MAGE-D1对大鼠牙胚来源外胚间充质干细胞增殖迁移能力的影响  

The effects of MAGE-D1 on proliferation and migration of ectomesenchymal stem cells originated from rat teeth germ

作　　者：杨琨[1] 李骏[2] 丰奇昊 沈梦杰 陈谦谦 罗雅馨 刘琪[1] 温秀杰 Yang Kun;Li Jun;Feng Qihao;Shen Mengjie;Chen Qianqian;Luo Yaxin;Liu Qi;Wen Xiujie(Periodontal Department,Affiliated Stomatological Hospital of Zunyi Medical University,Zunyi Guizhou 563099,China;Oral Planting Department,Affiliated Stomatological Hospital of Zunyi Medical University,Zunyi Guizhou 563099,China;Department of Stomatology,Daping Hospital,Army Medical University,Chongqing 400000,China)

机构地区：[1]遵义医学院附属口腔医院牙周科,贵州遵义563099 [2]遵义医学院附属口腔医院种植科,贵州遵义563099 [3]陆军军医大学大坪医院口腔科,重庆400000

出　　处：《遵义医学院学报》2018年第5期569-575,共7页Journal of Zunyi Medical University

基　　金：国家自然科学基金资助项目(NO:81760199);贵州省科技厅计划项目(NO:黔科合基础[2018]1185)

摘　　要：目的研究黑色素瘤相关抗原D1(Melanoma associated antigens,MAGE-D1)对大鼠牙胚来源的外胚间充质干细胞(Ectomensenchymal stem cells,EMSCs)增殖及迁移能力的影响。方法取SD大鼠孕19. 5 d胎鼠牙胚组织,组织块法培养牙胚来源外胚间充质干细胞,流式细胞仪进行外胚间充质干细胞表面分子鉴定CD14、CD29、CD44、CD45、CD90、CD105、CD146、CD166和p75NTR,MAGE-D1 siRNA转染入外胚间充质干细胞,实验组分为:对照组(Control)和MAGE-D1 siRNA组(MAGEsiRNA-EMSCs),CCK8法和划痕法分别检测两组细胞增殖能力与迁移能力,Real Time-PCR及Western blot检测迁移及WNT通路相关基因MAGE-D1、β-catenin、E-cadherin的mRNA及蛋白表达量。结果组织块法成功培养出牙胚来源的外胚间充质干细胞,经流式细胞仪检测细胞阳性表达CD14(92. 16%)、CD29(94. 53%)、CD44(99. 51%)、CD90(95. 18%)、CD105(34. 22%)、CD146(92. 39%)、CD166(98. 15%)和p75NTR(88. 56%),阴性表达CD45(0. 5%);CCK8法检测MAGE-D1 siRNA组EMSCs增殖能力受到抑制;划痕试验法检测MAGE-D1 siRNA组细胞48 h迁移量高于对照组细胞;Real Time-PCR检测Mage-D1、β-catenin和E-cadherin表达量,MAGE-D1 siRNA组EMSCs MAGE-D1和E-cadherin表达较对照组低,而β-catenin表达较高,Western blot检测MAGE-D1、β-catenin和E-cadherin表达水平趋势与Real Time-PCR结果相一致。结论抑制EMSCs中MAGE-D1的表达上调β-catenin,下调E-cadherin,能够提高细胞的迁移能力,但是其导致增殖能力降低,MAGE-D1可能作为EMSCs迁移增殖能力的关键因子。Objective To study the effects of MAGE-D1 on the proliferation and migration of ectomesenchymal stem cells originated from rat teeth germ. Methods Ectomesenchymal stem cells （EMSCs） were cultures by tissue mass technique.The mesenchymal membrane surface molecular CD14,CD29,CD44,CD45,CD90,CD105,CD146,CD166 and p75NTR of ectomesenchymal stem cells were detected.This study was divided into non-treatment EMSCs group （Control） and MAGE-D1 siRNA group （MAGE siRNA-EMSCs）.Cell proliferation was detected by CCK8 assay.The migration ability of EMSCs was analyzed by scratch wound healing assay.Real-time PCR and western blot assay was applied to detect the mRNA and protein level of migration and WNT related genes MAGE-D1,β-catenin and E-cadherin. Results EMSCs were successfully cultured by tissue mass technique.The cell membrane molecular expression were CD14 （92.16%）,CD29 （94.53%）,CD44 （ 99.51% ）,CD90 （95.18%）,CD105 （34.22%）,CD146 （92.39%）,CD166 （98.15%）,p75NTR（ 88.56% ）and CD45（0.5%）in teeth germ originated EMSCs.The proliferation ability in MAGE siRNA-EMSCs group was lower than that in control group.A higher wound-healing rate was shown in MAGE siRNA-EMSCs group compared to control group.Lower expressions of MAGE-D1 and E-cadherin in MAGE siRNA-EMSCs group and the higher expression of β-catenin were indicated. Conclusion The inhibition of MAGE-D1 expression leads to upregulating β-catenin and downregulating E-cadherin and further promoting the migration rate of EMSCs,but EMSCs proliferation ability is decreased,which suggesting that MAGE-D1 might be the key factor of proliferation and migration in EMSCs.

关 键 词：黑色素瘤相关抗原D1 外胚间充质干细胞 增殖 迁移 WNT信号通路 

分 类 号：R781[医药卫生—口腔医学]