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- 2018
过表达DDIT3对人牙髓干细胞增殖和分化能力影响的研究
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Abstract:
摘要 目的:通过构建DDIT3过表达质粒,进一步验证过表达DDIT3对人牙髓干细胞(human dental pulp cells,HDPCs)增殖和分化能力的影响。方法:构建带有绿色荧光标记的DDIT3慢病毒载体,分为3组:空白(A组)对照组,绿色荧光对照组(B组),DDIT3过表达组(C组);荧光显微镜观察病毒感染效率;qRT-PCR和Westernblot检测过表达效果;MTT检测三组细胞的增殖能力;ALP、茜素红、vonKossa染色和qRT-PCR检测DDIT3对HDPCs成骨/成牙本质分化能力的影响。结果:荧光显微镜下观察C组绿色荧光表达面积＞90%,qRT-PCR和Westernblot结果表明,C组DDIT3mRNA和蛋白表达明显升高;MTT结果显示,与对照组相比,过表达DDIT3在72 h和96 h可明显抑制HDPCs的增殖;qRT-PCR结果表明,DDIT3过表达可明显增加矿化相关基因OSX,DSPP,DMP1和OCNmRNA表达水平,同时增加DSPP蛋白表达;ALP染色结果表明,3组细胞在成骨/成牙本质分化第7天,均可见明显的ALP染色阳性细胞,但染色面积无明显差异。然而,在成骨/成牙本质分化第14天,茜素红染色和vonKossa染色结果表明,过表达DDIT3可明显增加钙沉积。结论:DDIT3可促进HDPCs晚期的成骨/成牙本质分化
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