摘要目的 检测Wnt5a及Frizzled-1(Fzd-1)基因在肛门直肠畸形(anorectal malformations,ARM)胎鼠直肠肠壁的胚胎发育过程中的表达变化,探讨其可能与肠壁神经肌肉发育的关系.方法 将Wistar大鼠随机分为正常组与乙烯硫脲致畸的ARM组,应用免疫组化、Western Blot方法检测正常与ARM组胎鼠直肠末端组织Wnt5a及Fzd-1蛋白的定位及定量表达,RT-PCR方法检测Wnt5a及Fzd-1 mRNA的表达差异.结果 Wnt5a/Fzd-1在正常大鼠胚胎直肠发育过程中,在肠壁肌间神经丛(myenteric nerve plexus,MP)、肠壁平滑肌及黏膜层均有表达,随胚胎发育,表达强度逐渐增强,胚胎晚期主要集中在肠壁平滑肌及MP处;ARM组:越接近直肠末端的肠壁内阳性表达细胞明显减少.western blot及RT-PCR结果显示正常胎鼠直肠末端组织中Wnt5a及Fzd-1总蛋白量及mRNA明显高于畸形组(E21:Wnt5amRNA:0.81±0.14比0.21±0.03;Fzd-1mRNA:0.72±0.11比0.20±0.04;Wnt5a:1.12±0.11比0.48±0.05;Fzd-1:0.91±0.10比0.32±0.04,P<0.05).结论 Wnt5a/Fzd-1基因在肛门直肠畸形的神经肌肉发育过程中表达下调,可能与其发育不良有一定关系.

abstractsObjective To assess the roles of Wnt5a and Frizzled-1 (Fzd-1)in the neuromuscular development of the anorectal unit in fetal rats with anorectal malformations (ARM).Methods The ARM was induced by ethylenethiourea (ETU)in fetal rats.The RNA and protein expression level of Wnt5a and Fzd-1 were studied by immunohistochemistry,reverse transcription polymerase chain reaction (RT-PCR)and Western blotting.Results Immunohistochemical staining demonstrated Wnt5a and Fzd-1 expressed in myenteric nerve plexus (MP),smooth muscle layers and the mucosa of the rectum in the normal rat fetuses.From E17 to E21,the expression became stronger and stronger in normal fetuses.In the late fetal phase,Wnt5a and Fzd-1 expression was mostly found in the MP and smooth muscle layers.However,in ARM embryos,the expression of Wnt5a and Fzd-1 were only found in the rectum terminus.During gestational day 19 to 21,the mRNA and protein expression of Wnt5a and Fzd-1 significantly decreased in the rectum of ARM rat embryos comparing with normal rat embryos (E21:Wnt5amRNA:0.81 ± 0.14 vs 0.21 ± 0.03;Fzd-1mRNA..0.72 ± 0.11 vs 0.20 ±0.04;Wnt5a:1.12 ± 0.11 vs 0.48 ± 0.05;Fzd-1:0.91 ± 0.10 vs 0.32 ± 0.04,P<0.05).Conclusions The expressions of Wnt5a and Fzd-1 were downregulated in anorectal unit of fetal rats with ethylenethiourea induced anorectal malformations,which may play important roles in the pathogenesis of neuromuscular development.