银杏内酯B通过调控miR-24-3p对人牙周膜干细胞增殖、成骨分化的影响  

Effects of ginkgolide B on proliferation and osteogenic differentiation of human perimembrane stem cells by regulating miR-24-3p

作　　者：李彦浇 梁雷 金钫 王智伟 Li Yanjiao;Liang Lei;Jin Fang;Wang Zhiwei(Department of Orthodontics,School of Stomatology,the Fourth Military Medical University,Shaanxi Clinical Research Center for Oral Diseases,National Clinical Research Center for Oral Diseases,State Key Laboratory of Oral&Maxillofacial Reconstruction and Regeneration,Xi'an 710032,China)

机构地区：[1]口颌系统重建与再生全国重点实验室,国家口腔疾病临床医学研究中心,陕西省口腔疾病临床医学研究中心,第四军医大学口腔医院正畸科,西安710032

出　　处：《中华细胞与干细胞杂志（电子版）》2024年第4期229-235,共7页Chinese Journal of Cell and Stem Cell（Electronic Edition）

基　　金：国家自然科学基金(82170988)。

摘　　要：目的探究银杏内酯B(GB)调控miR-24-3p对人牙周膜干细胞增殖和成骨分化的影响。方法体外分离培养人牙周膜干细胞,使用10、25、50μmol/L GB处理细胞;按照脂质体法将anti-miR-NC、anti-miR-24-3p转染细胞内;将miR-NC、miR-24-3p转染细胞内,用50μmol/L GB处理。用MTT检测细胞增殖。将各组细胞进行成骨诱导后,试剂盒检测ALP活性;Western blot检测PCNA、Osx、OPN和OCN蛋白表达;RT-qPCR检测miR-24-3p表达水平。两组间比较采用独立样本t检验,多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。结果GB呈剂量依赖性增加人牙周膜干细胞增殖活性[48 h:(0.52±0.05、0.64±0.05、0.80±0.08比0.33±0.05);72 h:(0.72±0.05、0.85±0.04、1.08±0.11比0.51±0.06)]、ALP活性(0.45±0.06、0.75±0.08、1.12±0.08比0.27±0.05)、PCNA(0.41±0.05、0.57±0.06、0.78±0.06比0.26±0.04)、Osx(0.45±0.06、0.68±0.05、0.79±0.03比0.33±0.04)、OPN(0.56±0.06、0.75±0.05、0.92±0.06比0.42±0.05)、OCN蛋白表达(0.48±0.05、0.59±0.04、0.76±0.08比0.30±0.06),降低miR-24-3p表达水平(0.35±0.04、0.52±0.06、0.87±0.06比1.00±0.09)(P均<0.05)。干扰miR-24-3p可增加48、72 h时人牙周膜干细胞增殖活性[(0.57±0.06比0.35±0.04)、(0.90±0.10比0.47±0.06)]、ALP活性(0.68±0.05比0.30±0.04)、PCNA(0.47±0.05比0.29±0.04)、Osx(0.50±0.04比0.35±0.04)、OPN(0.62±0.05比0.39±0.03)、OCN蛋白表达(0.75±0.07比0.42±0.06)(P均<0.05)。与GB+miR-NC比较,GB+miR-24-3p干预的细胞48、72 h的OD值[(0.67±0.05比0.82±0.07)、(0.84±0.05比1.10±0.09)]、ALP活性(0.56±0.04比1.15±0.15)、PCNA(0.46±0.06比0.79±0.07)、Osx(0.52±0.03比0.80±0.05)、OPN(0.63±0.04比0.91±0.09)、OCN蛋白表达(0.45±0.06比0.78±0.08)降低(P<0.05)。结论GB可能通过降低miR-24-3p促进人牙周膜干细胞增殖和成骨分化。Objective To investigate the effect of ginkgolide B(GB)regulating miR-24-3p on human peripheral stem cells'proliferation and osteogenic differentiation.Methods Human periodontal stem cells were isolated and cultured in vitro.The cells were treated with 10,25,50μmol/L GB.Anti-miR-NC and anti-miR-24-3p were transfected into cells by liposome method,and miR-NC and miR-24-3p were transfected into cells and treated with 50μmol/L GB.MTT detected cell proliferation;After osteogenic induction of cells with different treatments,the ALP activity was detected by the kit;the protein expressions of PCNA,Osx,OPN and OCN were detected by Western blot;and RT-qPCR detected the expression level of miR-24-3p.Results GB increased the proliferative activity(48 h,72 h)[(0.52±0.05,0.64±0.05,0.80±0.08 vs 0.33±0.05),(0.72±0.05,0.85±0.04,1.08±0.11 vs 0.51±0.06)],ALP activity,PCNA(0.45±0.06,0.75±0.08,1.12±0.08 vs 0.27±0.05),PCNA(0.41±0.05,0.57±0.06,0.78±0.06 vs 0.26±0.04),Osx(0.45±0.06,0.68±0.05,0.79±0.03 vs 0.33±0.04),OPN(0.56±0.06,0.75±0.05,0.92±0.06 vs 0.42±0.05),OCN(0.48±0.05,0.59±0.04,0.76±0.08 vs 0.30±0.06)protein expression of human periodontal ligament stem cells in a dose-dependent manner,and decreased the expression level of miR-24-3p(0.35±0.04,0.52±0.06,0.87±0.06 vs 1.00±0.09)(P<0.05).Interfering with miR-24-3p increased the proliferative activity[48 h:(0.57±0.06 vs 0.35±0.04);72 h(0.90±0.10 vs 0.47±0.06)],ALP activity(0.68±0.05 vs 0.30±0.04),PCNA(0.47±0.05 vs 0.29±0.04),Osx(0.50±0.04 vs 0.35±0.04),OPN(0.62±0.05 vs 0.39±0.03),OCN(0.75±0.07 vs 0.42±0.06)protein expressions of human periodontal ligament stem cells(all P<0.05).Compared with GB+miR-NC group,the OD value[48 h:(0.67±0.05 vs 0.82±0.07);72 h:(0.84±0.05 vs 1.10±0.09)],ALP activity(0.56±0.04 vs 1.15±0.15),PCNA(0.46±0.06 vs 0.79±0.07),Osx(0.52±0.03 vs 0.80±0.05),OPN(0.63±0.04 vs 0.91±0.09),OCN(0.45±0.06 vs 0.78±0.08)protein expression of GB+miR-24-3p group decreased(all P<0.05).Conclusion GB may promote prolifer

关 键 词：银杏内酯B miR-24-3p 人牙周膜干细胞 增殖 成骨分化 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]