Heat shock-induced three-dimensional-like proliferation of mouse fibroblasts mediated by hydroxyapatite

本研究的目的是确定热处理有效的最小条件和增强三维（3D）样细胞增殖的最佳加热条件。C3H10T1/2小鼠成纤维细胞在40、41.5、43、44或45℃条件下分别加热2、10、15、20、30、45、60、90、180和360 min，羟基磷灰石颗粒培养10周。然后确定诱导细胞3d样增殖的最小和最佳热处理温度和持续时间条件。诱导3d样细胞增殖的最小热处理条件为43℃保温2 min，最佳热处理条件为43℃保温10 min。43℃热处理2 min和10 min的细胞平均3d样增殖率显著高于未处理细胞（分别高出1.7倍和3.7倍）（p < 0.05）。我们还观察到，在40℃下加热90或180分钟，在41.5℃下加热15分钟和44℃下加热10分钟，热处理对3d样细胞增殖的影响更大。我们发现，在43℃热处理10 min和30 min后1周，细胞凋亡率分别为7.5%和87.0%。Western blot分析显示，43℃热处理10 min后，p38丝裂原活化蛋白激酶（MAPK）磷酸化水平明显升高。这些发现表明，热休克激活p38 MAPK与3d样细胞增殖有关。本研究结果对进一步研究热疗和热疗的生理机制有一定的指导意义。

The aim of this study was to determine the minimal conditions in which heat treatment is effective for and the optimal heat conditions for enhancing three-dimensional (3D)-like cell proliferation. C3H10T1/2 mouse fibroblasts were cultured with hydroxyapatite granules for 10 weeks after heat treatment at 40, 41.5, 43, 44 or 45℃ for 2, 10, 15, 20, 30, 45, 60, 90, 180 and 360 min. Then minimal and optimal conditions of temperature and duration of heat treatment for induction of 3D-like proliferation of cells were determined. The minimal conditions of heat treatment to induce 3D-like proliferation were 43℃ for 2 min and the optimal conditions were 43℃ for 10 min. The mean rates of formation of 3D-like proliferation patterns by cells heat-treated at 43℃ for 2 and 10 min were significantly higher (1.7-and 3.7-fold higher, respectively) than that by untreated cells (p < 0.05). We also observed significantly greater effects of heat treatment on 3D-like proliferation at 40℃ for 90 or 180 min and at 41.5℃for 15 min and 44℃ for 10 min. We found that apoptosis had occurred in 7.5% and 87.0% of the cells at one week after heat treatment at 43℃ for 10 min and 30 min, respectively. Western blot analysis demonstrated that phosphorylation of p38 mitogen-activated protein kinase (MAPK) was markedly increased by heat treatment at 43℃ for 10 min. These findings suggest that activation of p38 MAPK by heat shock is associated with 3D-like cell proliferation. The results of this study should be useful for further studies aimed at elucidation of the physiologic mechanisms underlying thermotherapy and hyperthermia.