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Study for the induction of the periodontal ligament cell apoptosis by mechanical stress

机械应力诱导牙周膜细胞凋亡的研究

基本信息

批准号：
15592163
负责人：
TSUBOI Yoshiko
金额：
$ 2.24万
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：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15592163/
关键词：
mechanical stress periodontal ligament cells apoptosis macroarray clone

项目摘要

In the orthodontic treatment, the tooth moves in the alveolar bone by the mechanical stress induced by various devices. However, it still remains unclear that how the physical mechanical stress is replaced into the various biological events. To find a clue to clarify this mechanism, we tried to identify the genes expressed by mechanical stress in the human periodontal ligament(PDL) primary culture cells, using the cDNA array method.The patient, who was diagnosed as tooth extraction from orthodontic reasons, was thoroughly informed about the objective of the experiments, and their consent was obtained. PDL tissue derived from a premolar of the patient was excised, and was placed onto flexible bottom plate. One month later, the heterogeneous cells reached confluence, and continuous mechanical stress was loaded onto the cells for 1, 6, 24 hours. Then, total RNA was extracted and purified. The total RNA was reverse transcribed into cDNA, and amplified by PCR method. Afterwards, it was labe … More led by ^<32>P and hybridized to the human array membrane. As a result, among 1176 genes studied, many genes were up-regulated or down-regulated within a wide range after 1 hour of stretching ; however, almost all of them returned to the control level by 6 or 24 h. The up-regulated or down-regulated genes at 1h stretching were related to cell cycle arrest and apoptosis, as well as cytokines, hormones, extracellular matrixes, adhesion moleclues, protein kinases, and others. Quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) for selected genes confirmed the results of the cDNA array analysis. We also demonstrated that the gene most markedly induced by continuous stretching and related to the cell cycle or apoptosis was in part activated via the p38 mitogen-activated protein kinase (MAPK) pathway, by Western blotting and the inhibitor experiment. This work has established an important baseline for clarifying the mechanisms whereby the PDL responds to mechanical stresses, using cDNA array analysis. Less

在正畸治疗中，牙齿在各种装置引起的机械应力作用下在牙槽骨内移动。然而，物理机械应力是如何转化为各种生物事件的，目前尚不清楚。为了找到这一机制的线索，我们尝试用cDNA阵列方法鉴定机械应力在人牙周韧带（PDL）原代培养细胞中表达的基因。被诊断为因正畸原因拔牙的患者被充分告知实验目的，并征得其同意。从患者的前磨牙中取出PDL组织，并放置在柔性底板上。1个月后，异质细胞汇合，对细胞连续施加机械应力1、6、24小时。然后提取总RNA并纯化。将总RNA逆转录为cDNA，用PCR法扩增。然后用^<32>P进行标记，并与人阵列膜杂交。因此，在所研究的1176个基因中，拉伸1小时后，许多基因在很大范围内上调或下调；在拉伸1h时，上调或下调的基因与细胞周期阻滞和凋亡有关，也与细胞因子、激素、细胞外基质、粘附分子、蛋白激酶等有关。所选基因的实时定量逆转录聚合酶链反应（RT-PCR）证实了cDNA阵列分析的结果。我们还通过Western blotting和抑制剂实验证明，连续拉伸诱导最明显的基因，与细胞周期或凋亡相关，部分通过p38丝裂原活化蛋白激酶（MAPK）途径激活。这项工作为阐明PDL响应机械应力的机制建立了一个重要的基线，利用cDNA阵列分析。少