The role of basement membrane type heparan sulfate proteoglycan, perlecan, in epithelial morphogenesis -transgenic mice overexpressing perlecan in the epithelial cells-

Perlecan是一种基底膜型硫酸肝素蛋白多糖，存在于牙釉质器官的细胞间隙。因此，它在牙齿的形态发生中起着重要的作用。为了阐明perlecan在牙形成中的作用，我们利用角蛋白5启动子在上皮细胞中培养了过表达perlecan的转基因小鼠，并研究了perlecan过表达对牙釉质器官形成的影响。将具有上述DNA结构的C57BL/C3H F1卵母细胞原核微注射获得转基因小鼠，在牛角蛋白5启动子的调控下表达完整的perlecan核心蛋白。采用电镜、免疫组织化学、RT-PCR等方法对两组大鼠的磨牙胚芽进行检测。免疫组织化学证实，Perlecan在Tg小鼠的上皮组织（包括牙釉质器官）中表达强烈。胚期（E13-E18）磨牙星状网细胞间增宽，牙层厚度增大，形状不规则。出生后第1天（P1） Tg小鼠，牙釉质器官明显缺乏细胞密度，血管化程度较低。下颌磨牙牙冠呈钝端状，结晶不完全，牙根分叉。P1时Tg牙胚中FGF-2、TGF-β1和PKC-a mRNA表达水平升高。综上所述，perlecan可能通过结合和释放生长因子来控制基质的空间排列，并可能作为营养物质在上皮环境中扩散运输的载体。在成牙过程中，粗聚糖可能有助于形成高度水化的环境，以实现牙釉质器官的细胞生长和分化。然而，perlecan在上皮内表达的时间安排似乎受到严格控制，因为perlecan的持续过度表达干扰了正常的牙齿形态发生。少

Perlecan, a basement membrane-type heparan sulfate proteoglycan, is localized in the intercellular space of the enamel organ. Hence, it has been suggested to play an important role in tooth morphogenesis. To elucidate the function of perlecan in odontogenesis, we generated transgenic mice overexpressing perlecan in the epithelial cells using a keratin 5 promoter, and examined the effect of perlecan overexpression on enamel organ formation. Transgenic mice were generated by pronuclear microinjection of fertilized C57BL/C3H F1 oocytes with the DNA construct, and they express the entire perlecan core protein under the control of a bovine keratin 5 promoter. Their molar tooth germs were studied by EM, immunohistochemistry, and RT-PCR for perlecan and tooth germ-related molecules.Perlecan was immunohistochemically confirmed to be expressed strongly in epithelial tissues, including the enamel organ of Tg mice. Morphologically, the stellate reticulum of the Tg molars showed widened intercellu … More lar spaces and thick, irregularly shaped dental laminae at the embryonic stage (E13-E18). In postnatal day 1 (P1) Tg mice, the enamel organ was obviously lacking cell density and was less vascularized. Finally, the crown shape of Tg molars became dull-ended, with incomplete crystallization and divergent tooth roots in mandibular molars. mRNA expression levels for FGF-2, TGF-β1, and PKC-a were elevated in Tg tooth germs at P1.From these results, it was suggested that perlecan may control the space arrangement of stromata by binding and releasing of growth factors, and it may act as a carrier for nutrient transport by diffusion within the epithelial milieu. Also in the process of odontogenesis, perlecan might contribute in forming highly hydrated circumstances to realize cell growth and differentiation in the enamel organ. However, the time schedule of the intraepithelial expression of perlecan seems to be critically controlled, because a constant overexpression perlecan has interfered normal tooth morphogenesis. Less