Objective To determine P63 expressions during anorectal development in normal and anorectal malformation (ARM) embryos, and to investigate its role in the pathogenesis of ARM. Methods Pregnant Wistar rats (n =20) were fed with ethylenethiourea (ETU) to establish the anorectal malformation model. Rats were sacrificed to obtain fetal at E13,E14,E15 and E16 (Ex referring to the X day of pregnancy). Continuous sections were made in median sagittal plane,and were stained by P63 immunohistochemical method. Dynamic observation was made to compare the spatial distribution of cloacal chamber and the development of anorectum between rats with ARM and normal rats. P63 protein and mRNA expressions were studied in the hindgut and cloaca of normal and ARM embryos at different gestational days by western blot and quantitative real-time PCR(qRT-PCR). Results By immunohistochemical staining,P63 expression was confined to the ep-ithelia of the urorectal septum (URS)and the hindgut,cloaca membrane and urogenital sinus in the control group. However,in the ARM embryos, the epithelia of the cloaca,URS and anorectum was negative or weak positive for P63 from E13 to E16. By western blot and qRT PCR.P63 protein and P63 mRNA expression showed time-dependent changes in the developing hindgut,the expression level reached the peak when the anus was forming (at 14 d and 15 d). Once the anal was formed,P63 expression gradually decreased. The expression level of P63 in the ARM group was significantly lower than that of the control group (P < 0.05) at 14 d to 15 d. Conclusion In ARM embryos, spatiotemporal expression of P63 was imbalanced during the anorectal morphogenesis;during the critical period of anus forming (from 13 d to 16 d),the down regulation of P63 may be related to the development of ARM.%目的 检测P63在正常和畸形胎鼠肛门直肠胚胎发育过程的表达,探讨其表达的意义.方法 应用乙烯硫脲(ETU)致畸Wistar孕鼠(n=20),建立肛门直肠畸形动物模型,分别于E13、E14、E15、E16(Ex表示大鼠妊娠第X天)剖宫取胎.正中矢状面连续切片,P63免疫组化染色,连续动态观察P63在正常和畸形大鼠胚胎泄殖腔及肛门直肠发育过程中的空间表达情况.通过Western blot和荧光实时定量PCR(qRT-PCR)方法分别研究P63 mRNA和蛋白在正常和畸形大鼠胚胎泄殖腔及肛门直肠的表达.结果 免疫组化结果:正常大鼠胚胎中,P63表达于尿直肠隔以及后肠、泄殖腔膜和尿生殖窦的上皮层.肛门直肠畸形组大鼠胚胎中,在E13～E16,P63在尿直肠隔间质、后肠上皮层、泄殖腔膜或瘘管上皮层的表达减弱.Western blot和qRT-PCR定量检测结果:在发育期的后肠中P63蛋白和P63 mRNA的表达表现出时间依赖性,在肛门形成的关键时期,即14d和15d时P63的表达水平达到高峰,一旦肛门形成其表达量随即开始降低,畸形组与正常组比较,P63 mRNA和蛋白的表达在14～15 d明显降低,差异有统计学意义(P＜0.05).结论 在正常大鼠泄殖腔和直肠胚胎发育过程中,P63存在着时空表达不平衡性的特点；在肛门直肠形成的关键时期(13～16 d),先天性肛门直肠畸形大鼠胚胎中P63基因表达下调可能与肛门直肠畸形的发生有关.
展开▼
