Analysis of genes induced in human periodontal ligament cells respond to the mechanical stress

人PDL组织从前磨牙或第三磨牙上切除，由于正畸原因取出，并放置在含有10%胎牛血清的Dalbecco改良鹰培养基（DMEM）的柔性底板上。拉伸成纤维细胞10 ~ 14天后，1 ~ 3个月后，对原代培养细胞施加张力24h。从同一患者无应激的PDL组织中提取的细胞作为对照。从两个细胞中提取总RNA，逆转录成cDNA，然后进行PCR。用微阵列法分析了1176种基因的表达水平差异。对另外3例患者的牙周组织进行相同的实验，并在机械应力早期和晚期的某些时间点采集mRNA。鉴定出受机械应力增强的基因和受机械应力抑制的基因。有些基因在早期表达强烈，但随着时间的推移表达减弱，有些基因则表现出相反的模式。另一方面，对传代至3代的细胞施加机械张力。结果与原代培养细胞的实验结果基本相同，但对应激反应的基因数量较少，应激引起的表达变化量较小。这些结果表明，随着培养时间的延长或继代培养时间的延长，老化降低了对机械应力的敏感性或导致了对机械应力无反应的细胞的选择。目前，我们正在准备将这些观察结果的论文提交给国际期刊，其中一些已经被接受。

Human PDL tissue was excised from a premolar or a third molar, which were extracted from orthodontic reasons, and placed onto flexible bottom plate with Dalbecco's modified eagle medium (DMEM) containing 10 % fetal bovine serum. After 10 to 14 days fibroblastic cells were extended and after 1 or 3 months later, tension force was applied on the primary culture cells for 24h. The cells derived from the PDL tissue from the same patient without stress were used for control. Total RNA was extracted from both cells and reverse transcribed into cDNA, followed by PCR. The difference of the expression level of 1176 kinds of genes was analyzed by Micro Array method. Same experiments were done with other 3 patient's periodontal tissue and mRNA was collected at some points from early stage to late stage after mechanical stress. The genes reinforced by the mechanical stress, and those suppressed were identified. Some genes expressed strongly in early stage transiently, that decreased with time, and some showed reverse pattern. On the other hand, the mechanical tension force was applied using the cells that were subcultured to 3 passages. The results were almost the same to the experiments using the primary culture cells, although the number of genes responded to the stress were less and the amount of the expression change by stress was smaller. These results suggested that the aging accompanied with the long culture days or subculture lowed down the sensitivity for the mechanical stress or caused the selection for the cells that cannnot respond to the stress.Now, we are preparing the paper about these observations to submit to the international journals, and some of them have been accepted.