Pax9调控小鼠帽状期和钟状期牙乳头细胞的增殖和分化的初步研究

目的 观察Pax9对小鼠帽状期和钟状期牙胚牙乳头细胞的作用.方法 培养胚胎14.5d和16.5d小鼠下颌第一磨牙牙胚的牙乳头细胞.转染 Pax9 siRNA至牙乳头细胞中敲低 Pax9的表达,CCK8检测转染后24、48、72和96h细胞的增殖情况,Real-time PCR检测转染后Msx1、Bmp2、Bmp4的表达变化.在转染Pax9 siRNA的同时进行矿化诱导培养7d,然后检测Alp、Dmp1和Dspp的表达情况.结果 敲低Pax9表达后,牙乳头细胞的增殖能力减弱;Msx1的表达水平下降,而Bmp2和Bmp4的表达水平升高;牙本质形成相关基因-Alp、Dmp1和Dspp的表达水平升高,牙乳头细胞的矿化能力增强.结论 Pax9参与调控小鼠帽状期和钟状期牙乳头细胞的增殖和成牙本质分化,同时调控下游基因Msx1、Bmp2和Bmp4的表达.     

Temporal and spatial expression of Pax9 and Sonic hedgehog during development of normal mouse palates and cleft palates in TGF-beta3 null embryos

Transforming growth factor-beta (TGF-beta3) gene disruption causes cleft secondary palate. Pax9 and Sonic hedgehog (Shh) genes are involved in the patterning of vertebrate embryonic tissues, including the facial skeleton. We investigated the expression of Pax9 and Shh genes during normal mouse palate development and in the developing cleft palates of TGF-beta3 null embryos. Whole mount in situ hybridization was conducted with use of Pax9 and Shh riboprobes for TGF-beta3 null, heterozygous and wild type mice at E12.5-E16.5. Histological analysis was processed by section in situ hybridization. In the wild type, Pax9 and Shh were expressed in the palate between E12.5-E15.5. Shh expression in the secondary palate was restricted to the rugae and the soft palate. Pax9 expression was predominantly in the palatal medial edge between E14.5 and E15.5. These patterns suggest that Shh and Pax9 may have different functions during palate development. In TGF-beta3 null mice, both genes expression patterns in the palate were different to those in wild type mice. In TGF-beta3 null mice, Pax9 expression was much reduced in the palatal medial edge at the critical time of palatal fusion (E14.5-E15.5). Shh expression in the palates of TGF-beta3 null mice was reduced throughout E12.5-E15.5, whilst Shh expression in heterozygous did not appear down regulated compared with the wild type. These results indicate that Pax9 and Shh expression are altered when the TGF-beta3 gene is deleted and suggest that Pax9 and Shh may be involved in the TGF-beta3 regulation of normal palatal fusion.     

Fibroblast growth factor 9: cloning and immunolocalisation during tooth development in Didelphis albiventris

There are no reports in literature about functional roles of fibroblast growth factor 9 (FGF-9) in tooth development in animals with complete tooth pattern. The classical model for studying tooth development is the mouse, which has small number of teeth and distinctive incisor and molar patterns. The opossum Didelphis albiventris with five upper and four lower incisors, one canine, three premolars, and four molars, on each side of the jaw, seems to be a convenient model to test results obtained in the mouse. Molecular expression studies indicate that FGF-9 participates in murine tooth initiation and regulation of morphogenesis. Searching for similarities and differences in FGF-9 expression between the opossum and the mouse, amino acid sequence and expression pattern of FGF-9 in the developing first molars of D. albiventris were characterised. FGF-9 cDNA sequence was obtained using RT-PCR and expressed in bacterial system for recombinant protein production and analysis of immunoreactivity. FGF-9 expression during tooth development was investigated by immunoperoxidase method. FGF-9 protein consists in a 209-residue polypeptide with a predicted molecular mass of 23.5 kDa. FGF-9 amino acid sequence has 98% of sequence identity to human and 97% to rodents. During tooth development, epithelial FGF-9 expression was seen at the dental lamina stage. Mesenchymal expression was seen at the bud stage and at the cap stage. No significant expression was found in the enamel knot. While in rodents FGF-9 is involved in initiation and regulation of tooth shape, it is suggested that it is only involved in tooth initiation in D. albiventris.     

Tooth agenesis: from molecular genetics to molecular dentistry

Tooth agenesis may originate from either genetic or environmental factors. Genetically determined hypodontic disorders appear as isolated features or as part of a syndrome. Msx1, Pax9, and Axin2 are involved in non-syndromic hypodontia, while genes such as Shh, Pitx2, Irf6, and p63 are considered to participate in syndromic genetic disorders, which include tooth agenesis. In dentistry, artificial tooth implants represent a common solution to tooth loss problems; however, molecular dentistry offers promising solutions for the future. In this paper, the genetic and molecular bases of non-syndromic and syndromic hypodontia are reviewed, and the advantages and disadvantages of tissue engineering in the clinical treatment of tooth agenesis are discussed.     

11名少牙畸形病人Pax 9、Msx 1、Axin 2基因突变的检测

目的:对少牙畸形病人血样标本中提取的DNA进行Pax 9、Msx 1和Axin 2核苷酸单链构象多态性和突变的检测,探讨少牙畸形的发病机制。方法:收集11名少牙畸形病人外周血标本,提取DNA,采用聚合酶链反应(PCR)、聚合酶链反应-单链构象多态性分析(PCR-SSCP)技术结合DNA直接双向测序对病人DNA进行检测。结果:在11名病人中发现1个Msx 1单核苷酸多态位点,但没有发现有关Pax 9、Msx 1、Axin 2突变。结论:结合少牙畸形的高发病率及Pax 9、Msx 1、Axin 2基因突变的较低报告数值间的偏差,遗传因素在少牙畸形形成中的作用比原先预想的更加具有异质性。     

Mouse amelogenin exons 8 and 9: sequence analysis and protein distribution

Amelogenin is the major protein of the developing enamel. Two additional exons, termed 8 and 9, have been characterized in the rat. Our aim was: to identify the mouse amelogenin exons 8/9 sequences; to investigate the potential presence of the alternative spliced isoforms of amelogenin exons 8/9; and to immunolocalize proteins containing sequences encoded by exons 8/9 during odontogenesis. RT-PCR analysis with exon 9 anti-sense primer generated 2 major amplicons with the use of a mouse tooth cDNA library and dental cell lines. DNA sequence analysis showed 93% identify with the rat exons 8/9 sequence. Alternative splicing of exon 3 was also found, but only in cDNAs lacking exons 8 and 9. Immunohistochemistry localized exons 8/9-encoded proteins in ameloblasts, young odontoblasts, and stratum intermedium cells. Analysis of our data supports the hypothesis that: (1) AMELX contains 2 additional exons; (2) ameloblasts and odontoblasts synthesize amelogenin 8/9; and (3) amelogenin splice variants may have unique functions during tooth formation.     

小鼠牙胚发育帽状期Msx2、BMP—4基因的表达和意义

目的：探讨Msx2、BMP-4基因在小鼠牙胚发育帽状期的表达及其与牙胚形态发育的关系。方法：利用原位杂交的方法观察小鼠牙胚帽状期Msx2、BMP-4基因的表达方式;利用原位末端标记法（TUNEL法）研究牙胚细胞凋亡情况。结果：Msx2、BMP-4基因在牙胚的发育吸 表达。帽状期牙胚细胞的凋亡主要集中于釉结处。结论：Msx2、BMP-4参与了牙胚的发育。     

转录因子Msx2在犬恒牙牙根发育过程中的表达

目的：观察转录因子Msx2在犬恒牙牙根发育过程中的表达及时空变化，初步探讨其对牙根形态形成的调控作用。方法：本研究采用原位杂交技术，对犬恒牙牙根不同发育时期Msx2mRNA的表达进行观察。结果：在牙根发育启动期、生长期及成熟期，转录因子Msx2具有不同的时空表达方式。结论：Msx2作为牙齿发育过程中重要的转录因子，可能参与牙根形态发育的调控作用。     

Pax6基因纯合突变胎鼠上前牙区额外牙发生的初步研究

研究Pax6基因纯合突变胎鼠上前牙区额外牙的发生情况,为研究Pax6在上前牙发生、发育中的作用及额外牙的发生机制提供依据.方法收集E18.5 DEBA Pax6基因纯合突变胎鼠20只,制作胎鼠头部组织冠状面石蜡连续切片,HE染色,观察上前牙区额外牙的发生情况;E18.5 C57BL/6NCrlBR Pax6基因野生型胎鼠18只作为对照.结果 20只Pax6基因纯合突变胎鼠中,4只上前牙区显示有额外牙牙胚;18只Pax6基因野生型胎鼠上前牙区均无额外牙牙胚.Pax6基因纯合突变胎鼠和Pax6基因野生型胎鼠的下前牙区和上下颌第一、第二磨牙区牙胚的数目相同,形态结构未见明显差别.结论本实验结果提示Pax6基因在胎鼠上前牙发生过程中有非常重要的作用.     

Patterning of Molar Tooth Roots in Mammals

Tooth morphogenesis is regulated by reciprocal interactions between the dental epithelium and odontogenic mesenchyme. As tooth roots are fundamental structures of the tooth support system, the morphology and functions of the roots are very important. However, basic information on the morphology of tooth root patterning and the molecular mechanism of root morphogenesis is largely unavailable. Following tooth crown formation, the dental epithelium forms a double-layered Hertwig's epithelial root sheath (HERS) derived from inner and outer enamel epithelium. Previous studies have reported that HERS plays an important role in tooth root development. Here, we report the correlation between the number of major cusps of the tooth crown and number of tooth roots of first molar and last premolar teeth in several extant mammals. We also discuss the molecular mechanism of tooth root patterning by introducing studies of mouse mutants and human syndromes associated with an abnormal molar morphology.     

Fibroblast growth factor signaling in mammalian tooth development

In this review, we discuss the central role of fibroblast growth factor (FGF) signaling in mammalian tooth development. The FGF family consists of 22 members, most of which bind to four different receptor tyrosine kinases, which in turn signal through a cascade of intracellular proteins. This signaling regulates a number of cellular processes, including proliferation, differentiation, cell adhesion and cell mobility. FGF signaling first becomes important in the presumptive dental epithelium at the initiation stage of tooth development, and subsequently, it controls the invagination of the dental epithelium into the underlying mesenchyme. Later, FGFs are critical in tooth shape formation and differentiation of ameloblasts and odontoblasts, as well as in the development and homeostasis of the stem cell niche that fuels the continuously growing mouse incisor. In addition, FGF signaling is critical in human teeth, as mutations in genes encoding FGF ligands or receptors result in several congenital syndromes characterized by alterations in tooth number, morphology or enamel structure. The parallel roles of FGF signaling in mouse and human tooth development demonstrate the conserved importance of FGF signaling in mammalian odontogenesis.     

PAX9 polymorphism and susceptibility to sporadic non-syndromic severe anodontia: a case-control study in southwest China

Our research aimed to look into the clinical traits and genetic mutations in sporadic non-syndromic anodontia and to gain insight into the role of mutations of PAX9, MSX1, AXIN2 and EDA in anodontia phenotypes, especially for the PAX9.

Material and Methods:

The female proband and her family members from the ethnic Han families underwent complete oral examinations and received a retrospective review. Venous blood samples were obtained to screen variants in the PAX9, MSX1, AXIN2, and EDA genes. A case-control study was performed on 50 subjects with sporadic tooth agenesis (cases) and 100 healthy controls, which genotyped a PAX9 gene polymorphism (rs4904210).

Results:

Intra-oral and panoramic radiographs revealed that the female proband had anodontia denoted by the complete absence of teeth in both the primary and secondary dentitions, while all her family members maintained normal dentitions. Detected in the female proband were variants of the PAX9 and AXIN2 including A240P (rs4904210) of the PAX9, c.148C>T (rs2240308), c.1365A>G (rs9915936) and c.1386C>T (rs1133683) of the AXIN2. The same variants were present in her unaffected younger brother. The PAX9 variations were in a different state in her parents. Mutations in the MSX1 and EDA genes were not identified. No significant diferences were found in the allele and genotype frequencies of the PAX9 polymorphism between the controls and the subjects with sporadic tooth agenesis.

Conclusions:

These results suggest that the association of A240P with sporadic tooth agenesis still remains obscure, especially for different populations. The genotype/phenotype correlation in congenital anodontia should be verified.