Wnt2、Wnt5b和Wnt9a在先天性巨结肠症和肛门直肠畸形中的表达及意义

The expressions of Wnt2, Wnt5b and Wntga in colons of Hirschsprung disease and anorectal malformations

目的研究Wnt2、Wnt5b和Wnt9a在先天性巨结肠症（Hirschsprungdisease，HD）和肛门直肠畸形中（anorectalmalformations，ARMs）的表达情况，以期探讨其在HD和ARMs发生发展中的作用。方法利用荧光实时定量PCR（quantitative real—timePCR，qRT-PCR）和蛋白质印迹（westernblot）技术检测HD狭窄肠段和正常肠段肠管组织及ARMs直肠末端（高位、中位和低位组）和死于非胃肠道疾病（正常对照组）患儿各30例中Wnt2、Wnt5b和Wnt9a表达，对其表达进行定位和定量，并比较分析。结果Wnt2、Wnt5b和Wnt9a在HD狭窄段肠管中mRNA转录水平的表达分别为（10．19±0．27、10．38±0．19和11．43±0．51），均低于正常段肠管的相应表达水平（18．06±1．07、17．98±1．12、19．99±0．76）（P〈0．05）；Wnt2、Wnt5b和Wnt9a在ARMs高位组中mRNA转录水平的表达分别为（14．27±0．31、13．69±0．45、13．07±0．67），均低于正常对照组（24．98±2．03、26．43±1．77、28．11±1．44）（P〈0．05），中位组和低位组之间mRNA转录水平比较无统计学意义。westernblot结果显示，Wnt2、Wnt5b和Wnt9a在HD狭窄段肠管中蛋白表达水平分别为0．19±0．09、0．26±0．07、0．23±0．06，明显低于正常段肠管0．43±0．13、0．57±0．08、0．62±0．06，P〈0.05；Wnt2，Wnt5b和Wnt9a在ARMs高位组中蛋白表达水平分别为0．15±0．11、0．17±0．04、0．13±0．05明显低于正常对照组0．51±0．03、0．63±0．05、0．59±0．05，P〈0．05，而中位组和低位组之间蛋白表达水平无明显差异且与正常对照组比较无统计学意义（P〉0．05）。结论Wnt2、Wnt5b和Wnt9a在HD狭窄段和ARMs的高位组表达水平减低，可能在先天性消化道畸形的肠道发育中具有重要作用。

Objective To investigate the expressions of Wnt2, Wnt5b and Wnt9a in tissue samples from the patients with Hirschsprung disease （HD） and anorectal malformations （ARMs）. Methotis Ninety individuals were recruited in this study, including 30 with HD, 30 with ARMs, and 30 controls died of non-gastrointestinal diseases. According to the position of malformations, the 30 ARMs patients were subgrouped into high, intermediate and low ARMs groups. The terminal rectum from the ARMs group and the stenotic and normal appearance intestines from HD patients were collected during operation. The expression levels of Wnt2, Wnt5b and Wnt9a were studied using quantitative Real-time PCR （qRT-PCR） and western blotting. Results In the HD patients, the mRNA of Wnt2, Wnt5b and Wnt9a in stenotic intestine were significantly lower than those in the normal intestine （the relative quantification of mRNA 10. 19 ± 0.27, 10.38 ± 0. 19, 11.43 ± 0.51 vs 18.06 ± 1.07, 17. 98± 1.12, 19. 99± 0. 76, P〈0. 05）. In the ARMs patients, the mRNA of Wnt2, Wnt5b and Wnt9a in high ARMs patients were significantly lower than those in control patients （the relative quantification of mRNA 14. 27 ± 0. 31, 13. 69 ± 0. 45, 13. 07 ± 0. 67 vs 24. 98 ± 2. 03,26. 43 ± 1.77, 28. 11 ± 1.44, P〈0.05）. No significant difference of the mRNA expression was found between the low ARMs patients and intermediate ARMs patients （P〉0. 05）. The protein expressions of Wnt2, Wnt5b and Wnt9a in these patients changed in the same manners with what were found in the mRNA studies, in which Wnt2, Wnt5b and Wnt9a were decreased in the stenotic intestine of HD patients and high ARMs patients. Conclusions The expression of Wnt2, Wnt5b and Wnt9a changes in the tissue samples from the patients with HD and ARMs, which may play a role in the pathogenesis of congenital malformation of the alimentary tract.