机械压力诱导人牙周膜成纤维细胞表达白细胞介素6与p38丝裂原激活蛋白激酶阻断剂的影响

背景:牙周膜细胞对于维持牙周组织的形态和功能起着关键作用,不良应力可能引起其合成过量炎症因子从而最终引起牙周组织破坏。目的:分析p38丝裂原激活蛋白在压力诱导人牙周膜成纤维细胞合成炎症因子白细胞介素6过程中所起的作用。设计:观察对比实验。单位:解放军第四军医大学口腔生理实验室。材料:牙周膜成纤维细胞:取自因正畸需要拔除的20颗健康恒前磨牙的12～16岁青少年牙根中1/3牙周膜组织。试剂和仪器:白细胞介素6ELISA试剂盒(第四军医大学免疫学教研室);酶联免疫检测仪(华东电子管厂);p38p38丝裂原激活蛋白阻断剂特异阻断剂SB203580(Biochemical公司生产,第一军医大学病理生理教研室姜勇教授惠赠)。方法:采用常规组织块法将牙周膜组织细胞进行原代培养至第4、5代,将细胞随机分为4组:加压对照组:不对细胞加压及预处理;加压组:以200kPa持续加压细胞,不加预处理;预处理对照组:细胞加压前1h上清中加入10g/L二甲基亚砜,加压方式、时间同加压组;预处理组:细胞加压前1h预处理,即在上清中加入p38丝裂原激活蛋白阻断剂的特异阻断剂SB203580,浓度为1μmol/L,加压方式、时间同加压组。各组分别在持续加压后16,24h采集标本,通过酶标记免疫吸附测定法测定不同时间点各组细胞中白细胞介素6表达量。主要观察指标:压力对人牙周膜成纤维细胞产生的白细胞介素6的量的影响结果及p38丝裂原激活蛋白阻断剂对压力诱导人牙周膜成纤维细胞产生的白细胞介素6量的影响结果。结果:加压组细胞经持续加压16,24h后的白细胞介素6表达量分别为(143.1±0.42),(49.46±1.01)ng/L,明显高于加压对照组[(18.36±0.43),(18.78±0.50)ng/L,P<0.05];预处理组经持续加压16,24h后的白细胞介素6表达量分别为(56.39±0.72),(21.52±1.39)ng/L明显低于预处理对照组[(137.96±0.54),(48.74±0.79)ng/L,P<0.05]。结论:p38丝裂原激活蛋白是机械压力诱导人牙周膜成纤维细胞产生白细胞介素6的重要环节。

Expression of interleukin-6 stimulated by mechanical pressure in human periodontal ligament fibroblasts and the effect of p38 mitogen activated protein kinase inhibitor

BACKGROUND: Human periodontal ligament fibroblasts (HPLF) is the crucial cells in maintaining the configuration and function of periodontium. Adverse stress may cause HPLF to synthesize more inflammatory agents, which may cause the damage of periodontium.OBJECTIVE: To investigate the role of p38 MAPK of HPLF in the expres sion of inflammatory cytokine of interleukin-6 (IL-6) subjected to mechanical pressure, and explore the mechanism of the occlusal trauma to periodontium.DESIGN: A randomized and controlled trial.SETTING: Pathological Laboratory of the Fourth Military Medical Univer sity of Chinese PLA.MATERIALS: The HPLF were obtained from the middle part of 1/3 pe riodontium of 12 to 16-year-old youth whose 20 healthy permanent premo lar teeth should be extracted for orthodontic need. Main reagents and ap paratus: IL-6 enzyme-linked immunoabsorbent assay (ELISA) kit (Staff Room of Immunology of the Fourth Military Medical University of Chinese PLA); ELISA apparatus (Huadong Electronic Tube Factory); p38 MAPK specific inhibitor of SB203580 (produced by Biochemical Company, ob tained as a present from Professor Jiang, Staff Room of Pathophysiology, Southern Medical University).METHODS: The cells were primarily cultured till the 4-5 passages, and randomly divided into four groups: ①pressure-loading control group: the cell s were not subjected to pressure-loading and without pretreatment; ② pressure-loading group: the cells were subjected to continuous pressure-load ing (200 kPa) but without pretreatment; ③ pretreatment control group: the supernatant were added with 10 g/L dimathyl sulfoxide (DMSO, SB203580 solvent) at 1 hour before pressure-loading, the method and time of pressure loading were the same as those in the pressure-loading group; ④ pretreated group: the cells were pretreated with 1 μmol/L SB203580 (a specific in hibitor of the p38 MAPK) at 1 hour before pressure-loading, the method and time of pressure-loading were the same as those in the pressure-loading group. The cytosol and the supernatant in each group were sampled at 16 and 24 hours after pressure-loading respectively. The IL-6 expressions at different time points were detected with ELISA.MAIN OUTCOME MEASURES: The amount of IL-6 expression in HPLF induced by pressure with or without pretreatment by SB203580, a specific inhibiter of p38 MAPK.RESULTS: The expressions of IL-6 after continuous pressure-loading for 16 and 24 hours in the pressure-loading group were (143.1±0.42) and (49.46±1.01) ng/L, which were obviously higher than those in the pres sure-loading control group [(18.36±0.43), (18.78±0.50) ng/L, P ＜ 0.05]. The expressions of IL-6 after continuous pressure-loading for 16 and 24 hours in the pretreatment group were (56.39±0.72) and (21.52±1.39) ng/L, which were obviously higher than those in the pressure-loading control group [(137.96±0.54), (48.47±0.79) ng/L, P ＜ 0.05].CONCLUSION: p38 MAPK of HPLF acts as important cooperative mechanism to regulate IL-6 synthesis induced by mechanical pressure.