Wang Da-Jia, Bai Yu-Zuo, Zhang Shi-Wei, Gao Hong, Zhang Shu-Cheng, Zhang Dan, Zhang Tao, Yuan Zheng-Wei, Wang Wei-Lin
Department of Pediatric Surgery, Shengjing Hospital, China Medical University, Shenyang 110004, PR China.
J Pediatr Surg. 2009 Mar;44(3):592-9. doi: 10.1016/j.jpedsurg.2008.08.017.
The receptor tyrosine kinase of the Eph family is a large group of highly conserved molecules that function in diverse intercellular recognition events. It has been reported that EphB2 is related to caudal remodeling events. The aim of this study is to investigate EphB2 expression in anorectal development in normal and rat embryos with anorectal malformations (ARMs) and attempt to define its role in anorectal morphogenesis.
The ethylenethiourea (ETU) rat model of the ARMs was used in this study. Immunohistochemical analyses and real time quantitative polymerase chain reaction (PCR) were carried out to investigate EphB2 protein localizations and messenger RNA (mRNA) expression levels. (1) Rat embryos with ARMs were obtained by treating pregnant rats (n = 24) with administration of ETU on gestation day (Gd) 10. Normal rat embryos (n = 111) and embryos treated by ETU without ARMs (n = 90) were the control groups, and embryos with ARMs (n = 108) from Gd13 to Gd16 were divided according to the sections taken from specimens. (2) Embryos were sequentially sectioned in the sagittal and transversal planes before staining with a specific antibody to EphB2. Spatiotemporal study was carried out on EphB2 expression. (3) Individual frozen sections were used to manually microdissect the cloaca and anorectal specimens for total RNA extraction. EphB2 expression was evaluated by real time quantitative PCR.
On the immunologic labeling study, EphB2 expression was confined to the cloaca in control groups, whereas EphB2 expression was mainly located at the urorectal septum (URS) and cloacal membrane on Gd13 and Gd14. The increased positive expression was observed in the fused tissue of the URS and cloacal membrane on Gd15. On Gd16, the anal membrane broke down, and the rectum was able to be in contact with the anus, and EphB2 expression was then noted in mucous membrane of rectum. EphB2 expression was seen in the cloacal and anorectal tissues of embryos with ARMs. By integrated optical density (IOD) measurement, IOD value of EphB2 protein was significantly lower in the ARM group than that in the control groups on Gd13 to Gd16 (P < .05), respectively. As shown by real time quantitative PCR, EphB2 expression was detected in 3 groups. EphB2 mRNA level increased on Gd13 to Gd16 but gradually decreased after Gd16. The expression level of EphB2 mRNA in the ARM embryos was lower on Gd13 to Gd16 than that in control groups (P < .05).
EphB2 expression decreased in the ARM embryos and was confined to URS and cloaca, whereas it was higher in control group. Our data thus indicated that EphB2 molecules possibly contributed to the anorectal morphogenesis and the decreased expression of EphB2 might be related to the development of ARMs.
Eph家族的受体酪氨酸激酶是一大类高度保守的分子，在多种细胞间识别事件中发挥作用。据报道，EphB2与尾部重塑事件有关。本研究旨在探讨正常和患有肛门直肠畸形（ARM）的大鼠胚胎在肛门直肠发育过程中EphB2的表达情况，并试图确定其在肛门直肠形态发生中的作用。
本研究采用ARM的乙烯硫脲（ETU）大鼠模型。进行免疫组织化学分析和实时定量聚合酶链反应（PCR）以研究EphB2蛋白的定位和信使核糖核酸（mRNA）表达水平。（1）通过在妊娠第10天给怀孕大鼠（n = 24）施用ETU获得患有ARM的大鼠胚胎。正常大鼠胚胎（n = 111）和未患ARM的ETU处理胚胎（n = 90）为对照组，将妊娠第13天至第16天患有ARM的胚胎（n = 108）根据取材部位进行分组。（2）在用针对EphB2的特异性抗体染色之前，将胚胎在矢状面和横断面依次切片。对EphB2表达进行时空研究。（3）使用单个冰冻切片手动显微切割泄殖腔和肛门直肠标本以提取总RNA。通过实时定量PCR评估EphB2表达。
在免疫标记研究中，EphB2表达在对照组中局限于泄殖腔，而在妊娠第13天和第14天，EphB2表达主要位于尿直肠隔（URS）和泄殖腔膜。在妊娠第15天，在URS和泄殖腔膜的融合组织中观察到阳性表达增加。在妊娠第16天，肛门膜破裂，直肠能够与肛门接触，然后在直肠黏膜中观察到EphB2表达。在患有ARM的胚胎的泄殖腔和肛门直肠组织中可见EphB2表达。通过积分光密度（IOD）测量，在妊娠第13天至第16天，ARM组中EphB2蛋白的IOD值分别显著低于对照组（P <.05）。如实时定量PCR所示，在3组中均检测到EphB2表达。EphB2 mRNA水平在妊娠第13天至第16天升高，但在妊娠第16天后逐渐下降。在妊娠第13天至第16天，患有ARM的胚胎中EphB2 mRNA的表达水平低于对照组（P <.05）。
EphB2表达在患有ARM的胚胎中降低，并局限于URS和泄殖腔，而在对照组中较高。因此，我们的数据表明EphB2分子可能有助于肛门直肠形态发生，并且EphB2表达的降低可能与ARM的发生有关。
