生物活性玻璃45S5通过细胞自噬促进根尖牙乳头细胞成牙本质方向分化  

Bioactive glass 45S5 promotes odontogenic differentiation of apical papilla cells through autophagy

作　　者：刘炜林 苏灿 崔彩云 Liu Weilin;Su Can;Cui Caiyun(Dept.of Cariology and Endodontics,Binzhou Medical University Hospital,Binzhou 256600,China)

机构地区：[1]滨州医学院附属医院牙体牙髓科,滨州256600

出　　处：《华西口腔医学杂志》2025年第1期37-45,共9页West China Journal of Stomatology

基　　金：山东省自然科学基金(ZR2019PH083)。

摘　　要：目的45S5促进根尖牙乳头细胞(APCs)成牙本质方向分化作用机制仍不明确,本研究旨在探讨细胞自噬参与生物活性玻璃45S5促进APCs成牙本质方向分化的作用。方法体外分离培养APCs,流式细胞术鉴定细胞组织来源。配置1 mg/mL 45S5浸提培养液,检测pH和离子浓度。实验分为对照组、45S5组和3-甲基腺嘌呤(3-MA)45S5组,45S5组为1 mg/mL 45S5诱导培养APCs,3-MA 45S5组为1 mg/mL 45S5中加入自噬抑制剂(3-MA)。免疫印迹法(Western blot)检测各组诱导培养24 h后自噬相关蛋白微管相关蛋白1轻链3β(LC3B)和P62的表达,实时荧光定量聚合酶链反应(RT-qPCR)检测诱导培养7 d后骨涎蛋白(BSP)、Runt相关转录因子2(Runx2)、牙本质涎磷蛋白(DSPP)和牙本质基质蛋白-1(DMP-1)的表达,细胞碱性磷酸酶(ALP)染色分析诱导培养7 d细胞ALP活性,茜素红染色分析诱导培养21 d矿化结节形成。结果45S5浸提培养液的pH为8.65±0.01,与对照组差异无统计学意义(P>0.05);45S5诱导培养液的硅离子浓度为(1.56±0.07)mmol/L,高于对照组(0.08±0.01)mmol/L(P<0.05);45S5浸提培养液的钙离子浓度为(1.57±0.15)mmol/L,与对照组差异无统计学意义(P>0.05)。Western blot结果显示,与对照组相比,45S5组LC3B-Ⅱ/Ⅰ比值升高、P62表达降低(P<0.05);与45S5组相比,3-MA 45S5组LC3B-Ⅱ/Ⅰ比值降低、P62表达升高(P<0.05)。RT-qPCR结果显示,与对照组相比,45S5组BSP、Runx2、DMP-1和DSPP表达增加;与45S5组相比,3-MA 45S5组BSP、Runx2、DMP-1和DSPP表达降低(P<0.05)。ALP染色和茜素红染色分析结果显示,与对照组相比,45S5组ALP活性增强,矿化结节形成增多;与45S5组相比,3-MA 45S5组ALP活性降低,矿化结节形成减少。结论1 mg/mL 45S5体外通过细胞自噬促进APCs成牙本质方向分化。Objective The mechanism of the odontogenic differentiation of apical papillary cells(APCs)stimulated by bioactive glass 45S5 is still unclear.This study aims to investigate the effect of autophagy on the odontogenic differentiation of APCs stimulated by bioactive glass 45S5.Methods APCs were isolated and cultured in vitro,and the cell origin was identified by flow cytometry.The culture medium was prepared with 1 mg/mL 45S5,and its pH and ion concentration were determined.The experiments were divided into control,45S5,and 3-methyladenine(3-MA)45S5 groups.In the 45S5 group,APCs were induced to culture with 1 mg/mL 45S5.In the 3-MA 45S5 group,the autophagy inhibitor 3-MA was added to 1 mg/mL 45S5.Protein immunoblotting assay(Western blot)was used to detect the expression of autophagy-associated proteins of microtubule-associated protein 1 light-chain 3β(LC3B)and P62 after 24 h of induction culture in each group.Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression of bone sialoprotein(BSP),Runt-related transcription factor 2(Runx2),dentin sialophosphoprotein(DSPP),and dentin matrix protein-1(DMP-1)after 7 d of induction culture.Cellular alkaline phosphatase(ALP)staining analyzed cellular ALP activity at 7 d of induction,and alizarin red staining evaluated the formation of mineralized nodules at 21 d of induction.Results The pH of the 45S5 extract culture medium was 8.65±0.01,which was not significantly different from that of the control group(P>0.05).The silicon ion concentration of the 45S5 induction culture medium was(1.56±0.07)mmol/L,which was higher than that of the control group(0.08±0.01)mmol/L(P<0.05).The calcium ion concentration of the 45S5 induction culture was(1.57±0.15)mmol/L,which was not significantly different from that of the control group(P>0.05).Western blot results showed that LC3B-Ⅱ/Ⅰratio increased and P62 expression decreased in the 45S5 group compared with those in the control group(P<0.05).By contrast,the ratio decreased and the expression increas

关 键 词：成牙本质分化 根尖牙乳头细胞 自噬 生物活性玻璃45S5 

分 类 号：Q254[生物学—细胞生物学]