COL11A1基因突变引起先天缺牙的分子机制及运用CRISPR/Cas9技术进行基因修复的实验研究

Tooth agenesis brings serious functional, aesthetic and mental problems to the patients. COL11A1 encodes α1 chain of type XI collagen and plays an important role in the development of bone tissue. The expression of COL11A1 was reported to be correlated with Wnt pathway, which is a classical pathway regulating the development of teeth. We performed whole exome sequencing, bioinformatics analyses and genotype-phenotype coseparation analysis in patients with tooth agenesis. We found that COL11A1 mutations were correlated with tooth agenesis, but the mechanism remains elusive. In this study, we will screen for COL11A1 mutations in more patients with tooth agenesis. Dentinogenic/osteogenic ability of wild type and mutant stem cells from human exfoliated deciduous teeth (SHED) will be analysed. RNA sequencing and bioinformatics analyses will be used to search for differentially expressed genes, followed by GO and KEGG analyses. Key pathways involving in tooth development, such as Wnt/β-catenin pathway, will be analysed in detail to illuminate the mechanism of COL11A1 regulating tooth development. CRISPR/Cas9 gene editing system will be used to construct COL11A1 mutation knock-in mice. We will analyse the phenotype of the COL11A1 mutation knock-in mice and verify the mechanism by which COL11A1 regulates tooth development. We will try to correct the COL11A1 mutation of SHED from the patients, and restore its dentinogenic/osteogenic ability by using CRISPR/Cas9 system. This study will contribute to uncovering the mechanism of COL11A1 in tooth agenesis and provide new strategy for prevention and treatment of tooth agenesis.

先天缺牙严重影响患者口腔功能与身心健康。COL11A1基因编码XI型胶原蛋白α1链，对钙化组织形成与发育有重要影响,同时有研究报道该基因与经典Wnt通路高度关联。本课题组前期对先天缺牙家系进行全外显子测序和家系共分离分析，发现COL11A1基因的突变与先天缺牙密切相关，但具体机制不明。本研究拟进一步在先天缺牙病例中筛查COL11A1突变，分析COL11A1野生型和突变型乳牙干细胞的成牙/成骨能力差异，用RNA测序及生物信息学分析筛选差异表达基因，进行GO和KEGG通路富集，重点分析COL11A1突变对Wnt/β-catenin通路影响，阐明COL11A1影响牙发育的分子机制；应用CRISPR/Cas9技术构建COL11A1的突变敲入鼠模型，进一步明确突变与先天缺牙的关系；CRISPR/Cas9技术纠正突变型乳牙干细胞COL11A1基因，恢复其成牙/成骨能力，为先天缺牙防治提供新策略。

先天缺牙给患者口腔功能与身心健康造成严重的影响。COL11A1基因编码XI型胶原蛋白α1链，对钙化组织形成与发育有着重要影响,同时有研究表明该基因能调控经典Wnt通路。本课题组前期对先天缺牙家系进行全外显子测序和家系共分离分析，发现COL11A1基因的突变与先天缺牙密切相关，但具体机制不明。本课题通过大样本量里对包括COL11A1在内的候选致病基因进行致病突变的筛查，发现了EDA、WNT10A等基因的新突变，发现了KDF1基因突变引起的先天缺牙病例，因KDF1基因具有更高的确定性，因而后续采用KDF1基因替代COL11A1基因完成了细胞水平的实验、突变敲入鼠的构建和实验验证，发现KDF1在牙发育调控中的重要作用。本课题在探索KDF1基因在先天缺牙中的作用的同时，也完成了乳牙干细胞/骨髓间充质干细胞相关的成牙成骨相关实验，发现氧化石墨烯量子点通过Wnt/β-catenin通路促进乳牙间充质干细胞增殖和成骨分化。并且发现磁性氧化石墨烯联合静态磁场通过Wnt/β-catenin通路诱导BMSCs成骨分化。本课题基本阐明了KDF1基因是引起先天缺牙的重要因素，利用氧化石墨烯纳米衍生物等生物材料有望促进间充质干细胞成牙成骨分化，为先天缺牙防治提供新思路。