唇腭裂新基因Crispld1和Crispld2在小鼠颅颌面发育的作用和机制研究

Clefting Lip/Palate (CLP) is a common birth defect, occurs ~1 in 1000 live birth. This disease causes traumatic influence on patients and huge burden to patient families and the society. Sophisticated genome analyses have identified a number of putative disease-causing genes. Connecting these genes to mechanisms underlying facial malformation is a major task for biomedical researchers. In multiple Genome-wide Association Studies (GWAS), Crispld2 gene and its family member Crispld1 have been shown to be associated with non-syndromic clefting lip/palate (NSCLP). In preliminary study, we have discovered that FGF signaling regulates Crispld2 expression during facial morphogenesis. Here, we propose to generate conditionally knock-out mice models and transgenic mice models of Crispld2 and Crispld1 genes，respectively. Because mice undergo similar morphogenetic processes during growth and fusion of the facial primordia to humans, researches in these mice models will provide insight to understand the disease-causing mechanisms of Crispld gene family. In vitro, we propose to test CRISPLD proteins function at neural crest cell migration and adhesion processes in a microfluidic culture system. Finally, we will examine the interaction of CRISPLD proteins to heparin and FGF ligands, and the influence to FGF signaling transduction. Taken together, this proposal combines in vivo animal model and in vitro cell and biochemical experiments to investigate Crispld functions during facial morphogenesis.

唇腭裂是常见多发的新生儿畸形(1:1000),给患者和社会带来极大的负担。随着全基因组关联研究的开展,多个与唇腭裂发生相关的新基因位点被找到。亟需对相关的新基因位点在颌面发育的作用和唇腭裂中致病机制进行研究分析。在多个唇腭裂全基因组关联研究中,Crispld2及其蛋白家族成员Crispld1发现与唇腭裂发生相关联。同时在前期研究中,我们发现Crispld2受FGF信号通路调控。而Crispld基因的具体功能和在唇腭裂中致病机理尚不明了。本研究拟构建Crispld基因的条件性敲除和转基因过表达小鼠模型,探索它们在颌面发育中的作用和对唇腭裂发生的具体影响。本研究将在体外实验中,检测CRISPLD蛋白对细胞行为的作用。还有探索CRISPLD蛋白在细胞外基质对FGF信号传导通路的作用。本课题将结合动物模型和体外细胞生化实验探索Crispld基因在颅面发育的作用和它们的结构与功能。

在经费计划缩减至一年的情况下，将工作集中在Crispld1，Crispld2在哺乳动物细胞系的表达和功能研究。克隆了人源的Crispld1基因，并检测了其在多个哺乳动物细胞系表达和亚细胞定位。但是Crispld1的表达量很低，操作困难。而Crispld2的表达正常，现将工作转移至Crispld2对细胞生长和细胞迁移的影响上。