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目的研究肠易激综合征(IBS)患者结肠黏膜中促肾上腺皮质激素释放因子受体(CRFR)表达与肥大细胞的关系。方法选取2012年2月至2014年5月处于症状活动期的IBS患者74例作为实验组[其中便秘型(CIBS)29例,腹泻型(D-IBS)32例,混合(交替)型(A-IBS)13例],另选择痔疮便血及结肠息肉电切术术后的非IBS患者30例作为对照组,经结肠镜检查获取两组患者回盲部结肠黏膜组织。利用实时荧光定量酶链反应(real-time PCR)检测即刻早期基因(c-Fos)、CRFR1和CRFR2 mRNA表达,利用免疫组织化学染色和Western blot法检测CRFR1和CRFR2蛋白表达,利用甲苯氨蓝染色光镜下观察肥大细胞脱颗粒现象,采用免疫双标染色法分析肥大细胞数目及活化情况。结果 (1)real-time PCR检测发现,实验组C-IBS、D-IBS和A-IBS患者结肠黏膜组织中c-Fos mRNA表达量均高于对照组(P均<0.05),且D-IBS患者c-Fos mRNA表达量高于C-IBS和A-IBS患者(P均<0.05);实验组CRFR1 mRNA表达量均低于对照组(P均<0.05),以D-IBS组表达量最低;C-IBS和A-IBS患者CRFR2 mRNA表达量高于对照组(P均<0.05),D-IBS患者CRFR2 mRNA表达量低于对照组(P<0.05)。(2)免疫组织化学法和Western blot检测发现,C-IBS、D-IBS和A-IBS患者结肠黏膜组织中,CRFR1蛋白表达水平均低于对照组(P均<0.05),以D-IBS组表达水平最低;对照组CRFR2表达水平低于C-IBS组(P<0.05),但对照组、DIBS组和A-IBS组三者差异无统计学意义(P>0.05)。(3)甲苯氨蓝染色光镜下观察肥大细胞发现,IBS患者均存在一定程度的肥大细胞脱颗粒,其中以D-IBS组最严重,C-IBS和A-IBS次之;免疫双标染色法发现实验组肥大细胞计数和活化肥大细胞分数均显著高于对照组(P均<0.05)。结论 CRFR1和CRFR2表达水平的改变可能是介导肥大细胞活化的重要因素,在IBS的发病机制中可能发挥着重要作用。
Objective To investigate the relationship between the expression of corticotropin-releasing factor receptor (CRFR) and mast cells in colonic mucosa of patients with irritable bowel syndrome (IBS). Methods Totally 74 IBS patients with symptomatic active disease from February 2012 to May 2014 were selected as the experimental group [including 29 cases of constipation type (CIBS), 32 cases of diarrhea type (D-IBS), 32 cases of mixed type -IBS) 13 cases], another choice hemorrhoids and colorectal polyp resection of non-IBS patients 30 cases as a control group, by colonoscopy to obtain two groups of patients with ileocecal colon mucosal tissue. The expressions of c-Fos, CRFR1 and CRFR2 mRNA were detected by real-time PCR. The expressions of CRFR1 and CRFR2 protein were detected by immunohistochemistry and Western blot. The phenomenon of mast cell degranulation was observed under light microscope. The number of mast cells and the activation of mast cells were analyzed by double-stained immunofluorescence staining. Results (1) The real-time PCR results showed that the expression of c-Fos mRNA in colonic mucosa of C-IBS, D-IBS and A-IBS patients was higher than that of the control group (all P < The expression of c-Fos mRNA in IBS patients was higher than that in C-IBS and A-IBS patients (all P <0.05). The expression of CRFR1 mRNA in experimental group was lower than that in control group (all P <0.05) (P <0.05). The expression of CRFR2 mRNA in C-IBS and A-IBS patients was lower than that in control group (P <0.05). (2) Immunohistochemistry and Western blot showed that the expression of CRFR1 protein in colonic mucosa of C-IBS, D-IBS and A-IBS patients was lower than that of the control group (all P <0.05) The expression level of CRFR2 in control group was lower than that in C-IBS group (P <0.05), but there was no significant difference between control group, DIBS group and A-IBS group (P> 0.05). (3) Toluene Ammonia blue staining Mast cells were observed under light microscope. There was a certain degree of degranulation of mast cells in IBS patients, of which D-IBS group was the most serious, followed by C-IBS and A-IBS; The results showed that the number of mast cells and the number of activated mast cells in the experimental group were significantly higher than those in the control group (all P <0.05). Conclusion The changes of CRFR1 and CRFR2 expression may play an important role in mediating the activation of mast cells and may play an important role in the pathogenesis of IBS.