离子通道对牙齿发育起着重要调控。我们前期研究：由成熟型成釉细胞上的AE2、CFTR离子通道介导的对釉质空间pH调节以及由NKCX和NaPi离子通道所介导的钙磷离子转运机制对釉质发育至关重要。我们已证实：钠-钾-氯离子协同转运子-2在成熟型成釉细胞表达。而NKCC2 可能通过将NKCX和NaPi离子通道转运至釉质中的Cl-、Na+与K+重新转运至细胞内来影响上述离子通道。本课题探讨NKCC2在釉质发育中的作用：1）通过免疫组织化学及激光共聚焦技术研究NKCC2在釉质发育过程中时间和空间表达规律；2）成釉细胞体外培养模型中，利用呋塞米抑制NKCC2，通过RT-PCR等技术检测NKCC2对于与成熟型成釉细胞分化与功能状态相关基因、蛋白和离子转运影响；3）体内功能抑制的方式研究NKCC2对釉质发育作用表型。本项目明确NKCC2对釉质发育影响及相关离子疾病病理机制研究提供理论依据。

Different ion channels, which play an important role in the tooth development， are found to be expressed in the tooth germ and tissues. Our previous researches showed that the ion channels on the mature ameloblasts responsible for the pH regulation and the transport of calcium and phosphate to build the enamel prism are inevitable for the enamel formation. Furthermore, our data suggested that ameloblasts used NKCX4 and NaPi to transport mineral ions into enamel in cotransport or exchange with K+ and Na+，which are needed to be recycled back from enamel into the ameloblasts. NKCC2, which work as K+, Na+ and Cl- cotransporter is positively stained in mature ameloblast by immunostaining method. Thus, we hypothesize that NKCC2 affect the enamel formation by helping realizing the functions of other important ion channels, such as pH regulator and Ca and P transporters. In this proposal, we will use the following methods to investigate the effects of NKCC2 on the enamel formation. Firstly, localize the expression of NKCC2 on the mature ameloblasts by immunohistochemistry. Secondly, we will test the effect of NKCC2 on the differentiation and function of mature ameloblast in vitro. Thirdly, we will investigate the enamel phenotype by inhibiting the function of NKCC2 in vivo. The results of this project will facilitate understanding the pathology of NKCC2- related dental channelopathy.