不同作用时间下正弦波电磁场对大鼠骨髓间充质干细胞成骨分化的效应

Effects of sinusoidai electromagnetic fields on the osteogenic differentiation of bone marrow mesenchymal stem cells

目的探究15Hz正弦波电磁场体外诱导大鼠骨髓间充质干细胞（BMSCs）成骨方向分化的最佳时间窗。方法体外分离培养SD大鼠BMSCs，将长势良好的第三代细胞，按照1×10^5个／孔接种细胞于3．5cm细胞培养皿中，接种后第2天将所有培养皿编号。按随机数字表法随机分为对照组和暴磁组，再按暴磁总天数分为A组（7d）、B组（14d）、C组（28d），A组包括A0组（对照组）、A1组（暴磁每日1h）、A2组（暴磁每日4h）、A3组（暴磁每日8h）；B组包括B0组（对照组）、B1组（暴磁每日1h）、B2组（暴磁每日4h）、B3组（暴磁每日8h）；C组包括C0组（对照组）、C1组（暴磁每日1h）、C2组（暴磁每日4h）、C3组（暴磁每日8h）。每批次细胞取自同一只老鼠，暴磁组置于15Hz1mT正弦波电磁场中暴磁。用荧光定量PCR方法检测各组Runt相关转录因子2（RUNX2）、骨唾液酸蛋白（BSP）、骨桥蛋白（OPN）基因表达量的变化；用茜素红染色方法比较各组钙结节分布；用蛋白印迹法（Westernblot）检测比较各组RUNX2蛋白量的变化。结果15Hz正弦波电磁场刺激7d可以体外诱导大鼠BMSCs向成骨方向分化，以A，组的效应最为明显，并且以早期成骨指标RUNX2的效应最为突出；磁场刺激14d后，则以B．组效用明显，并且以晚期成骨指标OPN的效应明显。WesternBlot比较各组RUNX2蛋白量的变化，趋势相近。电磁场刺激14d和28d后，以B1组和C1组的钙结节量最多。结论15Hz正弦波电磁场诱导BMSCs成骨分化有明显的时间窗口效应；随着暴磁天数的增加．每天短时间（1h）暴磁即可达到较好的诱导效果。

Objective To explore in vitro the best time window for using sinusoidal electromagnetic fields to promote the osteogenic differentiation of bone marrow mesenchymal stem cells （BMSCs）. Methods BMSCs were isolated and cultured from 4-week-old Sprague-Dawley rats （male and female, 80-120 g）. The BMSCs （from passage 3） were exposed 0, 1, 4 or 8 h/d for 7 d, 14, or 28 d, respectively, to 15 Hz sinusoidal electromagnetic fields with a maximum amplitude of lmT. Those exposed 0 h/d served as the control. The relative expressions of runt related gene-2 （RUNX2）, bone sialoprotein （BSP） and osteopontin （OPN） were determined using real-time, quantitative reverse transcription-polymerase chain reactions （RT-PCRs）. The level of RUNX2 protein was determined by Western blotting after 14 d. Alizarin red staining was used to compare calcium distribution in each group. Results Obvious promotion of differentiation to osteoblasts was observed after 7 days of exposure to thel5 Hz sinusoidal electromagnetic fields, most obviously manifested by an outstanding increase of the early osteogenic index RUNX2 in those exposed 4 h/d. After 14 days of intervention, the 1 b/d exposure showed to be most effective, especially in inducing the changes of the late osteogenic index OPN. The trends of changes in RUNX2 protein were similar in all groups. After stimulating 1 h/d for 14 and 28 days, calcium deposition increased to the greatest extent. Conclusions Exposure to sinusoidal electromagnetic fields induces osteogenic differentiation to osteoblasts in rat BMSCs in vitro. There is an apparent window effect. The best results are observed with more days of exposure and shorter exposure time （1 h） every day.