低能量激光对衰老牙周膜干细胞增殖和成骨分化功能的影响  

Effect of low-level laser therapy on proliferation and osteogenic differentiation of aging periodontal ligament stem cells

作　　者：邵馨 王爽[1,2] 郭小梅 夜文敏 孔荣荣 宋扬 吴凡 王丽颖 SHAO Xin;WANG Shuang;GUO Xiaomei;YE Wenmin;KONG Rongrong;SONG Yang;WU Fan;WANG Liying(Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research,College of Stomatology,Xi’an Jiaotong University,Xi’an 710004,China;Department of Orthodontics,College of Stomatology,Xi’an Jiaotong University;Department of Pathology,College of Stomatology,Xi’an Jiaotong University;Department of Stomatology,Air Force 986 Hospital;Department of Cardiovascular Surgery,Air Force 986 Hospital)

机构地区：[1]西安交通大学口腔医院,陕西省颅颌面精准医学研究重点实验室,西安710004 [2]西安交通大学口腔医院正畸科 [3]西安交通大学口腔医院病理科 [4]空军第九八六医院口腔科 [5]空军第九八六医院心脏外科

出　　处：《山西医科大学学报》2022年第11期1452-1457,共6页Journal of Shanxi Medical University

基　　金：国家自然科学基金资助项目(82000999);陕西省自然科学基础研究计划项目(2020JQ-562)。

摘　　要：目的探究不同照射强度的低能量激光(low-level laser therapy,LLLT)对衰老牙周膜干细胞(periodontal ligament stem cells,PDLSCs)增殖和成骨分化功能的影响。方法将PDLSCs分为yPDLSCs组(年轻组,15~25岁,男3例,女3例,共10颗牙)和aPDLSCs组(老年组,40~55岁,男3例,女3例,共8颗牙)。待上述两组PDLSCs长至80%左右融合率时将细胞分为5组:yPDLSCs组(年轻组)、aPDLSCs对照组(老年对照组)、aPDLSCs 4 J/cm^(2)组、aPDLSCs 8 J/cm^(2)组和aPDLSCs 16 J/cm^(2)组,yPDLSCs组和aPDLSCs对照组不予以激光照射,其余3个实验组分别以4,8,16 J/cm^(2)LLLT处理aPDLSCs细胞,隔天照射1次,每次照射20 s,共照射3次。克隆形成实验检测各组细胞的增殖能力。茜素红染色检测各组细胞成骨结节形成量,碱性磷酸酶(ALP)活性检测细胞ALP活性。RT-PCR法检测细胞矮小相关转录因子2(runt-related transcription factor 2,Runx2)、ALP、骨钙素(osteocalcin,OCN)表达水平。ELISA法检测细胞TNF-α分泌量。结果与aPDLSCs对照组比较,4 J/cm^(2)组和8 J/cm^(2)组aPDLSCs的克隆形成率明显升高(P<0.05),16 J/cm^(2)组细胞的克隆形成率变化无统计学意义;与yPDLSCs组相比,aPDLSCs对照组克隆形成率明显下降(P<0.01)。与aPDLSCs对照组比较,4 J/cm^(2)组和8 J/cm^(2)组aPDLSCs的成骨结节形成明显增多(P<0.05),16 J/cm^(2)组aPDLSCs的成骨结节形成减少(P<0.05);与yPDLSCs组相比,aPDLSCs对照组的成骨结节形成量减少(P<0.01)。与aPDLSCs对照组比较,8 J/cm^(2)组aPDLSCs中ALP活性明显升高(P<0.01),16 J/cm^(2)组aPDLSCs中ALP活性降低(P<0.01);与yPDLSCs组相比,aPDLSCs对照组ALP活性降低(P<0.01)。与aPDLSCs对照组比较,8 J/cm^(2)组aPDLSCs的成骨相关基因Runx2、ALP、OCN表达水平明显升高(P<0.01),16 J/cm^(2)组aPDLSCs中Runx2、ALP、OCN表达水平变化无统计学意义;与yPDLSCs组相比,aPDLSCs对照组的成骨相关基因表达水平下降(P<0.01)。与aPDLSCs对照组比较,4 J/cm^(2)组和8 J/cm^(2)组aPDLSCs中TNObjective To investigate the effects of low-level laser therapy(LLLT)at different irradiation intensities on the proliferation and the osteogenic differentiation of aging periodontal ligament stem cells(PDLSCs).Methods PDLSCs were divided into yPDLSCs group(young group,15-25 years old,3 males and 3 females,a total of 10 teeth)and aPDLSCs group(aging group,40-55 years old,3 males and 3 females,a total of 8 teeth).When PDLSCs grew to about 80%confluence,the cells were divided into five groups:yPDLSCs group,aPDLSCs control group,aPDLSCs 4 J/cm^(2)group,aPDLSCs 8 J/cm^(2)group,and aPDLSCs 16 J/cm^(2)group.The cells in yPDLSCs group and aPDLSCs control group were not treated with laser irradiation.The aPDLSCs were treated with 4 J/cm^(2),8 J/cm^(2),16 J/cm^(2)LLLT in aPDLSCs 4 J/cm^(2)group,aPDLSCs 8 J/cm^(2)group,and aPDLSCs 16 J/cm^(2)group,respectively.The cells were irradiated once every other day,20 s each time and a total of 3 times.Colony-forming unit fibroblast assay was used to detect the proliferation ability of cells in each group.Alizarin red staining was used to detect the number of osteoblastic nodules,and alkaline phosphatase(ALP)kit was used to detect the ALP activity in cells.Real-time polymerase chain reaction(RT-PCR)was used to detect the expression levels of runt-related transcription factor 2(Runx2),ALP and osteocalcin(OCN)in different groups.The secretion of tumor necrosis factor-α(TNF-α)was detected by enzyme linked immunosorbent assay(ELISA).Results Compared with aPDLSCs control group,the adherent clonogenic cell clusters were increased in 4 J/cm^(2)group and 8 J/cm^(2)group(P<0.01),while the quantity of adherent clonogenic cell clusters had no obvious change in 16 J/cm^(2)group.The adherent clonogenic cell clusters were significantly lower in aPDLSCs control group than in yPDLSCs group(P<0.01).Compared with aPDLSCs control group,the osteogenic nodules were increased in 4 J/cm^(2)group and 8 J/cm^(2)group(P<0.05),while the osteogenic nodules were decreased in 16 J/cm^(2)group(P<0.05).The oste

关 键 词：牙周膜干细胞 低能量激光治疗 衰老 细胞增殖 成骨分化 

分 类 号：R781.4[医药卫生—口腔医学]