摘要
目的:通过过表达或抑制microRNA-675-5p(miR-675-5p),观察miR-675-5p对小鼠成牙骨质细胞OCCM-30分化的影响。方法:用矿化诱导培养基诱导OCCM-30细胞分化,检测各矿化指标及miR-675-5p的含量变化。而后利用Lipofectamine 3000脂质体分别将miR-675-5p的mimic和inhibitor及对应的negative control转染进OCCM-30细胞,采用real-time PCR技术验证转染是否成功,继而采用real-time PCR和Western Blot的方法检测矿化相关指标ALP,OSX,RUNX2,BSP的含量变化,利用ALP活性试剂盒检测碱性磷酸酶活性变化。结果:Real-time PCR结果显示在OCCM-30细胞分化过程中miR-675-5p表达下调,在mimic组miR-675-5p表达上调明显,同时ALP,OSX,RUNX2表达下降,Western Blot证明OSX,RUNX2与BSP在蛋白水平上表达同样明显降低,ALP活性试剂盒检测提示ALP活性被抑制。Inhibitor组结果与mimic组恰好相反。结论:miR-675-5p抑制OCCM-30细胞的分化能力。
Objective:To investigate the effects of microRNA-675-5 p(miR-675-5 p)on cementoblast differentiation.Methods:The expression of miR-675-5 p was tested by real-time PCR during cementoblast differentiation of murine cementoblast cell line OCCM-30.Cells were then transfected with miR-675-5 p mimics/inhibitors or the mimic/inhibitor negative control using Lipofectamine 3000 Reagent.Real-time PCR was used to examine the expression level of miR-675-5 p after the transfection.The protein levels of RUNX2,OSX and BSP were determined by Western Blot.In addition,a kit of ALP activity assay was used to determine the activity of alkaline phosphatase.Results:The expression of miR-675-5 p was decreased during the cementoblast differentiation of OCCM-30 cells.Over-expression of miR-675-5 p in the mimic group decreased the protein levels of RUNX2,OSX and BSP and inhibited ALP activity.Besides,knock-down of miR-675-5 p increased the protein levels of RUNX2,OSX and BSP and promoted ALP activity.Conclusion:miR-675-5 p inhibits the ability of cementoblast differ-entiation and mineralization.
作者
郝云茹
王云龙
曹正国
贺红
HAO Yunru;WANG Yunlong;CAO Zhengguo;HE Hong(The State Key Laboratory Breeding Base of Basic Science of Stomatology(Hubei?MOST)&Key Laboratory of Oral Biomedicine Ministry of Education,Wuhan 430079,Hubei,China;Dept.of Orthodontics,School&Hospital of Stomatology,Wuhan University,Wuhan 430079,Hubei,China;Dept.of Periodontology,School&Hospital of Stomatology,Wuhan University,Wuhan 430079,Hubei,China)
出处
《武汉大学学报（医学版）》
CAS
2019年第3期395-399,共5页
Medical Journal of Wuhan University
基金
国家自然科学基金资助项目(编号:81671020)
