Study on the mechanism for the periodontal ligament tissue regeneration using human periodontal ligament tissues derived from complex odontoma

复杂牙瘤来源的人牙周膜组织再生牙周膜组织的机制研究

• 批准号: 13672151
• 负责人: TSUBOI Yoshiko
• 金额: $ 2.5万
• 依托单位: OKAYAMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13672151/
• 关键词: complex odontoma; periodontal ligament cells; cloning; immortalize; regeneration

Orthodontic tooth movement depends on the active periodontal ligament remodeling and regeneration. To analyze this mechanism, we utilized odontoma PDL and tried to find the genes specific in odontoma PDL. Human PDL tissue derived from a premolar complex odotoma was excised, which was extracted from orthodontic reasons, and placed onto flexible bottom plate. One month later, the heterogeneous cells reached confluence and they were cloned. Each clone was collected at 6-8 passages and stocked. Cell morphology and growth and differentiation ability of each clone is analyzing.Total RNA was extracted from the heterogeneous primary culture cells from the odontoma PDL and from normal tooth PDL. To investigate the difference in the expression of Msx-1, Msx-2, which has been reported to control the tooth development, in odontoma PDL and normal tooth PDL, total RNA was reverse transcribed into cDNA, followed by quantitative real-tune PCR, using specific primers. As a result, all RNAs expressed the Msx-1 and Msx-2 mRNAs, though the expression level varied with the cells. The morphology of the cells also differed from each other.Furthermore, another periodontal ligament tissues from another complex odontoma were used and analyzed the expression of Msx-1 and Msx-2 and the same tendency was observed. These results suggested that the process for the formation of odontoma involves at least Msx-1 and Msx-2, but also other transcriptional factors. We are analyzing cDNA array method and differential display PCR method, to identity the genes that is expressed specific in odotoma PDL. We are also preparing to immortalize the cloned cells, and to identify the clone specific genes.

正畸牙齿移动依赖于牙周膜的主动改建和再生。为了分析这一机制，我们利用牙瘤PDL，并试图找到牙瘤PDL的特异性基因。从前磨牙复合体齿瘤中切除人PDL组织，其是从正畸原因中提取的，并放置在柔性底板上。一个月后，异质细胞达到汇合，并将它们克隆。在6-8代时收集每个克隆并储存。对各克隆的细胞形态、生长和分化能力进行分析。从牙瘤PDL和正常牙PDL的异种原代培养细胞中提取总RNA。为了研究Msx-1、Msx-2在牙瘤PDL和正常牙PDL中表达的差异，采用特异性引物，将总RNA逆转录成cDNA，然后进行定量实时PCR。结果，所有RNA都表达Msx-1和Msx-2 mRNA，尽管表达水平因细胞而异。此外，用另一种复杂性牙瘤的牙周膜组织进行Msx-1和Msx-2的表达分析，发现两者的表达趋势相同。这些结果表明，在牙瘤的形成过程中，至少涉及Msx-1和Msx-2，但也有其他转录因子。本研究采用cDNA芯片技术和差异显示PCR技术，分析了在Odotoma PDL中特异表达的基因。我们还准备使克隆细胞永生化，并鉴定克隆特异性基因。

项目成果

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Ichikawa H: "Osteopontin-immunoreactivity in the rat trigeminal ganglion and trigeminal sensory nuclei"Brain Res. 91・9. 147-154 (2001)

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