机械压应力条件下人牙周膜成纤维细胞炎症因子表达变化初探

Inflammation-related cytokines expression in periodontal ligament fibroblasts under mechanical stress

目的 探讨在一定机械压应力条件下人牙周膜成纤维细胞常见炎症相关细胞因子的表达谱变化,初步明确机械压应力对牙周膜成纤维细胞炎症相关细胞因子表达的影响.方法 取健康恒前磨牙牙根中1/3牙周膜组织,采用组织块法培养人牙周膜成纤维细胞,分为加压组（200 Pa持续加压）与未加压组培养24 h后,通过反转录聚合酶链反应（RT-PCR）及实时定量PCR法检测白细胞介素（IL）-1β、IL-2、IL-4、IL-6、IL-8、IL-10、IL-12、肿瘤坏死因子（TNF）-α、TNF-β、干扰素（interferon,IFN）-γ的mRNA表达量并与管家基因磷酸甘油醛脱氢酶进行对比.加压组与未加压组培养48 h后通过微球流式多因子方法检测上述细胞因子蛋白水平的表达量.结果 加压组人牙周膜成纤维细胞培养24 h后细胞中IL-1β、IL-6、IL-8、TNF-α、TNF-β及IFN-γ mRNA相对表达量（分别为0.3633±0.0874、0.4200±0.0285、0.1697±0.0284、0.0983±0.0131、0.2840±0.0676及3.1067±0.2857）显著高于未加压组（分别为0.1173±0.0176、0.1691±0.0174、0.0117±0.0021、0.0243±0.0050、0.0000±0.0000及0.1433±0.0125）,P＜0.05 加压组人牙周膜成纤维细胞培养48 h后细胞培养上清液中IL-1β、IL-6、IL-8、TNF-α、TNF-β及IFN-γ的蛋白表达量[分别为（18.21±1.01）、（1634.11±472.41）、（1461.47±50.53）、（20.71±2.52）、（884.11±118.86）以及（1461.47±333.37）ng/L也显著高于未加压组[分别为（5.32±4.97）、（373.56±155.92）、（679.11±141.42）、（4.32±0.73）、（3.56±0.92）及（204.11±35.36）ng/L],P＜0.05 IL-2、IL-4、IL-5、IL-10及IL-12的mRNA相对表达量及蛋白表达在两组中差异无统计学意义（P＞0.05）.结论 人牙周膜成纤维细胞在持续机械压应力作用下可显著上调IL-1β、IL-6、IL-8、TNF-α、TNF-β、IFN-γ等炎症相关细胞因子mRNA及蛋白水平的表达,从而进一步影响局部牙周组织微环境.

Objective To investigate the changes of inflammation-related cytokine expression profiles in human periodontal ligament fibroblasts （hPDLF） under mechanical stress. Methods The periodontal ligament attached to the mid-third part of the fresh root of young premolars extracted for orthodontic treatment was scalped and removed hPDLFs were cultured by the method of digesting by Ⅰ -type collagenase combined with tissue adhering, and then isolated and purified by cells passages. hPDLFs were then divided into two groups, group with mechanical force and group without mechanical force and then cultured for 24 h. Employing cytokine-microarray analysis to assess, in a comprehensive manner compared to the hPDLFs culture system without a static force. The quantity of different cytokine-related genes in hPDLFs was analyzed by means of quantitative with the special primers of up-and down-regulated genes. The mRNA of inflammation-related cytokines was examined by real-time PCR, and the expression of the cytokines in hPDLFs detected by cytokine flowcytomix assay. Results The relative expression of interleukin （IL）-1β,IL-6, IL-8, tumor necrosis factor（TNF）-α, TNF-β and interferon（IFN）-γ mRNA in the hPDLFs with 24 h persistent-pressure （0. 3633 ± 0. 0874, 0. 4200 ± 0. 0285, 0. 1697 ± 0. 0284, 0. 0983 ± 0. 0131, 0. 2840 ±0. 0676 and 3. 1067 ±0. 2857） was significantly higher than the group without mechanical force（0. 1173 ±0.0176, 0. 1691 ± 0.0174, 0.0117 ：± 0.0021, 0.0243 ± 0.0050, 0.0000 ± 0.0000 and 0.1433 ±0. 0125） ,P 〈0. 05. The cell culture supernatant cytokines expression of IL-1 β, IL-6, IL-8, TNF-α, TNF-βand IFN-γ after 48 h cultured [（18.21 ± 1.01）, （1634. 11 ± 472. 41）, （1461.47 ± 50. 53）, （20. 71 ±2. 52）, （884. 11 ± 118. 86） and （1461.47 ± 333.37） ng/L]was significantly higher than the group without mechanical force [（5. 32 ± 4. 97）, （373.56 ± 155.92）, （679. 11 ± 141.42）, （4. 32 ± 0. 73）, （3.56 ±0.92）and（204. 11 ±35.36） ng/L],P 〈0.05. The relative mRNA and protein expression of IL-2, IL-4,IL-5, IL-10 and IL-12 showed no significant difference between the both groups. Conclusions Persistent static mechanical force could regulate the expression of some inflammation-related cytokines. These upregulated cytokines may be invloved in remodeling of hPDLFs, bone resorption and periodontal microenvironment.