Dickkopfl在肛门直肠畸形大鼠泄殖腔发育中的差异表达研究

Differential expression of Dickkopfl in developing cloaca between normal rat embryos and counterparts with anoreetal malformations

目的 研究Wnt信号通路的细胞外拮抗物Dickkopf1（Dkk1）在正常胎鼠和肛门直肠畸形胎鼠的泄殖腔及后肠组织中的差异表达水平，探讨Dkk1与肛门直肠畸形（anorectal malformations，ARM）发生的相关关系。方法 用乙烯硫脲（ethylenethiourea，ETU）致畸Wistar孕鼠24只，制成ARM动物模型，根据切片观察，组成畸形组（n＝96），胎龄在12～17、19、21 d。取相同胎龄的正常对照组孕鼠16只，对应胎龄胎鼠作为正常对照组（n＝96）。常规制备胎鼠冰冻切片，解剖显微镜下手工显微切割方法准确切取泄殖腔及后肠组织。实时定量聚合酶链反应（real-time polymerase chain reaction，RT-PCR）方法和蛋白印迹（Western blot）方法，检测正常和ARM不同胎龄胎鼠的泄殖腔及后肠组织中Dkk1的mRNA表达情况和蛋白水平。应用随机区组设计的两因素方差分析，比较正常和肛门直肠畸形胎鼠泄殖腔和后肠组织的Dkk1表达水平的差异。结果 在对照组和畸形组大鼠胚胎泄殖腔及后肠组织中均能检测出Dkk1 mRNA和蛋白表达。对照组Dkk1 mRNA及Dkk1蛋白表达在胎龄16 d的大鼠中表达达到峰值分别为2.77±0.06和3.20±0.76。畸形组泄殖腔及后肠组织Dkk1 mRNA及Dkk1蛋白表达呈波动态势，平均水平分别为0.96±0.14和0.87±0.14，与对照组2.00±0.52和1.98±0.65比较，差异有统计学意义（P〈0.05）。结论 Dkk1在胎鼠的泄殖腔及后肠组织中的低表达可能与ARM的发生有关，可能在ARM的发生中具有重作用。

Objective Dickkopf1 （Dkk1） is an extracellular negative regulator of Wnt signaling pathway. This study was intended to explore the differential expression of Dkk1 in cloaca and hindgut in normal and anorectal malformation （ARM） rat embryos and examine potential association between Dkk1 and abnormal development of cloaca and hindgut in ARM.Methods Ethylenethiourea （ETU） was administered to 24 pregnant rats at gestational Day 10 via gastric gavage. And another 16 control pregnant rats received saline alone. The gestational ages of collected specimens were 12-17, 19 and 21 days respectively. Based upon anorectal morphology, the embryos were divided into two groups. Group ARM： ARM fetuses born from pregnant rats received ETU （n=96） and control group （n=96）. Normal fetuses were from pregnant rats receiving saline. Cloaca and hindgut tissues were dissected microscopically for extracting total RNA and protein. Real-time polymerase chain reaction （RT-PCR） and Western blot were performed to detect the expressions of Dkk1 mRNA and protein respectively. The experiment was analyzed by two-way ANOVA of randomized block design.Results Dkk1 mRNA and protein were detected in both groups. The expression of Dkk1 peaked at Day 16 in cloaca and hindgut of control group （2.77±0.06, 3.20±0.76） while the expression of Dkk1 fluctuated in ARM group. The expression levels of Dkk1 mRNA and protein were remarkably different during cloacal and hindgut development between normal （2.00±0.52, 1.98±0.65） and ARM embryos （0.96±0.14, 0.87±0.14, P〈0.05）.Conclusions A down-regulation of Dkk1 in cloaca and hindgut development may be partially related to maldevelopment of cloaca and hindgut in ARM.