【作者】 郭朝书；

【导师】 王承党；

【作者基本信息】 福建医科大学 ， 内科学， 2010， 硕士

【摘要】 目的:测定腹泻型肠易激综合征(IBS-D)病人升结肠肥大细胞的数量及类胰蛋白酶在结肠粘膜含量的改变,类胰蛋白酶对小鼠内脏感觉和脊髓背角神经递质(P物质(SP)和降钙素基因相关肽(CGRP)的影响,探讨类胰蛋白酶在腹泻型肠易激综合征发病机理中的作用。方法:1、符合RomeⅢ诊断标准的IBS-D病人24例、健康志愿者12例,肠镜检查时于升结肠取粘膜组织4块,采用甲苯胺蓝染色法计数粘膜肥大细胞数量、采用ELISA法检测结肠粘膜孵育上清液中类胰蛋白酶浓度,并与IBS-D病人胃肠道症状评分(GSRS)进行相关分析。2、C57BL/6系雄性6-8周龄小鼠56只随机分为7组,分别予健康对照组升结肠粘膜培养上清液100μl(A组)、IBS-D升结肠粘膜培养上清液100μl(B组)、IBS-D升结肠粘膜培养上清液100μl+丝氨酸蛋白酶抑制剂Fut-175(50μg /ml)(C组)、不同浓度类胰胰蛋白酶(0.6ng(D组)、2.2ng(E组)、2.2 ng+Fut-175(F组))、生理盐水灌肠(G组)。6小时后测定结肠气囊扩张时腹壁肌电变化;免疫组化法观察腰骶部脊髓背角SP、CGRP表达变化。结果:IBS-D病人升结肠MC数量(6.33±1.64)个/HP,健康对照组为(3.08±0.77)个/HP,差异有显著性意义(P=0.000)。IBS-D病人升结肠粘膜组织类胰蛋白酶浓度为(0.072±0.023) ng/mg组织,健康对照组为(0.026±0.014) ng/mg组织,差异有显著性意义(P=0.000)。类胰蛋白酶浓度与GSRS呈正相关(r=0.581,P=0.003),而肥大细胞数量与类胰蛋白酶浓度(r=0.010,P=0.963)、及GSRS(r=0.209,P =0.326)无显著相关性。各试验组小鼠在气囊内压为15mmHg时腹壁肌电差异无统计学意义。在气囊内压为30、45、60mmHg时,B组的腹壁肌电幅值(27.50±5.23,41.69±5.80,54.03±6.13uV)及E组(27.05±1.66,40.66±3.39,54.38±4.68 uV)较A组(22.49±3.43,31.45±3.62,47.68±5.78 uV)、C组(23.45±2.81,34.40±3.70,44.81±6.62 uV)、D组(22.92±3.31,33.81±3.73,45.73±6.16 uV)、F组(20.94±3.17,34.08±4.67,43.02±4.34 uV)、G组(22.24±3.49,32.67±6.99,47.82±6.79 uV)高(P均﹤0.05)。B组脊髓背角SP、CGRP之IOD值(121.72±4.67,123.54±4.32)与E组SP、CGRP之IOD值(124.69±5.06,126.32±5.09)较A组(114.76±3.02,105.79±5.22)、C组(113.62±4.04,110.06±4.89)、D组(115.75±3.99,106.93±4.85)、F组(112.04±5.92,107.79±5.43)、G组(114.74±3.90,110.59±4.63)高(P均﹤0.05)。各试验组小鼠在气囊内压为15mmHg时腹壁肌电差异无统计学意义(P=0.567>0.05)。在气囊内压为30、45、60mmHg时,B组及E组的腹壁肌电幅值较A、C、D、F、G组高(P均﹤0.05)。B组及E组脊髓背角SP、CGRP之IOD值较A、C、D、F、G组高(P均﹤0.05)。结论:IBS-D病人升结肠粘膜表达肥大细胞增加,释放类胰蛋白酶量增加;类胰蛋白酶可以导致小鼠内脏敏感性增高,类胰蛋白酶抑制剂FUT-175可以抑制此作用。更多还原

【Abstract】 Objective: To determine the numbers of mast cell and the content of tryptase in the ascending colon mucus in patients with diarrhea-predominant irritable bowel syndrome (IBS-D), to investigate the effect of tryptase on the visceral sensitivity and the expressions of substance P(SP), calcitonin gene-related peptide(CGRP) on cornu dorsale medullae spinalis in mince, and then to explore the roles of tryptase in the pathogenesis of IBS-D.Methods: Twenty-four patients with IBS-D and 12 healthy volunteers were included in this study. The mucosal biopsies were collected from ascending colon during colonoscopy, the mucosal mast cells were detected by toluidine blue stains, and the concentration of tryptase in biopsy-cultured supernatants was detected by ELISA.The relations of mast cell numbers, tryptase level with gastrointestinal symptoms Rating scale(GSRS) in IBS-D were analyzed respectivly.Fifty-six C57BL/6 mice were randomly divided into 7 groups and then were administrated intracolonically with volunteer’s biopsy-cultured supernatants (group A), IBS-D patient’s biopsy-cultured supernatants (group B) , supernatants of IBS-D biopsy-cocultured with FUT-175(group C), tryptase solution of 0.6ng(group D) and of 2.2ng(group E), tryptase + FUT-175(group F), and 0.9% sodium chloride as control (group G) respectively.The abdominal myoelectric activities (AMAs) during colorectal distension (CRD) were recorded, and the expressions of SP, CGRP in spine cord of these mice were detected by immunohistochemistry.Results: The numbers of mast cells (6.33±1.64 /HPE) in the ascending colon of patients with IBS-D were significantly higher than those in controls (p=0.000). The contents of tryptase (0.072±0.023ng/mg) from IBS-D patient’s biopsies were significantly higher than those in controls (p=0.000). A positive correlation was detected between the content of tryptase and the GSRS (r=0.581, p=0.003). However, there was not a correlation neither between the mast cell numbers and the tryptase contents (r=0.010,p=0.963), nor between the mast cell numbers and the GSRS (r=0.209, p =0.326).The AMAs in group B (27.50±5.23, 41.69±5.80, 54.03±6.13uV) and E (27.05±1.66, 40.66±3.39, 54.38±4.68 uV) with colonic distention by 30, 45, 60mmHg were significantly higher than those in group A (22.49±3.43, 31.45±3.62, 47.68±5.78uV), group C (23.45±2.81, 34.40±3.70,44.81±6.62uV), group D (22.92±3.31, 33.81±3.73,45.73±6.16 uV), group F (20.94±3.17, 34.08±4.67, 43.02±4.34uV) and group G (22.24±3.49, 32.67±6.99, 47.82±6.79 uV) respectively (p<0.05). The SP and CGRP average integrated option density(IOD) in group B (121.72±4.67, 123.54±4.32) and E (124.69±5.06, 126.32±5.09) were higher than in group A (114.76±3.02,105.79±5.22), group C (113.62±4.04, 110.06±4.89), group D (115.75±3.99, 106.93±4.85), group F (112.04±5.92, 107.79±5.43), group G (114.74±3.90, 110.59±4.63) respectively (p﹤0.05). There was no significant difference between group B and group E, and there was no significant difference among group A, C, D, F and group G.Conclusion: These results demonstrated that the increasing mast cell numbers and the abundant tryptase in the colon mucousa may be involved in the pathogenesis of IBS-D. Tryptase could stimulate the expressions of SP, CGRP in spine cord, which may alter the visceral sensitivity.更多还原