斑马鱼心脏发育强表达基因CXXC5调控心脏腔室发育的分子机制

Studies have shown that the expression of the human CXXC5 gene was related to the regulation of heart development. But the function of CXXC5 in heart development is not clear. The results of applicant preliminary study showed that the CXXC5 was expressed in the early developmental stages of zebrafish heart and regulated the developmental cardiac chamber size through negative regulation of cardiac precursor cell proliferation. And the TGF-β signaling pathway may be related to the regulation of the CXXC5 in the heart development. The applicant intends to study the molecular mechanism of CXXC5 gene regulation in cardiomyocyte proliferation or apoptosis and explore CXXC5 gene function in the TGF-β signaling pathway that regulate the cardiac chamber size, by establishing CXXC5 gene knockout and transgenic experimental model. On the basis of the the preliminary identification of the interaction between CXXC5 and Smad2 or Smad3, TGF-β regulatory proteins, the applicant intends to explore molecular mechanism of synergistic efficacy of the interaction between CXXC5 and Smad2 or Smad3 in the TGF-β signal regulation of the heart chamber size and myocardial cell proliferation or apoptosis. The research is of great significance for CXXC5 function of the human heart development.

人类CXXC5基因在心脏发育早期表达并调控心脏发育，但其具体机制未知。申请者前期研究发现：斑马鱼CXXC5在心脏发育早期强表达，该基因通过负调控心脏前体细胞的数目控制心脏腔室大小的发育，其对心脏发育的调控可能与TGF-β信号通路有关。为了进一步研究CXXC5调控心脏腔室大小的分子机制，申请者拟通过CXXC5基因敲除和转基因斑马鱼模型，研究CXXC5基因调控心肌细胞增殖或调亡的机理，探讨CXXC5基因是否通过TGF-β信号通路调控心脏腔室大小的发育过程。基于申请人鉴定的CXXC5与TGF-β信号激活蛋白Smad2和Smad3有相互作用的基础上，研究CXXC5与Smad2或Smad3通过TGF-β与细胞凋亡信号互作协同调控心脏腔室的大小及调控心肌细胞增殖的分子机理，该研究对揭示CXXC5调控人类心脏发育与疾病的机制具有重要意义。

CXXC5基因可能在心脏发育和心脏病过程中很可能发挥重要作用，但具体机制不明。本研究通过COIP和GST-pulldown实验证明了斑马鱼CXXC5与Smad通过ZF-CXXC结构和MH2结构实现相互作用，利用大鼠心肌细胞模型证实了CXXC5对TGF-β信号通路的激活作用。利用斑马鱼模型证明CXXC5在胚胎早期发育的时空定位与心脏细胞的发育定位一致。利用斑马鱼模型通过Morpholinos和mRNA拯救在体分析最直接最有效的方法鉴定CXXC5与心脏发育的关系，并探讨了其作用机理和调控的信号途径。敲低CXXC5的斑马鱼心脏表现为环化障碍、发育不良、心包水肿与心率不齐等类似于TGF-β信号紊乱的表型，同时伴随着TGF-β下游调控心脏环化相关基因nkx2.5、hand2、has2的显著下调，共同注射hand2 mRNA能部分拯救其环化缺陷。本研究利用斑马鱼模型首次提供了体内的证据证实CXXC5作为在心脏早期发育的调控因子，通过TGF-β信号调控心脏的环化过程。此外，本文通过检测临床法洛氏四联症（Tetralogy of Fallot，TOF）病人中CXXC5基因的表达，发现CXXC5的表达在TOF病人中显著下调，从而提示CXXC5为可为此类先天性心脏疾病（先心病）的早期诊断和治疗提供新的线索和可能的分子靶标。