在毛乳头细胞中MAD2B对TCF4的调控机制及其功能研究

Hair dermal papilla cells located at the bottom of hair follicles play pivotal roles in hair formation, growth and cycling. The biological dysfunction of hair dermal papilla cells is the main factor that cause disequilibrium of hair follicular cycles and alopecia. Recently we have firstly found that gene TCF4 gene differentially expressed in hair dermal papilla cells between anagen and telogen by the technique SSH. We have also proved that gene TCF4 may a pivotal role on the control of the biological function of hair dermal papilla cells　by by gene transfection and RNAs interference respectively.MAD2B was identified in a yeast two-hybrid screen for TCF4-interacting partners.The aim of this study is to to elucidate the biological functions of investigate the transduction of Wnt signaling pathway,especially the function of TCF4 interacting protein MAD2B in hair dermal papilla cells. Firstly, the role of the up-expression and silent expression of gene MAD2B and TCF4 will be investigated by gene transfection and RNAs interference respectively. Secondly, through rebuilding the hair follicle in vivo with nude mouse, the effects of the expressing of gene TCF4 on ability of inducing follicle formation will be observed.Finally, In this study we will determine whether MAD2B regulates TCF4-mediated biological processes.

毛乳头细胞在毛囊发育和周期性再生调控中起主导作用，其生物学功能异常变化是导致毛囊周期失衡和脱发性疾病的主要始动因素。申请者首次发现和证实TCF4基因在人毛囊毛乳头细胞生物学功能调控中起重要作用，并通过酵母双杂交及免疫共沉淀等技术首次证实在毛乳头细胞中MAD2B为TCF4相互作用蛋白。本课题拟通过基因转染和RNA干扰技术，获得可稳定高表达或下调表达MAD2B及TCF4的毛乳头细胞，研究MAD2B对TCF4的调控机制，观察MAD2B与TCF4相互作用对毛乳头细胞一般生物学功能的影响，利用裸鼠体内毛囊重建模型，明确MAD2B与TCF4相互作用对毛乳头细胞诱导毛囊形成能力的影响。这将有助于阐明TCF4基因在毛乳头细胞生物学功能调控中的具体分子机制，为深入研究MAD2B在毛囊发育和毛囊周期性再生调控中的作用奠定基础，也可为毛囊生物学和毛发疾病的研究提供新的思路。

本科室早期课题采用抑制性消减杂交方法首次发现Wnt信号通路关键信号分子Ｔ细胞因子4（T cell factor4，TCF4）基因在人毛囊毛乳头细胞中呈差异表达，在生长期毛乳头细胞中上调表达，通过基因转染及RNA干扰技术研究表明其对在毛乳头细胞生物学功能调控中起重要作用。前期课题通过构建凝集生长毛乳头细胞酵母双杂交文库，以TCF4 N 端和DNA 结合域为诱饵蛋白，筛选凝集生长毛乳头细胞中与TCF4相互作用的蛋白MAD2B。在此基础上，本课题成功构建pAdTrack-CMV-TCF7和pAdeno-MCMV-MAD2B腺病毒载体，通过正反向免疫共沉淀及细胞显微荧光图，进一步证实在凝集生长的毛乳头细胞中存在MAD2B和TCF4的相互作用。并首次发现MAD2B过表达于毛乳头细胞之后，其凝集性生长的特性明显减弱，通过荧光素酶活性分析、DPC增殖、细胞因子分泌变化，探讨了MAD2B过表达后对Wnt通路的影响。本课题深入探讨MAD2B基因过表达对TCF4的调控作用，以及对毛乳头细胞生物学功能的影响，这将有助于进一步阐明TCF4基因在毛乳头细胞生物学功能调控中的具体分子机制，为深入研究该基因在毛囊发育和毛囊周期性再生调控中的作用奠定基础，也可为毛囊生物学和毛发疾病的研究提供新的思路。