FGF10-FGFr2Ⅲb信号通路与先天性肛门直肠畸形发生关系的研究

Anorectal malformations(ARM) are among the most common congenital anomalies, occurring in approximately 1/1,500 to 1/1,000 live births, which influenced the quality of life of the patients severely. However, as ARM is a multigenerational complex disease, the etiology, embryology, and pathogenesis of ARM were poorly understood and controversial; it is impetuous to research into the developmental bases of normal and abnormal anorectal organogenesis. Fibroblast growth factor 10 (FGF10) and its main ligand fibroblast growth factor receptor 2Ⅲb (FGFr2Ⅲb) constitute FGF10-FGFr2Ⅲb signal pathway, which associates with instructive mesenchymal/epithelial interactions. FGF10-FGFr2Ⅲb signal pathway plays crucial roles in regulating cell proliferation, migration and differentiation during embryonic development. In our previous studies, the expression levels of FGF10 and FGFr2Ⅲb in terminal rectum with high, intermediate and low ARM were significant lower than that in normal controls. In ETU-induced ARM rat embryos, spatiotemporal expression of FGFr2Ⅲb was imbalanced during the anorectal morphogenesis. These suggest FGF10-FGFr2Ⅲb signal pathway may be related to the development of the hindgut and ARM. However, the exact roles of FGF10-FGFr2Ⅲb signal pathway in the development of the hindgut and the embryogenesis of ARM have yet to be elucidated. .To better understand the roles of FGF10-FGFr2Ⅲb signal pathway, we decide to perform systematic studies. Firstly, The expressions of FGF10 and FGFr2Ⅲb in terminal rectum are studied by immunohistochemical method、RT-PCR and western-blot in the patients of high、intermediate、low ARM and controls with no ARM. Secondly, we analyze the expression of FGF10、FGFr2Ⅲb in normal and ETU-induced ARM rat embryos with special emphasis on embryonic stages gestational day12 to 16, which are the critical time-points in the anorectal development. Thirdly, we construct the embryos using RNA interference(RNAi)to downregulate FGF10 、FGFr2Ⅲb genes. Genespecific shRNAs will be designed to target FGF10 、FGFr2Ⅲb genes. Then based on temporospatial expression of FGF10 、FGFr2Ⅲb, shRNA expression plasmids diluted in Ringer's solution are injected into the tail vein of pregnant rats and downregulate the expression of FGF10、FGFr2Ⅲb. Lastly, the percent of ARM are analysed in RNAi embryos. The proliferation and apoptosis, the development of vessel、muscle and nerve are stuied in the hindgut of normal and RNAi embryos. Meanwhile, we evaluate the development of striated muscle complex and the expression of other relevant genes in the hindgut of normal and RNAi embryos.We will therefore identify the functions of FGF10-FGFr2Ⅲb signal pathway and its impacting on the development of the hindgut and ARM. Our results about the roles of FGF10-FGFr2Ⅲb signal pathway in fetal development may facilitate better understanding of the pathogenesis and heredity intervence of ARM.

先天性肛门直肠畸形（anorectal malformations,ARM）是常见的消化道畸形，严重影响儿童的生活质量。ARM是多基因遗传病，目前其发病机制和预防措施仍不清楚。本课题前期研究发现FGF10-FGFr2Ⅲb信号通路参与后肠发育和ARM的发生，但是具体机制尚未明确。在此基础上本研究利用ARM患者和ETU致畸ARM胎鼠，研究FGF10、FGFr2Ⅲb基因在后肠的表达。采用跨胎盘RNAi技术，下调FGF10、FGFr2Ⅲb基因的表达，研究基因下调的程度与ARM类型、畸形率的关系。研究基因下调胚胎后肠细胞增殖-凋亡情况，后肠血管、肌肉、神经的发育，后肠周围横纹肌复合体的发育，是否影响其它相关基因的表达，目的在于明确FGF10-FGFr2Ⅲb信号通路对胚胎后肠发育的影响，及其与ARM发生的联系，探讨其在ARM发生中的作用，为ARM遗传干预、产前诊断和畸形防治奠定基础。

先天性肛门直肠畸形（ARM）是严重影响儿童生活质量及心理健康的常见消化道畸形，目前其发病机制不清。本课题组前期研究发现FGF10-FGFr2信号通路可能参与胚胎期后肠的发育和ARM的发生。课题组在前期研究的基础上拟进一步研究ARM患者直肠末端FGF10、FGFr2基因的表达情况；利用ETU致畸ARM动物模型，研究肛门直肠胚胎发育的关键时期FGF10、FGFr2基因在ARM后肠与正常胎鼠后肠的时空表达情况；利用RNAi技术下调FGF10、FGFr2基因的表达,建立ARM动物模型，研究FGF10、FGFr2基因对胚胎后肠发育的影响及其与ARM发生的联系。研究结果显示ARM患者直肠末端FGF10、FGFr2基因表达减低；胚胎期ARM胎鼠后肠、泄殖腔膜FGF10、FGFr2基因的表达较正常胎鼠明显减弱；利用RNAi技术干扰后肠FGF10、FGFr2基因的表达，目前未成功建立ARM动物模型。同时在实验中课题组发现胚胎期泄殖腔Bcl-2、Bax基因和后肠FGF9、FGFr3基因的表达与FGF10、FGFr2基因的表达密切相关，研究显示ARM胎鼠泄殖腔Bcl-2、Bax基因和后肠FGF9、FGFr3基因表达出现异常，影响胚胎期泄殖腔和后肠的发育。这些实验结果提示ARM 是多基因遗传病，FGF10-FGFr2信号通路参与肛门直肠的胚胎发育过程，是肛门直肠胚胎发育过程中的重要调控基因，FGF10-FGFr2信号通路的表达异常与ARM的发生密切相关，为ARM的发病机理和防治研究提供了理论基础。