Hypomaturation Enamel Defects in Klk4 Knockout/LacZ Knockin Mice
Hypomaturation Enamel Defects in Klk4 Knockout/LacZ Knockin Mice
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DOI：
10.1074/jbc.m109.013623
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发表时间：
2009-07-10
影响因子：
4.8
通讯作者：
Hu, Jan C. -C.
中科院分区：
文献类型：
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作者：
Simmer, James P.;Hu, Yuanyuan;Hu, Jan C. -C.
激肽释放酶4（KLK 4）被认为在釉质生物矿化中起重要作用，因为KLK 4的缺陷导致釉质发育迟缓。我们使用基因靶向产生敲入小鼠，其用lacZ报告基因替换Klk 4基因序列，从翻译起始位点开始。通过Southern印迹和PCR分析证实了转基因的正确靶向。组织化学X-gal（5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside）染色显示成熟期成釉细胞中β-半乳糖苷酶表达。在分泌期成釉细胞或成牙本质细胞中未观察到X-gal染色。在Klk 4缺失小鼠的成熟阶段釉质中观察到保留的釉质蛋白，但在Klk 4杂合或野生型小鼠中没有观察到。Klk 4缺失小鼠的牙釉质层厚度正常，并且含有交叉牙釉质棒，但在断奶后迅速磨损，尽管小鼠维持软食物。在功能上，釉质容易在覆盖牙本质-釉质连接的平行釉质上方的初始杆和杆间釉质内破裂。尽管缺乏Klk 4和釉质蛋白的保留，晶体成熟的显着水平发生（虽然延迟），和釉质实现的矿物质密度在某些地方大于骨和牙本质中检测到的。一个重要的发现是，萌出牙齿的单个釉质微晶未能生长在一起，互锁，并作为一个单位发挥作用。相反，当断裂时，单个微晶似乎从釉质中溢出。这些结果表明Klk 4对于釉质蛋白的去除和釉质晶体的适当成熟是必不可少的。
Kallikrein 4 (Klk4) is believed to play an essential role in enamel biomineralization, because defects in KLK4 cause hypomaturation amelogenesis imperfecta. We used gene targeting to generate a knockin mouse that replaces the Klk4 gene sequence, starting at the translation initiation site, with a lacZ reporter gene. Correct targeting of the transgene was confirmed by Southern blot and PCR analyses. Histochemical X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) staining demonstrated expression of beta-galactosidase in maturation stage ameloblasts. No X-gal staining was observed in secretory stage ameloblasts or in odontoblasts. Retained enamel proteins were observed in the maturation stage enamel of the Klk4 null mouse, but not in the Klk4 heterozygous or wild-type mice. The enamel layer in the Klk4 null mouse was normal in thickness and contained decussating enamel rods but was rapidly abraded following weaning, despite the mice being maintained on soft chow. In function the enamel readily fractured within the initial rod and interrod enamel above the parallel enamel covering the dentino-enamel junction. Despite the lack of Klk4 and the retention of enamel proteins, significant levels of crystal maturation occurred (although delayed), and the enamel achieved a mineral density in some places greater than that detected in bone and dentin. An important finding was that individual enamel crystallites of erupted teeth failed to grow together, interlock, and function as a unit. Instead, individual crystallites seemed to spill out of the enamel when fractured. These results demonstrate that Klk4 is essential for the removal of enamel proteins and the proper maturation of enamel crystals.