子宫腺肌病内膜间质细胞14-3-3ζ配体蛋白的差异蛋白质组学研究

Previous studies have suggested that 14-3-3 protein members participate in almost all of the phosphorylation process and some other biological processes in cells via binding to their respective ligand proteins. Our previous results have found and confirmed that 14-3-3ζ was the key protein that regulated the biological effects of stromal cells in adenomyosis. In this project, we will apply the combined techniques of TAP/2-D DIGE/MALDI-TOF-MS/2-D phospho-protein specific immuneblotting, to explore the differentially expressed proteomics of ligand proteins in eutopic and ectopic endometrial stromal cells under relevant biological effects, then construct the MAP profiles of ligand proteins and phosphorylation-associated ligand proteins of 14-3-3ζ using bioinformatic software, choose a representative ligand protein of 14-3-3ζ X with differential high expression, confirm its interreaction with 14-3-3ζ in vitro and in vivo; construct the corresponding transgenic cell strains with RNAi recombinant adenovirus vectors, to explore the effects of downstream ligand proteins and associated signaling pathways affected by the over-expression of 14-3-3ζ via relevant cellular and nude mice experiments, to further provide theoretical and experimental bases for further clarifying the molecularly biological mechanisms underlying the initiation and progression of adenomyosis.

本项目根据前人14-3-3必须通过与配体蛋白结合后，参与细胞内几乎所有磷酸化及其他生命过程结论；在前期工作中发现并证实14-3-3ζ为决定子宫腺肌病间质细胞特征性行为之蛋白的基础上，拟将TAP分离/2-D DIGE电泳/MALDI-TOF-MS分析/2-D磷酸化免疫印迹等技术有机结合，探讨14-3-3ζ对其在位与异位子宫内膜间质细胞产生相关生物学效应时配体蛋白组学的差异性，利用生物信息学专用软件绘制14-3-3ζ配体蛋白组以及参与磷酸化的14-3-3ζ配体蛋白组的MAP图；选择一个典型的差异性高表达14-3-3ζ配体蛋白X，通过体外、细胞学实验确认其与14-3-3ζ的相互作用；并采用RNAi腺病毒载体技术，建立相应的转基因细胞株，通过细胞学及裸鼠在体实验，探讨14-3-3ζ过表达对其下游配体蛋白及其相关信号转导通路的影响，为进一步阐明子宫腺肌病发生、发展的分子生物学机制奠定理论与实验基础。

14-3-3ζ蛋白在多种人类疾病发生发展中起着重要作用。本研究首先通过IHC方法，发现子宫腺肌病异位子宫内膜较在位内膜及对照内膜组织中的14-3-3ζ表达明显升高；随后分离病理确诊的子宫腺肌病异位内膜、在位内膜及对照个体在位内膜的原代子宫内膜间质细胞；半定量RT-PCR、WB证实了异位子宫内膜间质细胞中14-3-3ζ表达更高。利用siRNA敲低14-3-3ζ表达后，发现子宫内膜间质细胞的细胞增殖、迁移和侵袭能力减弱，细胞凋亡水平下降，这些结果表明子宫内膜间质细胞中14-3-3ζ的高表达能够促进子宫腺肌病的发生。利用串联亲和纯化结合蛋白飞行质谱鉴定出14-3-3ζ高表达异位内膜间质细胞中的81个14-3-3ζ相互作用蛋白，Co-IP实验证实14-3-3ζ能与其中的MAP3K2蛋白相互作用，提示MAP3K2可能参与14-3-3ζ高表达所致的子宫腺肌病的发病过程。这些结果有望为子宫腺肌病的分子靶向药物研究设计提供新思路。