神经导向因子-2及受体在先天性巨结肠中的表达

Expression of slit guidance ligand-2 and its receptor in Hirschsprung′s disease

目的观察先天性巨结肠(HSCR)患儿各段肠壁内神经导向因子-2(SLIT-2)及受体(ROBO-1)的表达、分布,探讨其在HSCR发病中的作用关系。方法 30例结肠标本选自2018年1月至2019年1月在山东大学齐鲁儿童医院外科手术切除的HSCR患者,根据术中结肠形态及术后病理结果分为正常段组(n=30)、移行段组(n=30)和痉挛段组(n=30)。应用免疫组织化学染色、蛋白质印迹法(Western blot)及实时定量反转录聚合酶链反应(RT-qPCR)方法检测SLIT-2和ROBO-1在各组中的表达。组间比较采用多因素方差分析。结果 (1)免疫组织化学染色结果显示SLIT-2和ROBO-1在痉挛段组中的阳性细胞表达量分别为53.767±14.395和42.933±14.034,明显低于移行段组和正常段组(89.667±14.822和93.867±15.874;126.733±9.770和119.674±12.933,t=11.437、6.902,P<0.05)。(2)Western blot结果显示SLIT-2及ROBO-1蛋白在痉挛段组肠管中相对表达量分别为0.348±0.086和0.225±0.069,明显低于移行段组和正常段组(0.541±0.085和0.392±0.081;0.702±0.123和0.634±0.081,t=5.532、8.622,P<0.05)。(3)RT-qPCR结果显示SLIT-2及ROBO-1 mRNA在痉挛段组肠管中相对表达量分别为0.574±0.159和0.477±0.169,明显低于移行段组和正常段组(0.749±0.162和0.663±0.175;0.914±0.146和0.834±0.116,t=4.128、8.624,P<0.05),与蛋白表达结果一致。结论 SLIT-2和ROBO-1在HSCR痉挛段肠管的表达量低于移行段组和正常组,提示其可能参与了结肠神经系统的发育成熟过程,并可能与HSCR的发生有关。

Objective To investigate the expression and distribution of slit guidance ligand-2(SLIT-2)and its receptor roundabout-1(ROBO-1)in the intestinal wall of each segment in children with Hirschsprung′s disease(HSCR),and to explore their roles in the pathogenesis of HSCR.Methods Totally,30 patients with HSCR surgically excised in Qilu Children′s Hospital of Shandong University from January 2018 to January 2019 were selected and divided into normal segment group(n=30),transitional segment group(n=30)and spastic segment group(n=30)according to the intraoperative colon morphology and postoperative pathological results.Immunohistochemical staining,Western blotting and real-time quantitative reverse transcriptase-polymerase chainreaction(RT-qPCR)were used to detect the expression of SLIT-2 and ROBO-1 in each group.Multivariate analysis of variance was used for comparison between groups.Results(1)Immunohistochemical staining results showed that the positive expression levels of SLIT-2 and ROBO-1 in the spastic segment were 53.767±14.395 and 42.933±14.034 respectively,which were significantly lower than those in the transitional segment group and normal segment group(89.667±14.822 and 93.867±15.874;126.733±9.770 and 119.674±12.933)(t=11.437,6.902,P<0.05).(2)Western blotting showed that the relative expression levels of SLIT-2 and ROBO-1 proteins in the spastic segment were 0.348±0.086 and 0.225±0.069,respectively,which were significantly lower than those in the transitional group and normal segment group(0.541±0.085 and 0.392±0.081;0.702±0.123 and 0.634±0.081,t=5.532,8.622,P<0.05).(3)The RT-qPCR results revealed that the relative mRNA expression levels of SLIT-2 and ROBO-1 in the spastic segment were 0.574±0.159 and 0.477±0.169 respectively,which were significantly lower than those in the transitional group and normal segment group(0.749±0.162 and 0.660±0.170;0.914±0.146 and 0.834±0.116,t=4.128,8.624,P<0.05),consistent with the results of protein expression.Conclusion The expression of SLIT-2 and ROBO-1 decreases significantly in the spastic segment of the intestinal tract in HSCR,suggesting that they may be involved in the development and maturation of the colonic enteric nervous system and may be related to the occurrence of HSCR.