MicroRNA-125b对人牙周膜干细胞成骨分化的影响

doi:10.19439/j.sjos.2018.01.003

摘要/Abstract

摘要： 目的: 探讨microRNA-125b（miR-125b）对人牙周膜干细胞（periodontal ligament stem cell, PDLSCs）成骨分化的影响。方法: 利用组织块法分离培养PDLSCs,流式细胞术检测细胞表面标志分子。PDLSCs经miR-125b模拟物或miR-125b抑制剂转染后,采用MTT法和LDH试剂盒分别检测细胞活性和细胞毒性,碱性磷酸酶（alkaline phosphatase,ALP）活性和细胞内钙离子水平均以相应试剂盒检测,Western 印迹法和RT-qPCR检测成骨相关基因水平。双荧光素酶基因报告实验检测miR-125b对细胞间隙连接蛋白43（connexin43,Cx43）的靶向调控作用。采用SPSS17.0软件包对数据进行统计学分析。结果: 体外分离培养的PDLSCs呈现间充质干细胞特性。转染miR-125b mimic后,PDLSCs的活性下降,毒性上升,ALP活性、钙离子水平以及成骨相关分子水平均显著下降;相反,anti-miR125b提高了PDLSCs的细胞活性、ALP活性、钙离子水平以及成骨相关分子的表达。双荧光素酶基因报告实验结果表明,miR-125b能够靶向降低Cx43基因水平。而在Cx43过表达的PDLSCs中,miR-125b模拟物对PDLSCs成骨分化的作用被反转。结论: miR-125b通过靶向下调Cx43而抑制PDLSCs的成骨分化。

关键词: 牙周膜干细胞, microRNA-125b, 细胞间隙连接蛋白43

Abstract: PURPOSE: To investigate the effect of microRNA-125b (miR-125b) on osteogenic differentiation of periodontal ligament stem cell (PDLSCs). METHODS: The surface factor of isolated PDLSCs was detected by flow cytometry. After transfected with miR-125b or anti-miR125b in PDLSCs, MTT and LDH were used to detect cell viability and cytotoxicity; ALP activity and calcium level were detected by ALP assay kit and calcium colorimetric assay kit. Western blot and RT-qPCR were used to determine the expression of osteogenesis-related genes. The interaction between miR-125b and connexin 43 (Cx43) was detected by dual luciferase gene reporter assay. The data were analyzed with SPSS 17.0 software. RESULTS: The cultured PDLSCs showed the characteristics of mesenchymal stem cells. After transfected with miR-125b in PDLSCs, the cell viability was decreased, cytotoxicity was increased; ALP activity, calcium level and the expression of osteogenesis-related genes were significantly decreased. On the contrary, cell viability, ALP activity, calcium level and the expression of osteogenesis-related genes in PDLSCs were increased after anti-miR-125b transfection. Dual luciferase gene reporter assay showed that miR-125b could target Cx43. In addition, the effect of miR-125b on osteogenic differentiation of PDLSCs was reversed after Cx43 overexpression. CONCLUSIONS: miR-125b may inhibit osteogenic differentiation of PDLSCs by targeting Cx43.

Key words: Periodontal ligament stem cell, MicroRNA-125b, Connexin43

中图分类号:

R781.4

杜莉, 曹伟靖, 田莹, 王原明. MicroRNA-125b对人牙周膜干细胞成骨分化的影响[J]. 上海口腔医学, 2018, 27(1): 11-17.

DU Li, CAO Wei-jing, TIAN Ying, WANG Yuan-ming. MicroRNA-125b regulates osteogenic differentiation of human periodontal ligament stem cells[J]. Shanghai Journal of Stomatology, 2018, 27(1): 11-17.

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