锌在腭裂发生中的作用及机制研究

Cleft lip and palate is a frequenly-see congenital malformation on oral and maxillofacial part.Recent research findings show that Wnt and Shh signal pathway directly regulate the normal developmental of palate，whereas they can not offer a exact pathogenesis of cleft palate for now.Preliminary studies of our research group find that neither high doses of zinc ion nor low doses of it is conductive to development of palate，what’s more，zinc acts an important role in antagonising the formation of cleft palate，it could be speculated that zinc ion plays a significant part in the development of palate.Zinc ion which works in the form of zinc finger protein serves on an important element in the cell differentiation，embryonic development and so on. Specificity proteins(Sp) transcription factor family which belongs to zinc finger protein superfamily has been demonstrated that direct relation between it and cleft palate exists by some researchs of scholars from home and abroad. Therefore，based on these pre-existing research，the object of the study is to sift out candidate genes for cleft palate -Sp gene, which is closely related to Wnt and Shh signal pathway，culture palate embryonic cells of mouse in vitro，detect the location of multiple components of Shh pathway under different zinc intaking and change of environmental factors.Then observe the effect Sp8 gene makes on cells proliferation and apoptosis， analyse the relation between Sp8 gene and Shh signal pathway，compare the expression of Fgf10 and Msx1 in wild type mouse and Sp8 knock-out mouse during palate fusion process by immunohistochemistry，scaning electron microscope，LSCM，RNAi and so on.To comprehensively analyze the mechanism of zinc ion influence palatal development from a new perspective.

唇腭裂是颌面部常见的先天畸形，现有研究发现wnt和shh信号通路直接调控腭的发育过程，但对于腭裂形成的机制目前尚不清楚。本课题组的前期研究及其他研究发现，过多过少的锌都不利于腭的发育，锌在腭裂发生中还表现出一定的拮抗作用，于是推测锌对腭的发育至关重要。锌离子以锌指蛋白形式在细胞分化及胚胎发育等方面具有重要作用。锌指蛋白超家族中的转录因子sp在腭裂发生中的作用已被有的研究所证实。本课题拟在已有研究基础上，筛选wnt和shh信号通路相关的SP基因，找出与wnt和shh信号通路相关的腭裂候选SP基因。通过体外培养胎鼠腭胚突细胞，检测不同锌离子浓度及致腭裂因素作用于孕鼠后，子代腭胚突细胞上wnt和shh信号通路相关受体和转录因子的定位及变化；通过失活小鼠Sp8等基因，观察腭突融合期细胞增殖和凋亡情况；比较Fgf10和Msx1等在正常腭与Sp8基因失活者的表达差异，综合分析锌在腭裂发生中的作用机制。

唇腭裂是颌面部常见的先天畸形，已有研究发现，过多过少的锌都不利于腭的发育，锌在腭裂发生中还表现出一定的拮抗作用；本课题主要研究内容：验证微量元素锌在高温、内毒素致小鼠腭裂发生过程中的保护作用及机制；重要结果：（1）母鼠在孕早期暴露于缺锌、低锌、高温、LPS等条件下，发现腭胚突组织细胞凋亡增加，增殖降低，HE染色显示孕中期母鼠腭板较对照组上抬明显迟缓；而富锌干预下，腭胚突组织细胞凋亡明显减少，且HE染色结果显示腭突发育正常。（2）通过构建两组基因芯片进行差异基因的筛选，发现缺锌组中Sp5-mRNA表达比常锌组表达多2.3倍以上，ED14.5缺锌腭裂组Sp5表达量比双侧腭板融合完全富锌组高6.78倍。（3)ED14.5缺锌组与常锌组相比，腭突组织中Sp5mRNA 的表达水平明显升高（P<0.05)，Sp8mRNA 的表达水平稍下降（P>0.05）；富锌组与常锌组相比，腭突组织中Sp5mRNA 的表达水平明显下降（P<0.05)，Sp8mRNA 的表达水平稍升高（P>0.05)。（4）沉默Sp8基因引起PTCH1相对表达量明显升高、GLI3A、GLI3R表达水平略有降低(P<0.05)，且GLI3A与GLI3R比值小于1；WNT3A和β-catenin表达水平显著降低(P<0.01)。（5）高温组、LPS组Wnt3a、GSK-3β、β-catenin、Lef-1、Sp5、MT蛋白的相对表达量降低，富锌干预后Wnt3a、GSK-3β、β-catenin、Lef-1、Sp5、MT蛋白的相对表达量升高。研究意义：富锌在一定程度上可以拮抗不良环境因素致子代小鼠腭裂的发生，其保护机制可能与信号通路Shh 、Wnt/β-catenin中SP5/8基因相关，从而为富锌预防腭裂的发生提供了一定理论基础。