长链非编码RNA POIR对多次传代后牙周膜干细胞成骨分化功能的影响  

Effect of long non-coding RNA POIR on osteogenic differentiation of periodontal ligament stem cells after multiple passages

作　　者：邵馨 王爽[1,2] 宋扬 吴凡 王丽颖 SHAO Xin;WANG Shuang;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 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]空军第九八六医院口腔科 [4]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6期731-737,共7页Journal of Shanxi Medical University

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

摘　　要：目的探究多次传代对牙周膜干细胞(periodontal ligament stem cells,PDLSCs)成骨分化的影响,以及牙周炎症来源牙周膜干细胞成骨相关长链非编码RNA(osteogenesis impairment-related long noncoding RNAs of periodontal mesenchymal stem cells from periodontitis patients,lncRNA POIR)对多次传代后PDLSCs成骨分化功能的影响。方法将PDLSCs细胞连续传代,取第3代、第15代、第30代细胞用于后续实验。碱性磷酸酶(alkaline phosphatase,ALP)染色、ALP活性和茜素红染色检测不同代数PDLSCs成骨分化能力。实时定量聚合酶链式反应(real-time polymerase chain reaction,RT-PCR)检测不同代数细胞lncRNA POIR表达水平。将第30代PDLSCs分为3组:空白对照组、无义序列转染对照组(转染sh-NC质粒)和lncRNA POIR下调组(转染sh-lncRNA POIR质粒)。ALP染色、ALP活性和茜素红染色检测各组PDLSCs成骨分化能力。RT-PCR、Western blot检测各组PDLSCs中矮小相关转录因子2(runt-related transcription factor 2,Runx2)、ALP、骨钙素(osteocalcin,OCN)表达水平。采用生物信息学分析lncRNA POIR和微小RNA-214(miR-214)的结合位点。为进一步确定lncRNA POIR与miR-214之间调控关系,RT-PCR检测lncRNA POIR下调组与对照组PDLSCs中miR-214表达水平的差异,从而评价lncRNA POIR对miR-214的作用。结果与第3代和第15代比较,第30代PDLSCs成骨结节形成量减少(P<0.05),ALP染色面积占比减少(P<0.01),ALP活性降低(P<0.01)。与第3代和第15代比较,第30代PDLSCs中lncRNA POIR的表达水平下降(P<0.01)。与空白对照组和无义序列转染对照组比较,lncRNA POIR下调组PDLSCs成骨结节形成减少(P<0.01),ALP染色面积占比减少(P<0.01),ALP活性降低(P<0.05)。与空白对照组和无义序列转染对照组比较,lncRNA POIR下调组PDLSCs中Runx2、ALP、OCN表达水平降低(P<0.05)。生物信息学分析结果显示lncRNA POIR与miR-214存在结合位点。与空白对照组和无义序列转染对照组比较,lncRNA POIR下调组miObjective To investigate the effects of multiple passages on osteogenic differentiation of periodontal ligament stem cells(PDLSCs),and the effects of osteogenesis impairment-related long noncoding RNAs of periodontal mesenchymal stem cells from perio-dontitis patients(lncRNA POIR)on the osteogenic differentiation of PDLSCs after passages.Methods PDLSCs were continuously subcultured,and the cells from passages 3,15 and 30 were used for follow-up experiments.The osteogenic differentiation ability of the different generations was detected by alkaline phosphatase(ALP)staining,ALP activity and Alizarin red staining,and the expression level of lncRNA POIR in different generations was detected by real-time polymerase chain reaction(RT-PCR).The 30th generation PDLSCs were divided into three groups:blank control group,nonsense sequence transfection control group(transfected with sh-NC plasmid)and lncRNA POIR down-regulation group(transfected with sh-lncRNA POIR plasmid).Then,the osteogenic differentiation ability of PDLSCs was detected by ALP staining,ALP activity and Alizarin red staining,and the expression levels of runt-related transcription factor 2(Runx2),ALP and osteocalcin(OCN)were detected by RT-PCR and Western blot.The binding sites of lncRNA POIR and microRNA-214(miR-214)were analyzed by bioinformatics.To determine the regulatory relationship between lncRNA POIR and miR-214,the expression level of miR-214 was detected by RT-PCR in lncRNA POIR down-regulation group and control group.Results Compared with the 3rd and 15th generations,the amount of osteogenic nodules in the 30th generation was decreased(P<0.05),the proportion of ALP staining area was decreased(P<0.01),and the activity of ALP was decreased(P<0.01).Compared with the 3rd and 15th generations,the expression level of lncRNA POIR in the 30th generation was decreased(P<0.01).Compared with blank control group and nonsense sequence transfection control group,the osteogenic nodules of PDLSCs in lncRNA POIR down-regulation group were decreased(P<0.01),the pro

关 键 词：牙周膜干细胞 长链非编码RNA miR-214 成骨分化 多次传代 

分 类 号：R781[医药卫生—口腔医学]