原花青素通过转录因子EB诱导的自噬-溶酶体途径促进人牙周膜干细胞成骨分化的研究  

Study of proanthocyanidin promotes osteogenic differentiation of human periodontal ligament stem cells through the transcription factor EB-induced autophagy-lysosome pathway

作　　者：刘卓[1] 李麒麟 巫雅欣 汪向垚 毛靖[1] 龚士强 Liu Zhuo;Li Qilin;Wu Yaxin;Wang Xiangyao;Mao Jing;Gong Shiqiang(Department of Orthodontics,Department of Stomatology,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology&School of Stomatology,Tongji Medical College,Huazhong University of Science and Technology&Hubei Province Key Laboratory of Oral and Maxillofacial Development and Regeneration,Wuhan 430030,China)

机构地区：[1]华中科技大学同济医学院附属同济医院口腔医学中心口腔正畸科、华中科技大学同济医学院口腔医学院、口腔颌面发育与再生湖北省重点实验室,武汉430030

出　　处：《中华口腔医学杂志》2024年第5期453-462,共10页Chinese Journal of Stomatology

基　　金：国家自然科学基金(81771121);湖北省自然科学基金(2021CFB367)。

摘　　要：目的研究原花青素(PA)调节人牙周膜干细胞(PDLSCs)成骨分化的具体机制,探讨PA对转录因子EB(TFEB)的表达和自噬-溶酶体途径的影响。方法将PDLSCs分为对照组和PA组,通过转录组测序(RNA Seq)分析差异表达基因,通过实时荧光定量PCR(RT-qPCR)、碱性磷酸酶(ALP)染色和透射电子显微镜(TEM)观察细胞成骨能力和自噬水平。通过划痕实验和Trasnwell实验检测PDLSCs的迁移能力,溶酶体荧光探针(Lysotracker)染色和免疫荧光染色检测溶酶体的生物发生,蛋白质印迹法检测TFEB总蛋白及其在细胞质和细胞核中的表达,激光扫描共聚焦显微镜(CLSM)观察TFEB的核易位。使用小干扰RNA(siRNA)技术敲低TFEB基因,进行PA处理或无处理。蛋白质印迹法检测细胞中自噬标志物苄氯素1(Beclin1)、微管相关蛋白1轻链3B(LC3B)和成骨标志物Runt相关转录因子2(RUNX2)、ALP和骨钙素的表达。结果与对照组相比,PA组中PDLSCs的成骨和自噬相关基因出现差异性表达(P<0.05)。PA组中成骨相关基因RUNX2(2.32±0.15)、Ⅰ型胶原蛋白ɑ1链(CoL1α1)(1.80±0.18)和自噬相关基因LC3B(1.87±0.08)、Beclin1(1.63±0.08)的mRNA表达水平均显著高于对照组(分别为1.01±0.16、1.00±0.10、1.00±0.07、1.00±0.06)(均P<0.001)。与对照组相比,PA组的ALP活性更高,TEM镜下自噬体和自噬溶酶体数目更多。PA显著促进了PDLSCs的迁移(P<0.05),并增加了溶酶体的数量和溶酶体相关膜蛋白1(LAMP1)的表达。PA组中TFEB总蛋白的相对表达水平(1.49±0.07)和TFEB在细胞核/细胞质中的相对表达水平(1.52±0.12)均显著高于对照组(分别为1.00±0.11、1.00±0.13)(均P<0.01)。PA组TFEB的细胞核/细胞质相对荧光强度(0.79±0.90)也显著高于对照组(0.11±0.08)(t=3.49,P<0.01)。敲低TFEB后,PDLSCs中TFEB(0.64±0.04)、LAMP1(0.69±0.09)、Beclin1(0.60±0.05)和LC3BⅡ/Ⅰ(0.73±0.07)蛋白的表达均显著低于阴性对照组(分别为1.00±0.15、1.00±0.10、1.00±0.05、1.00±0Objective To investigate the mechanism of proanthocyanidin(PA)in regulating the osteogenic differentiation of human periodontal ligament stem cells(PDLSCs),and to explore the effects of PA on the expression and nuclear translocation of transcription factor EB(TFEB)and on the autophagy-lysosome pathway.Methods PDLSCs were divided into control group and PA group,which were subjected to RNA sequencing analysis(RNA Seq)to detect differentially expressed genes.The osteogenic differentiation ability and autophagy level were observed by real-time fluorescence quantitative PCR(RT-qPCR)analysis,alkaline phosphatase(ALP)staining and transmission electron microscope(TEM),respectively.Scratch assay and Transwell assay were used to detect the migration ability of PDLSCs.Lysotracker and immunofluorescence staining were used to detect the biogenesis of lysosomes.The total protein expression of transcription factor EB(TFEB)as well as that in cytoplasm and nucleus were detected by Western blotting.Confocal laser scanning microscope(CLSM)was used to observe the nuclear translocation of TFEB.The PDLSCs were treated with small interfering RNA(siRNA)technology to knock down the expression levels of TFEB gene with or without PA treatment.Western blotting was used to analyze the expressions of autophagy-related proteins Beclin1 and microtubule-associated protein 1 light chain 3(LC3B),as well as osteogenic-related proteins runt-related transcription factor 2(RUNX2),ALP,and osteocalcin in PDLSCs.Results Compared with the control group,the osteogenic-related and autophagy-related genes showed differential expression in PDLSCs after PA treatment(P<0.05).The mRNA expression levels of osteogenic-related genes RUNX2(2.32±0.15)and collagen typeⅠalpha 1(COL1α1)(1.80±0.18),as well as the autophagy related genes LC3B(1.87±0.08)and Beclin1(1.63±0.08)were significantly increased in the PA group,compared with the control group(1.01±0.16,1.00±0.10,1.00±0.07,1.00±0.06,respectively,all P<0.01).Compared with the control group,the PA group had

关 键 词：原花青素类 人牙周膜干细胞 成骨分化 转录因子EB 自噬-溶酶体途径 核易位 

分 类 号：R781.4[医药卫生—口腔医学]