腹泻型肠易激综合征患者结肠黏膜类胰蛋白酶变化及其对内脏感觉的影响

The variation of tryptase expression in the colonic mucosa of patients with diarrhea-predominant irritable bowel syndrome and its effect on the visceral sensitivity

目的 观察腹泻型肠易激综合征（1BS-D）患者结肠黏膜类胰蛋白酶变化及其结肠黏膜孵育上清液对小鼠内脏感觉的影响.方法 IBS-D患者研究：肠镜检查获取24例非感染后IBS-D患者、12名健康志愿者的升结肠黏膜组织4块,采用甲苯胺蓝染色法计数黏膜肥大细胞数量、酶联免疫分析法检测结肠黏膜孵育上清液中类胰蛋白酶浓度.动物实验：56只6～8周龄C57BL/6系雄性小鼠分为7组,分别予健康小鼠升结肠黏膜培养上清液100μl（A组）、IBS-D升结肠黏膜培养上清液100 μl（B组）、IBS-D升结肠黏膜培养上清液100μl＋丝氨酸蛋白酶抑制剂Fut-175（50μg/ml,C组）、0.6 ng类胰蛋白酶（D组）、2.2 ng类胰蛋白酶（E组）、2.2 ng类胰蛋白酶±Fut-175（50μg/ml,F组）和0.9%氯化钠溶液（G组）灌肠.6 h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P物质、降钙素基因相关肽（CGRP）表达变化.结果 IBS-D患者研究：IBS-D患者升结肠肥大细胞（MC）数量为（6.33±1.64）个/高倍视野,健康对照组为（3.08±0.77）个/高倍视野,差异有统计学意义（P=0.000）.IBS-D患者升结肠黏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,差异有统计学意义（P=0.000）.动物实验发现在气囊内压为30、45、60 mmHg时,B组及E组小鼠的腹壁肌电幅值[（27.50±5.23）、（41. 69±5.80）、（54.03±6.13）μV和（27.05±1.66）、（40.66±3.39）、（54.38±4.68）μV]较A组、C组、D组、F组、G组高（P值均＜0.05）.B组及E组脊髓背角P物质和CGRP之积分吸光度值（121.72±4.67、123.54±4.32和124.69±5.06、126.32±5.09）较A组、C组、D组、F组、G组高（P值均＜0.05）.结论 IBS-D患者升结肠黏膜肥大细胞数量和类胰蛋白酶含量均明显增加,而且其结肠黏膜孵育上清液可导致小鼠内脏感觉过敏,提示类胰蛋白酶可能参与IBS-D患者内脏敏感性的改变.

Objective To observe the variation of tryptase expression in the colonic mucosa of patients with diarrhea-predominant irritable bowel syndrome（IBS-D） and the effects of their colonic mucosa cultured supernatants on mice visceral sensitivity. Methods The IBS-D patients study： of twenty-four patients with IBS-D and 12 healthy volunteers, four mucosal biopsies of each individual were collected at ascending colon under colonoscopy. The quantity of mucosal mast cells were detected by toluidine blue stains, and the tryptase concentration of colonic mucosa cultured supernatants were determined by enzyme-linked immunosorbent assay （ELISA）. Animal experiments： fifty-six male six to eight weeks old C57BL/6 mice were divided into 7 groups and then were administrated with healthy volunteer＇s ascending colonic mucosa-cultured supernatants 100 μl （group A）, IBS-D patient＇ s ascending colonic mucosa-cultured supernatants 100 μl （group B）, IBS-D ascending colonic mucosacultured supernatants 100 μl with Fut-175（50 μg/ml, group C）, tryptase with different concentration：0.6 ng （group D）, 2.2 ng （group E）, tryptase 2.2 ng with Fut-175 （group F）, and 0.9% sodium chloride as control （group G） respectively. After six hours, the abdominal myoelectric activities （AMAs） were recorded during colorectal balloon distension （CRD）, and the expressions of substance P （SP）, Calcitonin gene-related peptide （CGRP） in spinal dorsal horn of lumbosaeral region were detected by immunohistochemistry. Results The number of mast cells in the ascending colon of patients with IBS-D [6.33 ± 1.64） / HP] was higher than that in controls [（3.08 ± 0.77 ） / HP], the difference was statistical significant （P = 0.000）. The tryptase concentration of ascending colonic mucosa cultured supernatants in IBS-D patients [（0. 072 ± 0. 023） ng/mg] was higher than that in controls [（0.026 ± 0.014） ng/mg], the difference was statistical significant （ P = 0. 000）. In animal experiments, when the inside pressure of balloon was 30, 45, 60 mmHg, respectively, the AMAs value in group B [（27.50±5.23）, （41.69±5.80）, （54. 03±6.13） μV, respectively] and group E [（27. 05±1.66）, （40. 66±3.39）, （54. 38± 4.68） μV, respectively] were significantly higher than those in group A, group C, group D, group F and group G （P〈0. 05, respectively）. The average integrated option densities （IOD） of SP and CGRP in spinal dorsal horn in groupB （121.72 ± 4.67,123.54±4.32）and E （124.69±5.06,126.32±5.09） were higher than in group A, group C, group D,group F and group G （P〈0.05, respectively）. Conclusion The quantity of mast cell and the content of tryptase in the ascending colonic mucosa of IBS-D patients increased significantly. And their colonic mucosa cultured supernatants caused the mice visceral hypersensitivity, which indicated that tryptase may involved in the variation of visceral sensitivity in IBS-D patients.