慢传输性便秘儿童结肠壁内脂肪酰胺水解酶的检测

Expression and activity of fatty acid amide hydrolase in the colon in children with slow transit constipation

目的 检测脂肪酰胺水解酶（FAAH）在慢传输性便秘（STC）患儿结肠壁内的表达及活性情况,并探讨其与儿童STC发生的关系.方法 取便秘组（21例）和对照组（15例）患者手术切下的结肠标本,应用免疫组织化学、Western blot及实时荧光定量PCR方法,对两组儿童结肠壁内FAAH的表达进行分析,并联合应用免疫荧光双重染色技术观察FAAH与大麻素受体1（CB1）的位置与分布,联合应用高压液相色谱技术对两组患儿结肠壁内FAAH的生物活性进行定量检测和分析.结果 便秘组升结肠、降结肠和乙状结肠肠壁中,Western印迹检测FAAH的蛋白表达分别为8.7±3.4、8.2±1.2和8.0±7.2,均弱于对照组的10.5±3.7、10.0±6.4和9.8±3.4,两组比较,差异有统计学意义（P＜0.05）;免疫组化和荧光定量PCR检测结果相同.FAAH在升结肠、降结肠和乙状结肠肠壁中的水解活性分别为（0.51±0.23）nmol·min^-1·mg^-1、（0.39±0.25）nmol·min^-1·mg^-1和（0.35±0.37）nmol·min^-1·mg^-1,低于对照组的（0.84±0.24）nmol·min^-1·mg^-1、（0.55±0.44）nmol·min^-1·mg^-1和（0.58±0.48）nmol·min^-1·mg^-1,两组比较,差异有统计学意义（P＜0.05）.结论 STC患儿结肠壁内FAAH的表达降低,水解活性减弱;FAAH可能参与了儿童STC的发生.

Objective To investigate the expression and activity of fatty acid amide hydrolase （FAAH） in the colon and its role in children with slow transit constipation （STC）.Methods Patients were divided into constipation group（n=21） and control group（n=15）.The constipation group was consistent with the diagnostic criteria for STC.Western blotting,immunohistochemistry and real-time PCR were used to examine the FAAH expression in surgical specimen of colon.The location and distribution of FAAH and cannabinoid receptor type 1 （CB1）were detected by immunofluorescence double staining.The biological activity of colon FAAH was detected by high-performance liquid chromatography.Results Western blotting revealed that FAAH protein expression in the ascending colon,descending colon and sigmoid colon were significantly decreased in the myenteric neurons and absorption cells in the constipation group as compared with the control group（8.68±3.4 vs.10.47±3.7,8.21±1.2 vs.9.95±6.4,8.01±7.2 vs.9.79±3.4,all P〈0.05）.The same results were found by immunohistochemistry and real-time PCR.The FAAH hydrolysis activity in the ascending colon,descending colon or sigmoid colon decreased significantly in the constipation group,as compared with the control group [（0.51±0.23） nmol·min^（-1）.mg^（-1） vs.（0.84±0.24） nmol·min^-1·mg^-1,（0.39±0.25） nmol·min^-1·mg^-1 vs.（0.55±0.44） nmol·min^-1·mg^-1,（0.35±0.37） nmol·min^-1·mg^-1 vs.（0.58±0.48） nmol·min^-1·mg^-1,all P〈0.05].Conclusion FAAH expression and hydrolysis activity in the colon decrease in children with STC.FAAH may play a role in the pathogenesis of slow transit constipation in Children.