Cdx2-Hox信号通路与先天性肛门直肠畸形发生关系的研究

Anorectal malformations (ARMs) are among the most common congenital anomalies and adversely influence patient quality of life. As ARM is a multigenerational complex disease, its etiology, embryology, and pathogenesis are poorly understood and controversial. Our preliminary studies on the spatiotemporal expression pattern of Cdx2 in rat embryo have suggested that downregulation of Cdx2 at the time of cloaca separating into the rectum and urethra is related to anorectal development and ARM. But the mechanism how Cdx2 influence anorectal development and occurrence of ARM is not yet clear. Based on this study we will further study the influence of Cdx2-Hox signal pathway on anorectal development. In this study, we will analyze the spatiotemporal distribution of Cdx2 and Hox genes in normal and ethylenethiourea (ETU)-induced ARM rat embryos by immunohistochemistry, Real-time PCR and Western blot methods with emphasis on embryonic stages GD13 to GD16, which are the critical time-points in anorectal development. By using RNA interference technology, inject interference plasmid via uterus of pregnant Wistar rats at the critical time-points in anorectal development to downregulate the expression of Cdx2 gene in offspring fetal rats. Anorectal development, ARM type and deformity rate, Hox gene expression were detected in Cdx2 silencing offspring fetal rats. Then, restore Hox genes function of Cdx2 gene silencing fetal rats at different time points and observe anorectal development. The aim of this study is to clear the relationship of Cdx2-Hox signal pathway on anorectal development and ARM and establish a foundation for genetic intervention, prevention and treatment of ARM.

先天性肛门直肠畸形（ARM）是小儿外科常见的消化道畸形，严重影响儿童生活质量。由于ARM是复杂的多基因疾病，迄今其发病机制仍不清楚。本课题组前期研究发现Cdx2基因参与肛门直肠发育和ARM发生，但其发生机制尚未明确。此基础上本研究将进一步研究Cdx2-Hox信号通路对肛门直肠发育的影响。本研究通过免疫组化、RT-PCR和Western-blot方法研究肛门直肠胚胎发育的关键时期Cdx2、Hox基因在ARM胎鼠和正常胎鼠后肠的时空表达及差异；采用RNAi技术，在后肠发育关键时期经Wistar孕鼠宫内注射干扰质粒，下调子代胎鼠Cdx2基因的表达，检测Cdx2基因沉默胎鼠肛门直肠发育情况、ARM类型和畸形率、Hox基因的表达；恢复Cdx2基因沉默胎鼠中Hox基因功能，观察肛门直肠的发育情况，目的在于明确Cdx2-Hox信号通路与肛门直肠发育和ARM发生的关系，为ARM遗传干预和畸形防治奠定基础。

应用乙烯硫脲致畸妊娠期Wistar大鼠，制作肛门直肠畸形（ARM）动物模型，应用免疫组化，Real time RT-PCR和Western blot技术检测Pcsk5在妊娠第13-16天大鼠胚胎泄殖腔/后肠的表达情况。免疫组化结果提示胚胎13天对照组中Pcsk5广泛表达在泄殖腔上皮，胚胎14和15天时，阳性细胞分布在尿直肠隔和肛膜。在胚胎13天至15天，ARM组中泄殖腔上皮有少量Pcsk5表达。Western blotting和real time RT-PCR结果提示在后肠发育过程中Pcsk5表达逐渐增强，Pcsk5在ARM组的表达水平低于对照组。提示Pcsk5在泄殖腔发育过程中表达下调可能和ARM的发生有关。.应用乙烯硫脲致畸妊娠期Wistar大鼠，制作肛门直肠畸形（ARM）动物模型，应用免疫组化，Real time RT-PCR和Western blot技术检测Wif1/β-catenin在妊娠第13-16天大鼠胚胎泄殖腔/后肠的表达情况。免疫组化结果提示在胚胎13至16天Wif1持续表达在对照组泄殖腔上皮。在胚胎13和14天时，Wif1广泛表达在泄殖腔上皮；在胚胎15天时，Wif1主要表达在肛膜。在ARM组，胚胎14至16天，Wif1少量表达在后肠和尿直肠隔上皮。Western blot和real time RT-PCR结果提示，在胚胎14和 15天时，Wif1和β-catenin 蛋白和mRNA在ARM组的表达水平低于对照组。在泄殖腔发育过程中，Wif1和β-catenin在 ARM胎鼠中的表达失调，Wif1和β-catenin在泄殖腔分离成原始直肠和尿生殖窦时表达下调可能和ARM的发生有关。.根据Cdx2基因序列，使用siRNA设计软件并避开突变多发区域，设计3个不同的siRNA及阴性对照siRNA，构建Cdx2基因表达下调的RNA干扰片段，优选出最佳RNA干扰片段，使用优选后的RNA干扰片段转染大鼠肠上皮细胞（IEC6），观察IEC6的增殖、凋亡、迁移和靶基因的表达改变等。结果提示，Cdx2具有促进增殖、抑制凋亡和促进迁移的作用，Cdx2可能通过调控细胞的增殖、凋亡和迁移影响肛门直肠的发育，甚至导致ARM的发生。Cdx2基因沉默后，Hoxa13 mRNA和蛋白的表达明显下调，提示Cdx2可能通过Hoxa13影响肛门直肠的发育，甚至导致ARM的发生。