FUNCTIONAL ANALYSIS OF TOOTH GERM DEVELOPMENT-RELATED GENES

我们之前研究了小鼠下颌骨的发育，并证明了牙形成发生在胚胎10.5天（e10.5）和E12之间。根据组织学结果，我们在E10.5和E12下颌骨之间进行了cDNA减法，以检测可能参与牙形成起始的差异表达基因。我们发现来自E10.5下颌骨的Pgk-1、Ccte、Hsp86、Nucleolin、Hsc73、Frg1、N-ras、Set alpha和Hsj2，以及来自E12下颌骨的E25、ATPase6、Mum2、Thymosin β 4和L21是差异表达基因。原位杂交分析显示，Set α、热休克蛋白（HSPs:Hsc73、Hsj2和Hsp86）、Pgk-1、Nucleolin、ATPase6、Thymosin β 4在发育中的牙胚中均有表达，表明该基因与牙形成密切相关。Runx2/Cbfal基因敲除小鼠显示完全没有骨化和牙胚发育停滞。通过对培养小鼠下颌骨给予反义硫代寡聚二氧核苷酸（S-ODNs），研究Runx2/Cbfa1亚型特异性功能受体在牙胚发育中的作用。在培养基中添加II/III型Runx2/Cbfa1反义s - odn，可抑制11日龄胚胎培养的下颌骨的芽样生长，同时抑制15日龄胚胎培养的牙胚的成牙细胞向成釉细胞和成牙细胞分化。半定量RT - PCR结果显示，牙本质基质蛋白1、牙本质唾液磷蛋白、成釉原蛋白和成釉细胞蛋白的表达在培养的牙胚中受到明显抑制。与此同时，使用I型Runx2/Cbfal反义S-ODNs处理的样品未观察到牙胚发育阻滞、基因表达抑制或牙源性细胞分化。同样的结果也被观察到无论是对照或有意义和随机序列的S-ODNs处理的样品。这些数据表明，II/III型Runx2/Cbfa1亚型与牙胚的发育和分化密切相关。少

We previously examined the development of the mouse mandible, and demonstrated that odontogenesis occurs between embryonic day 10.5 (E 10.5) and E12. Based on the histological findings, we performed cDNA subtraction between the E10.5 and E12 mandibles to detect any differentially expressed genes which might be involved in the initiation of odontogenesis. We found Pgk-1,Ccte,Hsp86,Nucleolin,Hsc73,Frg1,N-ras,Set alpha and Hsj2 from the E10.5 mandible, and E25,ATPase6,Mum2, Thymosin beta4 and L21 from the E12 mandible to be differentially expressed genes. In situ hybridization analyses showed the expression of Set alpha, heat shock proteins (HSPs:Hsc73,Hsj2 and Hsp86), Pgk-1,Nucleolin, ATPase6, Thymosin beta4 to be detected in the developing tooth germ, thus indicating a close relationship of this gene to odontogenesis.Runx2/Cbfal knockout mice showed both a complete lack of ossification and the developmental arrest of tooth germ. We examined Runx2/Cbfa1 isoform-type specific functional r … More oles in the development of tooth germ by the administration of antisense phosphorothioate oligodioxynucleotides (S-ODNs) into cultured mouse mandibles. The administration of type II/III Runx2/Cbfa1 antisense S-ODNs into the culture media resulted in an arrest of tooth germ growth at the bud-like stage in cultured mandible taken from the 11-day-old embryos, while also causing the inhibition of the differentiation of odontogenic cells into ameloblast and odontoblast in cultured tooth germs taken from the 15-day-old embryos. The expression of dentin matrix protein 1, dentin sialophosphoprotein, amelogenin and ameloblastin was shown to be markedly suppressed in cultured tooth germ by the semi-quantitative RT PCR method. Meanwhile, no developmental arrest of tooth germ, no inhibition of gene expression, or differentiation of odontogenic cells was observed in samples treated with the type I Runx2/Cbfal antisense S-ODNs. The same findings were also observed in either the control or the sense and random sequence S-ODNs treated samples. These data indicate that the type II/III Runx2/Cbfa1 isoform is closely related to the development and differentiation of tooth germ. Less