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

在正畸治疗中，牙齿在各种装置引起的机械应力的作用下在牙槽骨中移动。然而，目前仍不清楚物理机械应力是如何被替换成各种生物学事件的。为了寻找阐明这一机制的线索，我们尝试使用cDNA阵列方法鉴定人牙周膜（PDL）原代培养细胞中受机械应力表达的基因，并对诊断为正畸原因拔牙的患者详细告知实验目的，并征得其同意。切除来自患者前磨牙的PDL组织，并将其放置在柔性底板上。一个月后，异质细胞达到汇合，并对细胞施加持续的机械应力1、6、24小时。然后，提取总RNA并纯化。将总RNA逆转录成cDNA，并通过PCR方法扩增。后来，是拉贝 ...更多信息 由^ P引导<32>并与人阵列膜杂交。结果表明，在1176个基因中，许多基因在拉伸1 h后出现了大范围的上调或下调，但几乎所有基因在拉伸6 h或24 h后都恢复到对照水平。拉伸1h后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。定量实时逆转录聚合酶链反应（RT-PCR）选定的基因证实了cDNA阵列分析的结果。我们还表明，最显着的基因诱导的连续拉伸和相关的细胞周期或凋亡的部分激活通过p38丝裂原活化蛋白激酶（MAPK）途径，通过蛋白质印迹和抑制剂实验。这项工作建立了一个重要的基线，澄清机制，PDL响应机械应力，使用cDNA阵列分析。少

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