Tcf4在肛门直肠畸形大鼠胚胎发育中的表达

Expression of Tcf4 in the development of anorectai malformations in rat embryos

目的观察Tcf4在大鼠正常和畸形肛门直肠发育过程中的表达，探讨Tcf4在正常和肛门直肠畸形的胚胎发生机制中的作用。方法用乙烯硫脲（ethylenethiourea，ETU）致畸Wistar孕鼠24只肛门直肠畸形动物模型，切片于显微镜下观察，确定肛门直肠畸形的存在。标本经显微镜下观察后分为正常组和肛门直肠畸形组。正中矢状面、连续切片，Tcf4免疫组化染色。连续动态对比观察Tcf4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分布情况。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标本，从中提取总RNA和总蛋白，通过RT-PCR和Western blot方法分别研究Tcf4 mRNA和Tcf4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①免疫组化结果：Tcf4在大鼠发育过程中正常胚胎泄殖腔中表达，在13d和14d主要表达于尿直肠隔和泄殖腔膜，胚胎15d时在尿生殖膈与泄殖腔膜融合处Tcf4表达较强，16d时肛膜破裂，肛门直肠形成，Tcf4在直肠黏膜层表达。Tel4在肛门直肠畸形胎鼠泄殖腔和直肠黏膜层表达较弱或呈现阴性表达。②Westernblot和RT-PCR半定量检测结果：在发育期的后肠中Tcf4蛋白和Tcf4mRNA的表达表现出时间依赖性的特点，在肛门形成的关键时期即14d、14．5d和15d时Tel4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，畸形组与正常组相比较，Tcf4蛋白和Tcf4 mRNA的表达在14～15d明显降低，有统计学意义（P〈0．05）。结论在肛门直肠畸形大鼠胚胎中，在肛门直肠形成的关键时期（13～16d），Tcf4存在着时空表达不平衡的特点；在正常大鼠胚胎期泄殖腔和直肠发育过程中，Tcf4可能起重要作用。泄殖腔／后肠Tcf4的表达下调可能与肛门直肠畸形的发生有关。

Objective To determine Transcription factor 4 （Tcf4） 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 were fed of Ethylenethiourea （ETU） with the dose of 125 mg/kg on the tenth gestational day （GD10）. From GD13 to GD21, rats were sacrificed to obtain fetal rats whose embryos were divided into normal and ARM group, and presence of ARM was determined under the microscope. Continuous sections were made in median sagittal plane, which were stained by Tcf4 immunohistochemical method. Dynamic observation was made to compare the spatial distribution of cloacal chamber and development of anorectum between rats with ARM and control. The developing hindgut of each embryo was dissected under magnification and made into frozen sections. Tcf4 protein and mRNA expression was studied in the hindgut and cloaca of normal and ARM embryos at different gestational days by western blot and reverse transcriptase polymerase chain reaction （RT-PCR）. Results Immunohistochemical staining revealed that in normal embryos, on GDI3 and GD14, Tcf4 expression was mainly located at the dorsal urorectal septum fURS）, cloacal membrane （CM） and hindgut. On GD15, the increased positive tissue could be noted on the fused tissue of URS, especially in the thin anal membrane. On GD 16, the anal membrane ruptured, thus the anore turn formation. However, in the ARM embryos, the epithelia of the cloaca, URS and anorectum was negative or weak positive for Tcf4. In western blot and RT PCR, Tcf4 protein and Tcf4 mRNA ex pression showed time-dependent changes in the developing hindgut： on GD14, GD14. 5 and GD15, the expression level reached the peak when the anus was forming. Once the anal was opened, Tcf4 expression gradually decreased. The expression level of Tcf4 in the ARM group was significantly lower than that of the control group （P〈0.05）. Conclusions In ARM embryos, spatiotemporal expression of Tcf4 was imbalanced during the anorectal morphogenesis from GD13 to GD16, which suggests that the downregulation of Tcf4 at the time of cloacal separation into rectum and urethra may be related to the development of ARM.