Rbp4基因敲除协同维生素A缺乏在小鼠唇腭裂发生中的作用与机制研究

Cleft lip and/or palate (CL/P) is the most common congenital malformation in oral and maxillofacial region which is affected by both genetic and environment factors. The applicant first reported that the serum RBP4 and vitamin A content of CL/P were significantly lower than the control. Besides we constructed Rbp4-gene-knockout mice in the previous work, and observed cleft lip and palate on Rbp4-/- mice after the intervention of low vitamin A diet. Therefore, we proposed that combined deficiency of RBP4 and vitamin A could increase the risk of CL/P. This project intends to use the established Rbp4 knockout mice to further clarify the cleft phenotype through the intervention of low-vitamin A diet. The effects on the proliferation and apoptosis of mouse lip and palatal cells were detected in vivo. In addition, the differences of palatal fusion between different groups were observed at the level of in vitro organs. At the molecular and cellular level, the effects of Rbp4 knockout and vitamin A deficiency on cell proliferation, apoptosis and migration as well as the changes of related signaling molecules were detected by primary culture of embryonic palatal mesenchymal cells from different groups. This application aims to clarify the effects of Rbp4 knockout combined with vitamin A deficiency on cleft lip and palate and its cellular and molecular mechanism, so as to make a beneficial exploration for the early prevention of cleft lip and/or palate in the future.

唇腭裂是口腔颌面部最常见的先天发育畸形，其病因涉及遗传、环境及其互作。申请人曾首次报道非综合征型唇腭裂患者血清中RBP4和维生素A含量均显著低于对照组，在前期研究中我们构建了视黄醇结合蛋白4（RBP4）基因敲除小鼠（Rbp4-/-），对其进行低维生素A饮食干预后出现明显的唇腭裂表型，据此我们提出Rbp4敲除协同维生素A缺乏增加唇腭裂发生风险的科学假设。本申请拟利用构建的敲除小鼠联合低维生素A饮食，进一步明确唇腭裂表型及发生比率；并从体内水平检测二者协同对小鼠唇腭部细胞增殖、凋亡等的影响。此外在离体器官层面，观察不同组腭板融合的差异。同时在分子细胞层面，通过原代培养不同组别胚胎腭间充质细胞，检测Rbp4敲除协同维生素A缺乏对细胞增殖、凋亡及迁移的影响及相关信号分子的变化。本申请希望明确Rbp4敲除协同维生素A缺乏对唇腭裂的影响及细胞分子机制，为未来唇腭裂的早期预防做出有益的探索。

项目负责人在既往研究中首次报道了唇腭裂患者血清中RBP4蛋白和维生素A含量均显著低于对照组，且本项目在前期实验中构建了Rbp4基因敲除小鼠，发现Rbp4基因敲除小鼠协同低维生素A饮食干预会显著增加小鼠唇腭裂的发生率。因此本项目的研究目的主要是探讨RBP4与维生素A协同降低引起小鼠唇腭裂的机制。继成功培育出Rbp4敲除小鼠后，本项目通过重复摸索唇腭裂动物模型构建条件，明确低维生素A饮食干预下Rbp4-/-雌鼠的后代产生的唇腭裂表型，并计算唇腭裂的发生率，得出Rbp4-/-小鼠进行低维生素A饮食干预后子代出现唇腭裂的比率约为87%，显著高于其他三组对照。并通过体内外实验得出实验组细胞增殖明显降低，TGF-β3、BMP4等信号因子表达明显降低；实验组的唇、腭部组织细胞内异常线粒体的数量和比例显著高于对照组。体外原代培养不同组别E13的胚胎腭间充质细胞，结果表明实验组细胞增殖明显减弱，TGF-β1/2,SMAD,AKT,ERK表达明显下调，Cyclin D1表达明显上调。以上实验结果均提示RBP4降低会增加小鼠对维生素A缺乏的敏感性，进而增加发生唇腭裂的风险；并提示Rbp4敲除协同低维生素A通过影响胚胎腭部组织发育、融合过程中的细胞增殖、凋亡及迁移等生物学过程从而造成唇腭裂表型。本项目所探讨出的RBP4先天性表达降低协同维生素A摄入不足这一环境因素协同作用可能对唇腭裂发病产生影响，恰巧可以解释遗传-环境因素互作在唇腭裂发病中扮演的角色。此外，本项目的结果提示通过RBP4基因筛查配合母体预防性营养策略可能特异性降低唇腭裂发生的风险，从而在“精准医学”层面指导孕妇妊娠期营养素的补充，为降低唇腭裂的发病率提供科学指导。