张应力刺激下人牙周膜成纤维细胞纤连蛋白、整合素、细胞骨架的变化  

Effect of tensile stress on fibronectin-integrins-cytoskeleton in cultured human periodontal ligament fibroblasts

作　　者：朱庆党[1] 刘丽[1] 杨艳丽[1] 

机构地区：[1]浙江大学医学院附属口腔医院修复科,浙江杭州310006

出　　处：《上海口腔医学》2010年第6期601-606,共6页Shanghai Journal of Stomatology

基　　金：国家自然科学基金(30700959);浙江省医药卫生科学研究基金(2008A113)~~

摘　　要：目的:研究不同动态张应力刺激下体外培养的人牙周膜成纤维细胞纤连蛋白、整合素β1和细胞骨架的变化。方法:刮取牙根中1/3牙周膜组织,采用Ⅰ型胶原酶消化结合组织块贴附法进行人牙周膜成纤维细胞(hPDLF)原代培养并传代。用四点弯曲加载装置,通过对体外培养的hPDLF分别施加不同动态的张应力,采用实时荧光定量PCR法研究机械力对人牙周膜成纤维细胞纤连蛋白和整合素β1亚单位mRNA表达的影响。经荧光倒置显微镜观察施加不同动态张应力后细胞形态的变化。采用SPSS13.0软件包对荧光定量PCR所得数据取常用对数后行ANOVA分析。结果:张应力的大小及加力时间都对hPDLF纤连蛋白mRNA表达产生影响。加力后,hPDLF整合素β1亚单位mRNA表达下调,这种下调变化与所施加应力大小和作用持续时间相关。机械力刺激越强,整合素β1表达越明显。加力后,细胞骨架形态发生变化。加力前呈梭形,排列呈漩涡状,F-actin纤维粗且分布均匀;加力后,形态不规则,沿力的方向排列整齐,F-actin纤维细且分布不均匀;加力12h后,形态恢复到加力前形态,细胞F-actin荧光染色强度也由正常到下降再到正常。结论:动态张应力刺激在本实验提供的微应力范围内可以促进纤连蛋白、整合素β1mRNA表达改变,hPDLF细胞骨架的形态结构也发生一定规律的变化。PURPOSE：To investigate the effect of different dynamic tensile stress on the expression of fibronectin,integrins β1 subunit and cytoskeleton in cultured human periodontal ligament fibroblasts（hPDLF）.METHODS：The periodontal ligament attached to the mid-third part of fresh root of young premolars extracted for orthodontics was scalped and removed.hPDLF was cultured by the method of digesting by Ⅰ-type collagenase combining with tissue adhering.Then hPDLF was isolated and purified by cells passage.The cells were planted on plates in a density of 2.0×105/plate before loading.＂Forcel＂ four point bending device was used for mechanical loading.Cells were loaded by three levels（1000μstrain,2000μstrain,4000μstrain） of tensile stress and collected at different time（0h,0.5h,1h,4h,8h,12h） after strain loading.The quantity of mRNA of fibronectin and integrins β1 subunit in each group was analyzed by means of quantitative real-time PCR with the special primers of up-and down-regulated genes.The morphological change of cytoskeleton was observed by fluorescence converted microscope at different time after loading tensile stress.The data was analyzed by ANOVA with SPSS 13.0 software package.RESULTS：Tensile stress of different magnitude and different time significantly changed the mRNA expression of fibronectin in hPDLF.Dynamic tensile stress down-regulated the mRNA expression of integrins β1 subunit in hPDLF and the difference in groups by different magnitude and different time of mechanical forces loading was statistically significant.The stronger stimulated forces,the more down-regulated expression.The change of morphological characteristics of cyto-skeleton was statistically significant among all the groups.After loading,the shape of cells became irregular,cell arranged along the direction of loaded forces,microfilament became thinner and arranged unevenly.As the effect of mechanical force became longer,the above change vanished.CONCLUSIONS：Dynamic tensile stress could regulate the expression o

关 键 词：牙周膜成纤维细胞 张应力 纤连蛋白 整合素 细胞骨架 

分 类 号：R780.2[医药卫生—口腔医学]