14-3-3β/Sox2交互作用在先天性食道闭锁的发生、发展中的作用

On the basis of predecessors’ and our works’ summary, we hypothesized that 14-3-3 beta was markedly up-regulated to arrest Sox2 from cytoplasmic against nuclear, blocking the downstream gene p63 expression in the abnormal development of the dorsal foregut epithelial cells. And then Nkx2.1 was disinhibited and disturbed normal cell differentiation and development by Sox2 inactivating. Finally, congenital esophageal atresia (EA /TEF) was occurred. First of all, we detected the expression, the location and the relationship between 14-3-3 beta and Sox2, and the expression of downstream gene p63 in the clinical specimens of EA /TEF. Next chose the primary normal EA/TEF cell and NES-B3T cells for cell culture and then confirmed the 14-3-3 beta /Sox2 interaction with Co-IP, GST-pull down and yeast two-hybrid assay. And then we studied the role of 14-3-3 beta /Sox2 positioning, the expression and function of downstream gene p63 by gene transfection and RNAi method. At then, we constructed EA/TEF rat model with adriamycin and changed the expression of 14-3-3 to observe the expression of Sox2, Nkx2.1 and p63 in the dorsal foregut cells and the tissue pathological changes. Finally we clarified the molecular regulation mechanism of 14-3-3 beta /Sox2 in congenital EA/TEF.

在总结前人与自身工作基础上，我们假设：异常发育的前肠背侧上皮细胞14-3-3β极度上调，Sox2滞留胞质阻止入核，阻断下游基因p63等表达；Sox2失活，Nkx2.1抑制解除，细胞异常分化和发育，导致先天性食道闭锁（EA/TEF）的发生发展。首先拟检测EA/TEF临床标本中14-3-3β/Sox2表达、定位、相互关系及下游p63等表达。选用EA/TEF/正常对照原代培养细胞及NES-B3T细胞，用Co-IP、GST-pull down、酵母双杂交法证实14-3-3β/Sox2相互作用；用基因转染、RNAi等调控相应基因表达，探讨14-3-3β/Sox2定位与作用，对下游p63等表达与功能的影响；构建阿霉素EA/TEF大鼠模型并改变14-3-3β表达，观察前肠背侧细胞内Sox2、Nkx2.1、p63等表达，及细胞组织学改变，最终阐明14-3-3β/Sox2对先天性EA/TEF中分子调节机制。

紧扣“14-3-3β”之关键，以“异常的食管上皮细胞内14-3-3β通过调节Sox2表达水平变化，进而调控食管发育基因p63、Keratin5、Keratin14及气管发育基因Nkx2.1、β-catenin、β-tubulin等表达水平，参与前肠上皮细胞的分化、发育过程，诱导EA/TEF疾病的发生、发展”为主线。本课题结果初步证实Sox2是14-3-3β的下游靶蛋白之一，14-3-3β下调Sox2表达水平，引起p63、Keratin5、Keratin14等食管发育基因的表达水平下降，气管发育基因Nkx2.1、β-catenin、β-tubulin表达水平上升，进而促进食管上皮细胞向气管上皮细胞转化，最终引起食管闭锁等病理进程。.本课题完成研究计划，已投稿学术论文1篇（标注资助），正在撰写2篇，并培养硕士生2名。