熊果酸干预人牙周膜干细胞成骨分化的效应  

Effect of ursolic acid on osteogenic differentiation of human periodontal ligament stem cells

作　　者：郑茜 刘萍萍 顾煜婕 谢蕾 Zheng Qian;Liu Pingping;Gu Yujie;Xie Lei(Department of Orthodontics,Affiliated Stomatological Hospital of Southwest Medical University,Luzhou 646000,Sichuan Province,China;Oral&Maxillofacial Reconstruction and Regeneration of Luzhou Key Laboratory,Luzhou 646000,Sichuan Province,China;Department of Stomatology,Nanchong Central Hospital,Nanchong 637000,Sichuan Province,China;Department of Chengbei Outpatient,Affiliated Stomatological Hospital of Southwest Medical University,Luzhou 646000,Sichuan Province,China)

机构地区：[1]西南医科大学附属口腔医院正畸科,四川省泸州市646000 [2]口颌面修复重建和再生泸州市重点实验室,四川省泸州市646000 [3]南充市中心医院口腔科,四川省南充市637000 [4]西南医科大学附属口腔医院城北门诊部,四川省泸州市646000

出　　处：《中国组织工程研究》2025年第1期80-86,共7页Chinese Journal of Tissue Engineering Research

基　　金：四川省科技厅自然科学基金青年项目(2023NSFSC1522),项目名称:光刺激对光基因化牙囊细胞成骨分化的影响及其机制研究,项目参与人:顾煜婕;西南医科大学附属口腔医院自然科学基金青年项目(201912),项目名称:基于BMP信号通路探讨熊果酸对牙周膜干细胞骨分化能力的影响,项目负责人:谢蕾。

摘　　要：背景:熊果酸可促进骨髓间充质干细胞向成骨细胞定向分化,但其对人牙周膜干细胞是否具有促成骨作用目前相关报道少见。目的:探讨熊果酸对人牙周膜干细胞增殖和成骨分化能力的影响。方法:分离、培养人牙周膜干细胞,取第3代细胞,分别采用含不同浓度(0,1,2,4,6,8μmol/L)熊果酸的普通培养基干预,干预1,3,5,7 d后,采用CCK-8法检测细胞增殖,筛选适宜干预浓度。取第3代人牙周膜干细胞,分别用含0,1,2,4μmol/L熊果酸的成骨诱导液干预,干预7 d后,采用qRT-PCR法检测碱性磷酸酶、Runx2、骨钙素m RNA的表达,干预14 d后,茜素红染色观察矿化结节形成。取第3代人牙周膜干细胞,对照组加入成骨诱导液,熊果酸组、拮抗剂组分别加入含熊果酸(2μmol/L)、骨形态发生蛋白信号通路拮抗剂Noggin的成骨诱导液,熊果酸+拮抗剂组加入含熊果酸(2μmol/L)、骨形态发生蛋白信号通路抑制剂Noggin的成骨诱导液,培养7 d后,采用qRT-PCR和Western blot检测骨形态发生蛋白2、Smad1、骨桥蛋白、Runx2的m RNA及蛋白表达。结果与结论:(1)1,2,4μmol/L熊果酸可促进人牙周膜干细胞的增殖,6,8μmol/L熊果酸可抑制人牙周膜干细胞的增殖,后续实验选择1,2,4μmol/L熊果酸进行干预;(2)与0μmol/L相比,1,2,4μmol/L熊果酸可促进碱性磷酸酶、Runx2、骨钙素m RNA的表达以及矿化结节的形成(P<0.05),其中以2μmol/L熊果酸效果最显著;(3)与对照组比较,熊果酸组骨形态发生蛋白2、Smad1、骨桥蛋白、Runx2的mRNA及蛋白表达均升高(P<0.05),拮抗剂组上述指标的mRNA及蛋白表达均降低(P<0.05);与熊果酸组比较,熊果酸+拮抗剂组上述指标的mRNA及蛋白表达均降低(P<0.05);(4)结果表明,熊果酸可通过激活骨形态发生蛋白信号通路促进人牙周膜干细胞的成骨分化。BACKGROUND:Ursolic acid can promote the directed differentiation of bone marrow mesenchymal stem cells into osteoblasts.However,there are few reports on whether ursolic acid has osteogenic effect on human periodontal ligament stem cells.OBJECTIVE:To investigate the effect of ursolic acid on proliferation and osteogenic differentiation of human periodontal ligament stem cells.METHODS:The human periodontal ligament stem cells were isolated and cultured.Passage 3 cells were selected and treated with ordinary medium containing different concentrations(0,1,2,4,6,8μmol/L)of ursolic acid.After intervention for 1,3,5,7 days,the cell proliferation was detected by CCK-8 assay and the appropriate intervention concentration was screened.Passage 3 human periodontal ligament stem cells were treated with osteogenic induction solution containing 0,1,2,4μmol/L ursolic acid,respectively.After 7 days of intervention,the mRNA expressions of alkaline phosphatase,Runx2,and osteocalcin were detected by qRT-PCR.After 14 days of intervention,the formation of mineralized nodules was observed by alizarin red staining.Passage 3 human periodontal ligament stem cells were taken and the control group was added with osteogenic induction solution;the ursolic acid group and the antagonist group were added with osteogenic induction solution containing ursolic acid(2μmol/L)and the bone morphogenetic protein signaling pathway antagonist Noggin,respectively.The ursolic acid+antagonist group was added with osteogenic induction solution containing ursolic acid(2μmol/L)and Noggin,the inhibitor of bone morphogenetic protein signaling pathway,and cultured for 7 days.qRT-PCR and western blot assay were used to detect the mRNA and protein expressions of bone morphogenetic protein 2,Smad1,osteopontin,and Runx2.RESULTS AND CONCLUSION:(1)1,2,4μmol/L ursolic acid could promote the proliferation of human periodontal ligament stem cells.6,8μmol/L ursolic acid could inhibit the proliferation of human periodontal ligament stem cells,and 1,2,4μmol/L ursolic a

关 键 词：人牙周膜干细胞 熊果酸 成骨 骨形态发生蛋白信号通路 碱性磷酸酶 骨钙素 骨形态发生蛋白2 Smad1蛋白 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R78