Fam83h在氟牙症发病机制中的作用研究

The incidence of dental fluorosis presents an ascending trend recently and the 4th International Dental Health Survey found that the prevalence of fluorosis among 12 year olds in mainland China was as high as 13.4%. However, the pathogenesis of fluorosis is still unclear. Fluorosis and hypocalcified amelogenesis imperfect (AI) with FAM83H mutations have the similar clinical and pathological feature. We previously found that the mouse ameloblast-like LS8 cells can respond to fluoride by down-regulating Fam83h expression and decreasing the mineralization level in mouse derived ameloblast-like cell lines LS8. Then we performed immunohistochemical staining of sections of F-group and control-group embryo mandible regions at stages of tooth development and found that the expression of Fam83h in the F group was significantly lower than that in the control group. Meanwhile, we found that Fam83h has certain relationship with CK1α. Based on these previous results, we hypothesize that excess fluoride will suppress the expression of Fam83h and CK1a in odontogenesis and delay in the removal of enamel matrix proteins (EMPs), subsequently, these EMPs accumulated in the ameloblasts stimulate the expression of CK1α as the enamel matures; however, F-suppresses the kinase groups of CK1α, which result in losing the activity of CK1α and then failing to activate the expression of Fam83h. In this project, multidisciplinary experimental methods and fluoride induced cell and mouse model are designed to explore the role of Fam83h in the pathogenesis of fluorosis and the regulatory mechanism by which CK1a regulates, wishing to make contributions to find out the pathogenesis of fluorosis and optimize overall treatments.

氟牙症发病率近年呈上升趋势，第4次国际口腔健康调查发现中国大陆12岁儿童氟牙症患病率高达13.4%，发病机制仍未明确。氟牙症和Fam83h突变所致牙釉质发育不全具有临床表型和病理结构的相似性。我们前期研究发现高氟抑制小鼠成釉细胞矿化及Fam83h表达，Fam83h在氟牙症组小鼠磨牙胚中的表达量明显低于正常组；同时发现Fam83h与CK1α在氟牙症牙胚发育过程中有相关性。据此我们提出科学假说:高氟抑制牙胚中Fam83h和CK1α表达，釉基质蛋白降解延迟，随着牙釉质进入成熟期，蓄积的釉基质蛋白刺激CK1α表达增加，但由于F-的抑制作用致CK1α失去激酶功能而无法激活Fam83h表达。本课题拟采用多学科的实验方法，结合高氟诱导细胞和动物模型，探索Fam83h在氟牙症发病机制中的作用及CK-1α对Fam83h的表达调控机制。本项目的完成可以为探明氟牙症的发病机制及优化治疗手段提供新思路。

氟牙症和FAM83H突变所致牙釉质发育不全具有临床表型和病理结构的相似性。本课题组构建Fam83h基因敲除小鼠探索该基因在牙釉质发育过程中发挥的作用，并通过高氟诱导成釉细胞和氟牙症小鼠模型，进一步探索Fam83h在氟牙症发病机制中的作用。Fam83h基因敲除小鼠大多在出生后3周死亡，且身体发育迟缓伴牙齿迟萌、毛发稀疏、活动力低、体型小伴牙齿较小。扫描电镜结果显示牙釉质超微结构在纯合小鼠和野生鼠之间无差异；免疫组化结果显示纯和小鼠分泌期釉基质分泌较少，且成熟期有较多的釉基质残留说明Fam83h基因敲除并不影响成釉细胞功能，但是会延迟牙釉质发育过程，Fam83h基因是生长发育的关键因子，对于维持牙釉质正常发育是必须的。接着我们课题组通过高氟诱导小鼠成釉细胞，发现高氟抑制小鼠成釉细胞矿化，并通过JNK和P38信号通路抑制Fam83h表达。构建氟牙症小鼠模型，发现氟牙症小鼠牙胚在蕾状期（E13.5）、帽状期（E15.5）、钟状早期（E18.5）和钟状晚期（PN3）的Fam83h表达均低于野生鼠。最后，课题组利用Motif Scan(http://myhits.isb-sib.ch/cgi-bin/motif-scan)和ELM(http://elm.eu.org/)发现在FAM83H（121-852）aa处有一个IMP dehydrogenase/GMP reductase domain功能域，可能是CK1α磷酸化位点。接着我们使用免疫共沉淀发现FAM83H和CK1α有结合能力，这提示二者在牙胚发育过程中有密切关系。氟牙症小鼠的免疫组化结果显示E18.5(钟状期早期)和PN3(钟状期晚期) CK1α表达水平升高，这与细胞结果一致，而且高氟是通过JNK信号通路增加成釉细胞中CK1α表达。以上研究结果说明，在牙釉质形成的后期分泌/早期成熟阶段，氟牙症牙釉质中滞留大量未降解釉基质蛋白激活JNK信号通路刺激 CK1α大量表达进而发挥激酶作用，同时抑制了与之结合的Fam83h表达，导致牙釉质矿化延迟。