摘要目的 探讨肠易激综合征(IBS)结肠黏膜是否存在黏附连接——E-钙黏蛋白(E-cad)、β-链蛋白(β-cat)的改变.方法 以结直肠扩张联合束缚应激建立SD大鼠IBS模型,模型组SD大鼠8只,正常对照组SD大鼠8只,取大鼠距回盲部5 cm结肠组织(I)和直肠乙状结肠交界处结肠组织(R),用实时荧光定量PCR、蛋白印迹和免疫荧光检测E-cad、β-cat mRNA和蛋白水平及黏附连接结构变化.根据罗马Ⅲ标准收集2016年于浙江中医药大学附属第一医院消化内镜中心接受肠镜检查的腹泻型IBS(IBS-D)患者17例及健康志愿者(健康对照)17名,取I处和R处结肠的活检标本,用蛋白印迹、免疫荧光和透射电镜检测结肠上皮细胞间E-cad、β-cat蛋白表达以及黏附连接结构的改变情况.结果 (1)IBS模型组大鼠I和R处E-cad的mRNA水平和蛋白表达均明显低于对照组大鼠(均P<0.05).IBS模型组大鼠I处和R处β-cat的mRNA水平与对照组大鼠比较差异无统计学意义,β-cat蛋白水平表达在I处低于对照组大鼠(P<0.05),在R处与对照组大鼠比较差异无统计学意义.免疫荧光结果可见IBS模型组大鼠I处和R处E-cad结构较对照组大鼠模糊;β-cat在对照组和IBS模型组大鼠I和R处无明显差异.(2)IBS-D患者的I处和R处上皮组织中E-cad的蛋白表达均低于健康对照(I处:0.85±0.30比1.24±0.34,P=0.00;R处:0.86±0.17比1.14±0.48, P=0.05);β-cat蛋白表达也均低于健康对照(I处:0.85±0.39比1.22±0.51, P=0.04;R处:0.92±0.22比1.16±0.31, P=0.02).免疫荧光结果可见E-cad和β-cat在健康对照的I处和R处结肠上皮细胞均沿细胞膜分布,呈蜂窝线性荧光,而IBS-D患者E-cad和β-cat结构较模糊,胞质内分布紊乱.透射电镜观察到在IBS-D患者I处和R处上皮细胞间连接间隙较健康对照增宽.结论 IBS-D患者存在细胞间连接间隙增宽,全结肠细胞间黏附连接蛋白E-cad和β-cat的降低伴随结构的紊乱.E-cad和β-cat的改变在IBS-D 患者结肠黏膜屏障破坏及肠上皮细胞间通透性的增加中发挥重要的重用,但其具体机制,有待进一步研究.

abstractsObjective To investigate whether there are changes in adherens junctions(AJ), including E-cadherin (E-cad) and β-catenin (β-cat), in the colonic mucosa of irritable bowel syndrome (IBS).Methods Colorectal dilatation combined with restraint stress was used to establish IBS SD rat model. There were 8 rats in the model group and 8 rats in normal control group. From each rat, the colon tissue 5 centimeters away from ileocecum (I) and the junction of the rectum and sigmoid colon (R) were taken, in which E-cad and β-cat mRNA,protein level and structural changes were detected using real-time quantitative-PCR, Western blot, and immunofluorescence. According to the Rome Ⅲ criteria, IBS with diarrhea (IBS-D) patients receiving colonoscopy (n=17) and healthy volunteers (n=17) were enrolled in Center of Endoscopy of the First Affiliated Hospital of Zhejiang Chinese Medicine University. The colon tissues of I and R locations were collected from them. Colonic epithelial cell AJ protein expression and AJ structural changes in the tissues were detected using Western blot, immunofluorescence and transmission electron microscope (TEM).Results (1)Both in I and R sites, the mRNA and protein expression of E-cad in IBS rats were both significantly lower than in normal SD rats (all P<0.05). The mRNA expression of β-cat showed no significant change in IBS SD rats compared with the normal SD rats;the protein expression of β-cat was decreased in I(P<0.05), no significantly different in R.(2)In I and R of IBS-D patients, the expression of E-cad protein were significantly decreased(I: 0.85±0.30 vs 1.24±0.34, P=0.00;R: 0.86±0.17 vs 1.14±0.48, P=0.05);the protein expression of β-cat also showed significant reduction both in I(0.85±0.39 vs 1.22±0.51, P=0.04)and R (0.92±0.22 vs 1.16±0.31, P=0.02) of IBS-D patients, compared with the healthy controls. Immunofluorescence results showed that E-cad and β-cat in colonic epithelial cells of I and R in the healthy controls were distributed along the cell membrane, demonstrating honeycomb linear fluorescence;however, in IBS-D patients, the structure of E-cad and β-cat were fuzzy, with disrupted distribution in the cytoplasm. Under TEM, the gaps of AJ in I and R of IBS-D patients appeared wider than in the healthy volunteers.Conclusions In IBS-D patients, there are wider gaps of AJ, and decreased expression with structural disorder of E-cad and β-cat in the colon. The change of AJ plays an important role in the damage of colonic mucosal barrier and the increase of intestinal epithelial permeability in IBS-D patients, but the mechanism is yet to be further explored.