14-3-3ε/Tbx1交互作用在圆锥动脉干畸形的发生、发展中的作用

On the basis of predecessors’ and our works’ summary, we hypothesized that 14-3-3 epsilon was markedly up-regulated to arrest Tbx1 from cytoplasmic against nuclear, blocking the downstream gene Nkx2.6 expression in the abnormal development of the conotruncal cells. And then it resulted in anomaly of separation, absorption and rotation of the conotruncal. Finally, conotruncal defects (CTDs) was occurred. First of all, we detected the expression, the location and the relationship between 14-3-3 epsilon and Tbx1, and the expression of downstream gene Nkx2.6 in the clinical specimens of CTDs. Next chose the H9c2(2-1) cells and primary normal CTDs cell for cell culture and then confirmed the 14-3-3 epsilon /Tbx1 interaction with Co-IP, GST-pull down and yeast two-hybrid assay. And then we studied the role of 14-3-3 epsilon /Tbx1 positioning, the expression and function of downstream gene Nkx2.6 by gene transfection and RNAi method. At then, we constructed CTDs rat model with fertilysin and changed the expression of 14-3-3 epsilon to observe the expression of Tbx1, Nkx2.6、HOD in the conotruncal cells and the tissue pathological changes. Finally, we clarified the molecular regulation mechanism of 14-3-3 epsilon /Tbx1 in CTDs.

在总结前人与自身工作基础上，我们假设：异常发育的圆锥动脉干细胞内14-3-3ε表达显著上调，Tbx1滞留在细胞质中阻止入核，阻断下游Nkx2.6等基因表达，导致圆锥动脉干分隔、吸收、旋转异常，最终引起圆锥动脉干畸形（CTDs）疾病的发生、发展。首先拟检测CTDs临床标本中14-3-3ε/Tbx1表达、定位、相互关系及下游Nkx2.6等表达。选用H9c2(2-1)细胞及CTDs正常对照原代培养细胞，用Co-IP、GST-pull down、酵母双杂交法证实14-3-3ε/Tbx1相互作用；用基因转染、RNAi等调控相应基因表达，探讨14-3-3ε/Tbx1定位与作用，对下游Nkx2.6等表达与功能的影响；构建佛替来辛CTDs大鼠模型并改变14-3-3ε表达，观察圆锥动脉干细胞内Tbx1、Nkx2.6、HOD等表达，及细胞组织学改变，最终阐明14-3-3ε/Tbx1在CTDs中的分子调节机制。

本项目将14-3-3ε/Cbx3有机结合，探讨相关信号通路在圆锥动脉干畸形（CTDs）中最常见的疾病法洛四联症（TOF）右心室流出道发育异常中的作用。TOF右心室流出道发育异常主要表现为流出道心肌组织肥厚，我们以TOF为研究对象，首先发现了14-3-3ε/Cbx3在TOF右心室流出道组织中的表达差异。第二，构建了相关腺病毒载体以及Cbx3不同表达转基因心肌细胞，发现Cbx3高表达可显著增强AC16细胞（人心肌细胞系）的增殖能力。第三，应用Co-IP及GST-pull down法证实了Cbx3为14-3-3ε配体蛋白及两者间的交互作用，荧光标记/Hoechst/Confocal法证实了14-3-3ε不同表达能改变AC16细胞内Cbx3蛋白的亚定位。第四，构建Cbx3高表达/14-3-3ε高表达或低表达转基因心肌细胞，发现转染Ad-14-3-3ε RNAi干扰14-3-3ε表达导致细胞增殖水平显著上升、MKI67和PCNA的mRNA水平显著上升、GATA4的mRNA水平显著上升。第六，我们还首次发现并证实川陈皮素及甜橙黄酮中药有效成分可导致AC16细胞14-3-3ε蛋白水平显著上调、细胞增殖水平显著下降，为类似防治药物的研发奠定理论与实验基础。本项目研究结果首次发现了14-3-3ε/Cbx3在TOF右心室流出道发育异常中的作用；首次证实了Cbx3为14-3-3ε配体蛋白，14-3-3ε蛋白是调控Cbx3蛋白亚定位的关键性蛋白；发现了14-3-3ε/Cbx3二者协同作用影响心肌细胞增殖。这有助于人们进一步深入了解TOF右心室流出道发育异常机制。