Mage-D1对敲除小鼠骨髓间充质干细胞的矿化调控作用  

Mineralization regulation of MAGE-D1 on bone marrowmesenchymal stem cells in knockout mice

作　　者：陆明洁 袁洪燕 徐丹 彭雪莲 邹续强 谢波 毛靖雯 温秀杰 LU Mingjie;YUAN Hongyan;XU Dan;PENG Xuelian;ZOU Xuqiang;XIE Bo;MAO Jingwen;WEN Xiujie(Department of Orthodontics,Luzhou Key Laboratory of Oral&Maxillofacial Reconstruction and Regeneration,Institute of Stomatology,Affiliated Stomatology Hospital of Southwest Medical University,Luzhou,Sichuan Province,646000,China)

机构地区：[1]西南医科大学附属口腔医院正畸科,口颌面修复重建和再生泸州市重点实验室,西南医科大学口腔医学研究所,四川泸州646000

出　　处：《陆军军医大学学报》2024年第18期2069-2080,共12页Journal of Army Medical University

基　　金：国家自然科学基金面上项目(81970906)。

摘　　要：目的探讨黑色素瘤相关抗原D1(melanoma associated antigen D1,Mage-D1)对小鼠股骨骨量和对大鼠骨髓间充质干细胞(bone mesenchymal stromal cells,BMSCs)矿化能力的影响及潜在分子机制。方法以雌性Mage-D1基因敲除杂合子小鼠和雄性野生型(wide type,WT)小鼠作为亲本小鼠,繁育Mage-D1基因敲除纯合子(Mage-D1 knock out,Mage-D1 KO)小鼠。PCR和琼脂糖凝胶电泳鉴定雄性Mage-D1基因敲除纯合子小鼠和同窝雄性野生型小鼠,micro-CT扫描用于分析小鼠股骨骨量,ELISA和化学法用于检测小鼠血清钙离子、磷离子、降钙素和甲状旁腺素水平。原代培养BMSCs并进行流式细胞术鉴定,免疫荧光染色观察Mage-D1在BMSCs中的表达。构建Mage-D1沉默慢病毒感染BMSCs,分为阴性对照组(sh-NC)和沉默组(sh-Mage-D1);细胞划痕实验检测BMSCs的迁移能力,流式细胞术和CCK-8检测BMSCs周期变化和增殖能力;矿化诱导后行碱性磷酸酶染色、茜素红染色;RT-qPCR和Western blot检测ALP、Runx2、Col1表达水平;RT-qPCR检测矿化相关基因p75NTR及MSX1以探讨潜在机制。结果与野生型小鼠相比,Mage-D1敲除纯合子小鼠股骨皮质骨厚度减小、皮质骨矿物含量降低、松质骨骨矿物含量降低、骨小梁数减少、松质骨骨表面密度降低、骨小梁分离度增大(P<0.05);敲除Mage-D1后,小鼠血钙、血磷、降钙素和甲状旁腺素水平无明显变化。Mage-D1表达于整个BMSCs内,在细胞核及胞核周围区域高表达。与sh-NC相比,sh-Mage-D1组细胞增殖能力降低(P<0.01),细胞迁移能力增强(P<0.01);矿化诱导后,sh-Mage-D1组ALP、Runx2、Col1基因(P<0.05)和蛋白(P<0.01)表达下降,碱性磷酸酶染色和茜素红染色更浅;sh-Mage-D1组细胞中p75NTR、Msx1表达水平比sh-NC组低。结论Mage-D1基因敲除可明显降低小鼠股骨骨量。Mage-D1可促进BMSCs增殖、抑制其迁移,正向调控其体外矿化,p75NTR-Dlx1/Msx1信号轴可能参与Mage-D1对骨代谢活动的调控。ObjectiveTo investigate the effect of melanoma associated antigen D1(Mage-D1)on mouse femoral bone mass and mineralization ability of mouse bone marrow mesenchymal cells(BMSCs)and its potential molecular mechanism.MethodsFemale Mage-D1 gene knockout heterozygous mice and male wild-type(WT)mice were subjected as parent mice to breed Mage-D1 gene knockout homozygous(Mage-D1 KO)mice.PCR and agarose gel electrophoresis were used to identify male Mage-D1 knockout(Mage-D1 KO)mice and littermate male wild-type(WT)mice.Micro-CT scanning was performed to observe mouse femoral bone mass,and ELISA and chemical assay were employed to detect serum levels of calcium,phosphorus,calcitonin,and parathyroid hormone in mice.After primary cultured BMSCs were identified with flow cytometry,immunofluorescence staining was utilized to detect the expression of Mage-D1 in BMSCs.BMSCs were infected by Mage-D1 silencing lentivirus,and then the cells were divided into negative control group(sh-NC)and silencing group(sh-Mage-D1).Cell scratch assay was conducted to detect the migration ability of BMSCs,and flow cytometry and CCK-8 assay were conducted to detect the cycle change and proliferation ability of BMSCs.After mineralization induction,alkaline phosphatase(ALP)staining and alizarin red staining were performed;RT-qPCR and Western blotting were used to measure the expression levels of ALP,Runx2 and Col1.RT-qPCR was used to detect mineralization-related genes p75NTR and Msx1.ResultsCompared with the WT mice,the femoral cortical bone thickness,cortical bone mineral content,cancellous bone mineral content,trabecular number,and cancellous bone surface density were decreased,and trabecular separation was increased in the Mage-D1 knockout homozygous mice(P<0.05).There were no significant changes in the serum levels of calcium,phosphorus,calcitonin and parathyroid hormone in mice after Mage-D1 knockout.Mage-D1 was expressed in the whole BMSCs and was highly expressed in the nucleus and perinuclear regions.Compared with the sh-NC BMSCs,the sh-Ma

关 键 词：Mage-D1 骨髓间充质干细胞 基因敲除 矿化调控 

分 类 号：R322.71[医药卫生—人体解剖和组织胚胎学] R329.24[医药卫生—基础医学] R394.2