科學的依據(jù)。結果共發(fā)放問卷8000份,回收有效問卷7472份,回收率93.4%;蘇州市區(qū)1～6年級小學生有癥狀符合羅馬Ⅱ診斷標準的IBS患病率為10.81%。男、女IBS患病率差異無統(tǒng)計學意義(10.3%vs11.3%)。多因素logistic回歸模型分析提示:有食物過敏史(OR=1.53,95%CI:1.13～2.07)、兒童期有腸炎史(OR=1.29,95%CI:1.00～1.63)、喜食油炸食物(OR=1.62,95%CI:1.34～1.96)、心情焦慮(OR=1.49,95%CI:1.16～1.93)、兒童幼年時有意外打擊史(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)為男女兒童IBS發(fā)病最可能的危險因素。兒童年齡越大,IBS發(fā)病危險有逐漸下降的趨勢(OR=0.94,95%CI:0.89～0.99)結論IBS是蘇州市區(qū)小學生的常見病。影響蘇州市區(qū)小學生IBS可能的危險因素是:年齡小、有食物過敏史、兒童期有腸炎史、喜食油炸食物、心情焦慮、兒童幼年時有意外打擊史及父母有便秘史。因此,提示臨床醫(yī)師應重視此病,積極防治腸炎,調(diào)整兒童飲食,避免多食油炸食品及過敏的食物,以更好地防治本病。 第二部分幼鼠腸易激綜合征模型的建立及鑒定 目的建立適合兒童腸易激綜合征(IBS)研究的幼鼠IBS動物模型并鑒定。方法應用20只SD新生大鼠,隨機分為IBS模型組和正常對照組,IBS模型組進行母子分離、改良機械束縛、結腸芥子油刺激等綜合因素干預幼鼠建立IBS的動物模型;正常組不作干預。用自制的擴張器對幼鼠進行直腸擴張,評估不同壓力下腹部收縮反射(AWR)閾值,測定幼鼠腹壁肌電活動;利用血管球囊擴張導管以5ml、10ml分別擴張IBS模型組、對照組幼鼠直腸,分別采集兩組鼠腦部的fMRI數(shù)據(jù),采用腦功能成像分析軟件進行數(shù)據(jù)處理,應用血氧水平依賴性功能性MRI(BOLD-fMRI)技術,研究直腸球囊擴張刺激下腸易激綜合征模型幼鼠腦內(nèi)臟痛覺中樞興奮情況及其興奮區(qū)分布情況;取乙狀結腸降段結腸用分層刮片技術刮取由外向內(nèi)取1-4層細胞,丫啶橙熒光染色觀察幼老細胞的比值并對腸粘膜上皮細胞進行美蘭還原反應時間測定;并行結腸組織病理檢測;干預后稱兩組幼鼠體重,觀察糞便性狀,查糞常規(guī),并收集糞便稱濕重和干重,應用Bristol分型觀察各組糞便性狀進行評分。結果直腸擴張時,在不同壓力下模型組腹壁收縮反射(AWR)評分較對照組閾值顯著降低(P值均0.01);不同擴張壓力下,腹壁肌電活動隨壓力增加明顯增加(P0.01);直腸球囊刺激后可引起IBS模型組幼鼠腦內(nèi)與內(nèi)臟相關的腦區(qū)(腦島皮質(zhì)、額前皮質(zhì)、丘腦及扣帶前皮質(zhì))神經(jīng)元活動增強,表現(xiàn)為高興奮區(qū),而對照組幼鼠無相應興奮區(qū)表現(xiàn);結腸黏膜分層刮片后丫啶橙染色見IBS模型組粘膜上皮最外層細胞幼老細胞比值較正常對照組明顯減少,美藍還原時間延長(P0.05);結腸病理檢查兩組均未見明顯異常;IBS模型組腹瀉癥狀明顯較正常對照組增加(P0.05),模型組糞便多為軟的團塊或泥漿樣,而對照組為柔軟的香腸狀或團塊狀,兩組糞便濕重存在顯著差異(P0.05),而體重、糞便常規(guī)檢查兩組間無明顯差異。兩組Bristol分型評分比較有顯著性差異(P0.05)。結論用母子分離、改良機械束縛、結腸芥子油刺激等綜合因素干預幼鼠可成功建立適合兒童的幼鼠IBS動物模型,其機制可能為內(nèi)臟敏感性增高。功能MRI是一種較為直觀且準確的觀察腦功能活動的腦顯像技術,本研究顯示IBS幼鼠中樞內(nèi)腦島皮質(zhì)、額前皮質(zhì)及丘腦可能是參與內(nèi)臟感覺的主要部位。幼鼠IBS模型結腸粘膜上皮細胞功能處于抑制狀態(tài)。 第三部分ICC細胞和腦腸肽在幼鼠IBS模型中作用機制的實驗研究 目的旨在通過檢測幼鼠結腸、脊髓和丘腦組織中腦腸肽NPY和cGRP的mRNA表達和結腸ICC細胞c-kit陽性細胞的表達,探討腦腸肽、腦腸軸及ICC細胞在幼鼠IBS模型發(fā)病機制中的作用。方法根據(jù)簡單數(shù)字法隨機將乳鼠分成IBS模型組和正常對照組二組,各10只。IBS模型組用母子分離、改良機械束縛、結腸芥子油刺激等綜合因素干預的方法造模,對照組則不做任何干預。采用半定量逆轉(zhuǎn)錄多聚酶鏈式反應法檢測二組幼鼠結腸、脊髓、丘腦組織中NPY和cGRP mRNA的表達。另取結腸組織通過免疫組化檢測肌間ICC細胞c-kit陽性表達細胞數(shù)。結果IBS模型組中NPY在結腸、脊髓、丘腦組織中mRNA表達明顯較正常對照組降低,t值分別為9.66,8.55,12.65,(均P0.01);而cGRP則在腦腸軸各個層面(結腸、脊髓、丘腦)的表達中,IBS組明顯較正常對照組增加,t值分別為14.41,12.85,12.41,(均P0.01)。二組幼鼠結腸ICC細胞c-kit陽性表達細胞數(shù)有差異(t=5.294,P0.01)。結論ICC細胞、腦腸肽可能在幼鼠IBS模型的發(fā)生、發(fā)展中起重要作用,本研究為深入研究兒童IBS發(fā)病機制提供了實驗依據(jù)。 第四部分馬來酸曲美布汀干預幼鼠IBS模型的療效觀察 目的探討馬來酸曲美布汀干預幼鼠腸易激綜合征(IBS)模型后的治療療效。方法用母子分離、改良機械束縛、結腸芥子油刺激等綜合的方法建立幼鼠IBS模型共20只,隨機選10只在干預同時用馬來酸曲美布汀3mg/kg.d灌服治療為治療組,另10只為模型組,取同期正常生長幼鼠10只為正常對照組。分別對三組幼鼠行肛門直腸球囊擴張刺激下測腹壁肌電活動,AWR評分;處死后取結腸對其腸粘膜上皮細胞進行分層刮片、熒光染色和還原能力測定,RT-PCR檢測結腸、脊髓、丘腦中NPY和cGRP的mRNA表達。結果IBS模型組和正常對照組間不同壓力下AWR評分均有顯著性差異(P0.05),而治療組和正常對照組間差異無顯著性(P0.05);在8mmHg壓力時,IBS模型組腹壁收縮次數(shù)增加達(6.65±1.04)次/3 min ,肌電圖振幅增高,與另二組比較有顯著性(P0.01),而治療組、正常對照組間腹壁收縮活動和肌電圖振幅差異無顯著性(p0.05);在12mmHg、15mmHg擴張壓力下,IBS模型組腹壁收縮活動增加,肌電圖振幅增高(P0.05),但正常對照組與治療組間無顯著差異(p0.05);在28mmHg壓力擴張下,三組幼鼠腹壁收縮次數(shù)及肌電圖振幅增高差異均無顯著性(P0.05);三組結腸粘膜由外向內(nèi)1-4層取材標本中,IBS模型組的腸粘膜上皮細胞美藍還原反應時間最長(11.8±1.45)min ;NPY在IBS模型組結腸、脊髓、丘腦三個部位的表達均較另二組低,F值分別為11.29,34.15,27.13(均P0.05),而治療組和對照組間差異無顯著性;cGRP在模型組三個部位的表達均較正常對照組和治療組增強,F值分別為98.58,39.12,82.67(均P0.01),而治療組和對照組間表達無顯著性差異(P0.05)。結論馬來酸曲美布汀治療幼鼠IBS模型有效,可改善其腸道運動功能及內(nèi)臟高敏感性。
[Abstract]:Part one: epidemiological risk factors for irritable bowel syndrome (IBS) in children in Suzhou.
Objective To investigate the prevalence and risk factors of irritable bowel syndrome (IBS) among pupils in Suzhou. Methods A cross-sectional epidemiological study was conducted among the pupils of grade 1 to 6 in Canglang, Pingjiang and Jinlong primary schools. According to Rome II diagnostic criteria, IBS cases were diagnosed after excluding other organic diseases, and other non-IBS cases were compared. Unconditional logistic multiple regression model was fitted to analyze the possible risk factors affecting the incidence of IBS and provide scientific basis for the prevention of IBS in children. There was no significant difference in the prevalence of IBS between males and females (10.3% vs 11.3%). Multivariate logistic regression analysis showed that there was a history of food allergy (OR = 1.53, 95% CI: 1.13-2.07), a history of enteritis in childhood (OR = 1.29, 95% CI: 1.00-1.63), and a preference for food. Fried food (OR = 1.62,95% CI: 1.34-1.96), mood anxiety (OR = 1.49,95% CI: 1.16-1.93), history of unintentional attack in childhood (OR = 1.47,95% CI: 1.02-2.20) and history of constipation in parents (OR = 1.81,95% CI: 1.46-2.24) were the most likely risk factors for IBS in boys and girls. Conclusion IBS is a common disease among pupils in Suzhou. The possible risk factors affecting IBS among pupils in Suzhou are: younger age, history of food allergy, history of enteritis in childhood, preference for fried food, anxiety, history of accidental attack in childhood and history of constipation in parents. We should actively prevent and treat enteritis, adjust children's diet, avoid eating more fried food and allergic food, so as to prevent and cure the disease better.
The second part is the establishment and identification of irritable bowel syndrome model in young rats.
Methods Twenty SD neonatal rats were randomly divided into IBS model group and normal control group. The infant rats in IBS model group were intervened by maternal-fetal separation, improved mechanical restraint and colon mustard oil stimulation to establish an IBS animal model. Intervention: Rectal dilatation was performed with a self-made dilator to evaluate the abdominal contractile reflex (AWR) threshold under different pressures, and the abdominal wall electromyographic activity was measured. The IBS model group was dilated with a balloon dilator catheter of 5ml and 10ml respectively, and the control group was dilated with rectum. The fMRI data of the brains of the two groups were collected and analyzed by functional brain imaging. Data were processed by software and blood oxygen level-dependent functional MRI (BOLD-fMRI) technique was used to study the excitability of visceral pain center and the distribution of excitatory areas in the brains of irritable bowel syndrome (IBS) rats induced by rectal balloon dilatation. The ratio of young and old cells was observed by fluorescence staining and the methylene blue reduction reaction time of intestinal mucosal epithelial cells was determined. The colon histopathological examination was carried out. After intervention, the body weight of the two groups of young rats was weighed, the stool characteristics were observed, the stool routine was checked, and the wet and dry weights were collected. The stool characteristics of each group were scored by Bristol typing. The abdominal wall systolic reflex (AWR) score in the model group was significantly lower than that in the control group at different pressure (P 0.01); the abdominal wall electromyographic activity was significantly increased with the increase of pressure (P 0.01); rectal balloon stimulation could induce the viscera-related brain areas (insular cortex, prefrontal cortex, thalamus and anterior cortex) in the brain of the IBS model group. The activity of neurons in the anterior cingulate cortex was enhanced, showing the excitatory zone, but there was no corresponding excitatory zone in the control group. The symptoms of diarrhea in IBS model group were significantly higher than those in normal control group (P There was significant difference (P 0.05). Conclusion Infant mice with IBS can be successfully established by the intervention of maternal-fetal separation, modified mechanical restraint and colon mustard oil stimulation. The mechanism may be increased visceral sensitivity. Functional MRI is a more intuitive and accurate brain imaging technique for observing brain function. It is suggested that the insular cortex, prefrontal cortex and thalamus may be the main sites involved in visceral sensation in young rats with IBS.
The third part is the experimental study on the mechanism of ICC cell and brain gut peptide in the IBS model of young rats.
Objective To investigate the role of brain-gut peptide, brain-gut axis and ICC cells in the pathogenesis of infant rat IBS model by detecting the expression of brain-gut peptide NPY and cGRP mRNA in colon, spinal cord and thalamus and the expression of c-kit positive cells in colon ICC cells. Rats in the IBS model group were established by the method of maternal-fetal separation, modified mechanical restraint and colon mustard oil stimulation, while those in the control group were given no intervention. The expressions of NPY and cGRP mRNA in colon, spinal cord and thalamus were detected by Semi-quantitative reverse transcriptase polymerase chain reaction. Results The expression of NPY in colon, spinal cord and thalamus of IBS model group was significantly lower than that of normal control group, with T values of 9.66, 8.55 and 12.65 respectively (all P 0.01), while the expression of cGRP in all levels of brain-gut axis (colon, spinal cord, thalamus) was significantly higher in IBS model group than that of normal control group. There were significant differences in the number of c-kit positive cells between the two groups (t = 5.294, P 0.01). Conclusion ICC cells and brain-gut peptides may play an important role in the development of IBS model in young rats. This study provides experimental basis for further study of the pathogenesis of IBS in children.
The fourth part of trimebutine maleate intervened in the IBS model of young rats.
Objective To investigate the therapeutic effect of trimebutine maleate on irritable bowel syndrome (IBS) in young rats. Methods Twenty IBS models were established by maternal-fetal separation, modified mechanical restraint and colon mustard oil stimulation. Ten rats were randomly selected as the treatment group and 10 rats were treated with trimebutine maleate at the same time. The abdominal myoelectric activity and AWR score were measured under anorectal balloon dilatation stimulation, and the intestinal mucosal epithelial cells were scraped, stained and reduced by fluorescence. NPY and cGRP in colon, spinal cord, thalamus were detected by RT-PCR. Results There was significant difference in AWR score between IBS model group and normal control group (P 0.05), but there was no significant difference between treatment group and normal control group (P 0.05). At 8 mmHg pressure, the number of abdominal wall contractions in IBS model group increased by (6.65 (1.04) times/3 minutes, and the amplitude of electromyogram increased significantly (P 0.0). 1) There was no significant difference in abdominal wall contraction and EMG amplitude between the treatment group and the control group (p0.05); the abdominal wall contraction and EMG amplitude increased in the IBS model group under 12 mmHg and 15 mmHg dilatation pressure (P 0.05), but there was no significant difference between the normal control group and the treatment group (p 0.05); the abdominal wall contraction of the three groups of young rats under 28 mmHg dilatation pressure. There was no significant difference in frequency and amplitude of EMG (P 0.05). Among the three groups, methylene blue reduction reaction time of intestinal epithelial cells in IBS model group was the longest (11.8 The expression of cGRP in the three parts of the model group was higher than that of the normal control group and the treatment group, F values were 98.58, 39.12 and 82.67 respectively (all P 0.01), but there was no significant difference between the treatment group and the control group (P 0.05). Conclusion Trimebutine maleate is effective and can be modified in the treatment of IBS model in young rats. Good bowel movement function and visceral Gao Min sensibility.
【學位授予單位】：蘇州大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R725.7;R-332
[Abstract]:Part one: epidemiological risk factors for irritable bowel syndrome (IBS) in children in Suzhou.
Objective To investigate the prevalence and risk factors of irritable bowel syndrome (IBS) among pupils in Suzhou. Methods A cross-sectional epidemiological study was conducted among the pupils of grade 1 to 6 in Canglang, Pingjiang and Jinlong primary schools. According to Rome II diagnostic criteria, IBS cases were diagnosed after excluding other organic diseases, and other non-IBS cases were compared. Unconditional logistic multiple regression model was fitted to analyze the possible risk factors affecting the incidence of IBS and provide scientific basis for the prevention of IBS in children. There was no significant difference in the prevalence of IBS between males and females (10.3% vs 11.3%). Multivariate logistic regression analysis showed that there was a history of food allergy (OR = 1.53, 95% CI: 1.13-2.07), a history of enteritis in childhood (OR = 1.29, 95% CI: 1.00-1.63), and a preference for food. Fried food (OR = 1.62,95% CI: 1.34-1.96), mood anxiety (OR = 1.49,95% CI: 1.16-1.93), history of unintentional attack in childhood (OR = 1.47,95% CI: 1.02-2.20) and history of constipation in parents (OR = 1.81,95% CI: 1.46-2.24) were the most likely risk factors for IBS in boys and girls. Conclusion IBS is a common disease among pupils in Suzhou. The possible risk factors affecting IBS among pupils in Suzhou are: younger age, history of food allergy, history of enteritis in childhood, preference for fried food, anxiety, history of accidental attack in childhood and history of constipation in parents. We should actively prevent and treat enteritis, adjust children's diet, avoid eating more fried food and allergic food, so as to prevent and cure the disease better.
The second part is the establishment and identification of irritable bowel syndrome model in young rats.
Methods Twenty SD neonatal rats were randomly divided into IBS model group and normal control group. The infant rats in IBS model group were intervened by maternal-fetal separation, improved mechanical restraint and colon mustard oil stimulation to establish an IBS animal model. Intervention: Rectal dilatation was performed with a self-made dilator to evaluate the abdominal contractile reflex (AWR) threshold under different pressures, and the abdominal wall electromyographic activity was measured. The IBS model group was dilated with a balloon dilator catheter of 5ml and 10ml respectively, and the control group was dilated with rectum. The fMRI data of the brains of the two groups were collected and analyzed by functional brain imaging. Data were processed by software and blood oxygen level-dependent functional MRI (BOLD-fMRI) technique was used to study the excitability of visceral pain center and the distribution of excitatory areas in the brains of irritable bowel syndrome (IBS) rats induced by rectal balloon dilatation. The ratio of young and old cells was observed by fluorescence staining and the methylene blue reduction reaction time of intestinal mucosal epithelial cells was determined. The colon histopathological examination was carried out. After intervention, the body weight of the two groups of young rats was weighed, the stool characteristics were observed, the stool routine was checked, and the wet and dry weights were collected. The stool characteristics of each group were scored by Bristol typing. The abdominal wall systolic reflex (AWR) score in the model group was significantly lower than that in the control group at different pressure (P 0.01); the abdominal wall electromyographic activity was significantly increased with the increase of pressure (P 0.01); rectal balloon stimulation could induce the viscera-related brain areas (insular cortex, prefrontal cortex, thalamus and anterior cortex) in the brain of the IBS model group. The activity of neurons in the anterior cingulate cortex was enhanced, showing the excitatory zone, but there was no corresponding excitatory zone in the control group. The symptoms of diarrhea in IBS model group were significantly higher than those in normal control group (P There was significant difference (P 0.05). Conclusion Infant mice with IBS can be successfully established by the intervention of maternal-fetal separation, modified mechanical restraint and colon mustard oil stimulation. The mechanism may be increased visceral sensitivity. Functional MRI is a more intuitive and accurate brain imaging technique for observing brain function. It is suggested that the insular cortex, prefrontal cortex and thalamus may be the main sites involved in visceral sensation in young rats with IBS.
The third part is the experimental study on the mechanism of ICC cell and brain gut peptide in the IBS model of young rats.
Objective To investigate the role of brain-gut peptide, brain-gut axis and ICC cells in the pathogenesis of infant rat IBS model by detecting the expression of brain-gut peptide NPY and cGRP mRNA in colon, spinal cord and thalamus and the expression of c-kit positive cells in colon ICC cells. Rats in the IBS model group were established by the method of maternal-fetal separation, modified mechanical restraint and colon mustard oil stimulation, while those in the control group were given no intervention. The expressions of NPY and cGRP mRNA in colon, spinal cord and thalamus were detected by Semi-quantitative reverse transcriptase polymerase chain reaction. Results The expression of NPY in colon, spinal cord and thalamus of IBS model group was significantly lower than that of normal control group, with T values of 9.66, 8.55 and 12.65 respectively (all P 0.01), while the expression of cGRP in all levels of brain-gut axis (colon, spinal cord, thalamus) was significantly higher in IBS model group than that of normal control group. There were significant differences in the number of c-kit positive cells between the two groups (t = 5.294, P 0.01). Conclusion ICC cells and brain-gut peptides may play an important role in the development of IBS model in young rats. This study provides experimental basis for further study of the pathogenesis of IBS in children.
The fourth part of trimebutine maleate intervened in the IBS model of young rats.
Objective To investigate the therapeutic effect of trimebutine maleate on irritable bowel syndrome (IBS) in young rats. Methods Twenty IBS models were established by maternal-fetal separation, modified mechanical restraint and colon mustard oil stimulation. Ten rats were randomly selected as the treatment group and 10 rats were treated with trimebutine maleate at the same time. The abdominal myoelectric activity and AWR score were measured under anorectal balloon dilatation stimulation, and the intestinal mucosal epithelial cells were scraped, stained and reduced by fluorescence. NPY and cGRP in colon, spinal cord, thalamus were detected by RT-PCR. Results There was significant difference in AWR score between IBS model group and normal control group (P 0.05), but there was no significant difference between treatment group and normal control group (P 0.05). At 8 mmHg pressure, the number of abdominal wall contractions in IBS model group increased by (6.65 (1.04) times/3 minutes, and the amplitude of electromyogram increased significantly (P 0.0). 1) There was no significant difference in abdominal wall contraction and EMG amplitude between the treatment group and the control group (p0.05); the abdominal wall contraction and EMG amplitude increased in the IBS model group under 12 mmHg and 15 mmHg dilatation pressure (P 0.05), but there was no significant difference between the normal control group and the treatment group (p 0.05); the abdominal wall contraction of the three groups of young rats under 28 mmHg dilatation pressure. There was no significant difference in frequency and amplitude of EMG (P 0.05). Among the three groups, methylene blue reduction reaction time of intestinal epithelial cells in IBS model group was the longest (11.8 The expression of cGRP in the three parts of the model group was higher than that of the normal control group and the treatment group, F values were 98.58, 39.12 and 82.67 respectively (all P 0.01), but there was no significant difference between the treatment group and the control group (P 0.05). Conclusion Trimebutine maleate is effective and can be modified in the treatment of IBS model in young rats. Good bowel movement function and visceral Gao Min sensibility.
【學位授予單位】：蘇州大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R725.7;R-332
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