低能量激光对机械压力作用下牙周膜成纤维细胞成骨分化作用的研究  

Study on Effect of Low-Level Laser on Osteogenic Differentiation of Periodon⁃tal Ligament Fibroblasts under Mechanical Stress

作　　者：柳强 刘亚辉 李利坤 郭建茹 LIU Qiang;LIU Yahui;LI Likun;GUO Jianru(Department of Oral Periodontology,Tangshan Vocational&Technical College,Tangshan 063004,China;Department of Laboratory,Tangshan Vocational&Technical College,Tangshan 063004,China;Department of Pathology,Tangshan Vocational&Technical College,Tangshan 063004,China)

机构地区：[1]唐山职业技术学院附属医院口腔牙周科,唐山市063004 [2]唐山职业技术学院附属医院检验科,唐山市063004 [3]唐山职业技术学院附属医院病理科,唐山市063004

出　　处：《中国激光医学杂志》2024年第4期181-186,237,238,共8页Chinese Journal of Laser Medicine & Surgery

基　　金：河北省医学科学研究课题(20211544)。

摘　　要：目的探究低能量激光(low-level laser,LLL)对机械压力作用下人牙周膜成纤维细胞(human periodontal ligament fibroblasts,hPDLFs)成骨分化的作用。方法研究对象为体外培养hPDLFs,将细胞分为空白组、模型组和干预组,其中空白组细胞不给予机械压力或LLL干预;模型组细胞给予2 g/cm^(2)的机械静压力干预;干预组细胞给予机械压力后再行LLL干预。LLL激光照射波长为810 nm,光纤直径400μm,频率2.46 Hz,功率100 mW,光斑面积9.6 cm^(2),功率密度10 mW/cm^(2),能量密度值为3.75 J/cm^(2),干预24 h后,qPCR检测细胞成骨基因Runt相关转录因子2(Runt-related transcription factor 2,Runx2)、骨钙素(osteocalcin,OCN)、骨形态发生蛋白2(bone morphogenetic protein 2,BMP2)的mRNA水平,碱性磷酸酶(alkaline phosphatase,ALP)及茜素红(alizarin red,ARS)染色检测细胞成骨分化情况,Western blotting检测细胞BMP2蛋白及Smad1/5/9蛋白磷酸化水平。结果分离培养的hPDLFs表达波形丝蛋白及细胞角蛋白,符合hPDLFs特征。模型组和干预组细胞Runx2、OCN、BMP2的mRNA水平、ALP及ARS染色面积、BMP2蛋白及Smad1/5/9蛋白磷酸化水平显著高于空白组(P<0.05)。此外,干预组上述指标水平均显著高于模型组(P<0.05)。进一步对干预组细胞给予BMP/smad通路抑制剂LDN-193189处理,发现LDN-193189干预显著减少了细胞ALP及ARS染色面积(P<0.05)。结论LLL能够通过BMP2/Smad1/5/9通路促进机械压力作用下hPDLFs成骨分化。Objective To investigate the effect of low-level laser(LLL)on the osteogenic differentiation of human periodontal ligament fibroblasts(hPDLFs)under mechanical stress.Methods The hPDLFs cultured in vitro were used as the object in this research.The hPDLFs were cultured in vitro and divided into a blank group,a model group and an intervention group.The cells in the blank group were given neither mechanical stress nor LLL intervention,the cells in the model group were given 2 g/cm^(2) mechanical static stress intervention,and the cells in the intervention group were given both mechanical stress and LLL intervention.The parameters of the LLL laser used were set as below:wavelength:810 nm,fiber diameter:400 nm,frequency:2.46 Hz,power:100 mW,spot area:9.6 cm^(2),power density:10 mW/cm^(2) and energy density:3.75 J/cm^(2).After 24 hours’intervention,the mRNA levels of osteogenic genes Runt-related transcription factor 2(Runx2),osteocalcin(OCN)and bone morphogenetic protein 2(BMP2)were detected with qPCR.The osteogenic differentiation of cells was detected with alkaline phosphatase(ALP)and alizarin red staining(ARS).The phosphorylation levels of BMP2 protein and Smad1/5/9 protein were detected with Western blotting.Results The isolated and cultured hPDLFs expressed vimentin and cytokeratin,which were consistent with the characteristics of hPDLFs.The mRNA levels of RUNX2,OCN and BMP2,ALP and ARS staining area and BMP2 protein and Smad1/5/9 protein phosphorylation levels in the model group and the intervention group were significantly higher than those in the blank group(P<0.05).In addition,the levels of above indexes in the intervention group were significantly higher than those in the model group(P<0.05).It was found that,when the cells in the intervention group were further treated with BMP/Smad pathway inhibitor LDN-193189,LDN-193189 intervention significantly reduced the ALP and ARS staining areas(P<0.05).Conclusions LLL will promote the osteogenic differentiation of hPDLFs under mechanical stress through BMP2/Smad1

关 键 词：低能量激光 机械压力 牙周膜成纤维细胞 成骨分化 

分 类 号：R454.2[医药卫生—治疗学] R781.4[医药卫生—临床医学]