摘要
目的:探讨micro RNA-125b(mi R-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mi R-125b模拟物或mi R-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-q PCR检测成骨相关基因水平。双荧光素酶基因报告实验检测mi R-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mi R-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,antimi R125b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,mi R-125b能够靶向降低Cx43基因水平。而在Cx43过表达的PDLSCs中,mi R-125b模拟物对PDLSCs成骨分化的作用被反转。结论:mi R-125b通过靶向下调Cx43而抑制PDLSCs的成骨分化。
PURPOSE： To investigate the effect of micro RNA-125 b（mi R-125 b） on osteogenic differentiation of periodontal ligament stem cell（PDLSCs）. METHODS： The surface factor of isolated PDLSCs was detected by flow cytometry. After transfected with mi R-125 b or anti-mi R125 b 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-q PCR were used to determine the expression of osteogenesis-related genes. The interaction between mi R-125 b and connexin 43（Cx43） was detected by dual luciferase gene reporter assay. The data were analyzed with SPSS17.0 software. RESULTS： The cultured PDLSCs showed the characteristics of mesenchymal stem cells. After transfected with mi R-125 b 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-mi R-125 b transfection. Dual luciferase gene reporter assay showed that mi R-125 b could target Cx43. In addition, the effect of mi R-125 b on osteogenic differentiation of PDLSCs was reversed after Cx43 overexpression. CONCLUSIONS： mi R-125 b may inhibit osteogenic differentiation of PDLSCs by targeting Cx43.
出处
《上海口腔医学》
CAS
CSCD
北大核心
2018年第1期11-17,共7页
Shanghai Journal of Stomatology
