Zhang Tao, Bai Yu Zuo, Zhang Dan, Zhang Shi Wei, Wang Da Jia, Jia Hui Min, Yuan Zheng Wei, Wang Wei Lin
Department of Pediatric Surgery, Shengjing Hospital, China Medical University, Shenyang 110004, PR China.
J Pediatr Surg. 2009 Aug;44(8):1568-74. doi: 10.1016/j.jpedsurg.2008.10.002.
The aim of this study was to determine caudal-type homeobox gene-1 (Cdx1) expressions during anorectal development in normal and anorectal malformation (ARMs) embryos and investigate the possible role of Cdx1 in the pathogenesis of ARM.
Anorectal malformation was induced by ethylenethiourea on the 10th gestational day (GD10) in rat embryos. Cesarean deliveries were performed to harvest embryos from GD13 to GD21. The temporal and spatial expression of Cdx1 was evaluated in normal rat embryos (n = 334) and ARM embryos (n = 328) from GD13 to GD20 using immunohistochemistry staining, reverse transcriptase polymerase chain reaction (RT-PCR), and Western blot analysis.
Immunostaining revealed that in normal embryos, on GD13.5, Cdx1 expression was mainly located on the epithelium of the dorsal urorectal septum (URS), cloacal membrane, and the hindgut. On GD15, increased positive tissue staining was noted on the fused tissue of URS, especially in the very thin anal membrane. In the ARM embryos, however, the epithelium of the cloaca, URS, and anorectum was negative or faint for Cdx1. In Western blot and RT-PCR, in the normal group, Cdx1 protein and Cdx1 messenger RNA expression showed time-dependent changes in the developing hindgut, on GD14, GD14.5, and GD15. The expression level reached a peak when the anus was forming. Once the anus was open, Cdx1 expression gradually decreased. In addition, the expression level of Cdx1 in the ARM group from GD13 to GD16 was significant lower than that of the normal group (P < .05).
In ARM embryos, an imbalance of spatiotemporal expression of Cdx1 was noted during anorectal morphogenesis from GD13 to GD16. This suggests that downregulation of Cdx1 at the time of cloacal separation into rectum and urethra might be related to the development of ARM.
本研究旨在确定正常胚胎和肛门直肠畸形（ARM）胚胎在肛门直肠发育过程中尾型同源盒基因-1（Cdx1）的表达情况，并探讨Cdx1在ARM发病机制中的可能作用。
在大鼠胚胎妊娠第10天（GD10）用乙硫脲诱导肛门直肠畸形。剖宫产获取妊娠第13天（GD13）至第21天（GD21）的胚胎。采用免疫组织化学染色、逆转录聚合酶链反应（RT-PCR）和蛋白质印迹分析，评估从GD13至GD20的正常大鼠胚胎（n = 334）和ARM胚胎（n = 328）中Cdx1的时空表达。
免疫染色显示，在正常胚胎中，妊娠第13.5天，Cdx1表达主要位于尿直肠隔（URS）、泄殖腔膜和后肠的上皮。妊娠第15天，URS融合组织上阳性组织染色增加，尤其是在非常薄的肛膜处。然而，在ARM胚胎中，泄殖腔、URS和肛门直肠的上皮Cdx1呈阴性或弱阳性。在蛋白质印迹和RT-PCR中，正常组中，Cdx1蛋白和Cdx1信使核糖核酸表达在发育中的后肠于妊娠第14天、第14.5天和第15天呈现时间依赖性变化。当肛门形成时表达水平达到峰值。一旦肛门开放，Cdx1表达逐渐下降。此外，妊娠第13天至第16天ARM组中Cdx1的表达水平显著低于正常组（P < .05）。
在ARM胚胎中，从妊娠第13天至第16天肛门直肠形态发生过程中，Cdx1的时空表达存在失衡。这表明在泄殖腔分离为直肠和尿道时Cdx1的下调可能与ARM的发生有关。
