CAJ | 학술논문

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1×105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(Western blot)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A2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B1组效用明显,并且以晚期成骨指标OPN的效应明显.Westem Blot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.
목적 탐구15Hz정현파전자장체외유도대서골수간충질간세포(BMSCs)성골방향분화적최가시간창.방법 체외분리배양SD대서BMSCs,장장세량호적제삼대세포,안조1×105개/공접충세포우3.5cm세포배양명중,접충후제2천장소유배양명편호.안수궤수자표법수궤분위대조조화폭자조,재안폭자총천수분위A조(7d)、B조(14d)、C조(28d),A조포괄A0조(대조조)、A1조(폭자매일1h)、A2조(폭자매일4h)、A3조(폭자매일8h)；B조포괄B0조(대조조)、B1조(폭자매일1h)、B2조(폭자매일4h)、B3조(폭자매일8h)；C조포괄C0조(대조조)、C1조(폭자매일1h)、C2조(폭자매일4h)、C3조(폭자매일8h).매비차세포취자동일지로서,폭자조치우15Hz1mT정현파전자장중폭자.용형광정량PCR방법검측각조Runt상관전록인자2(RUNX2)、골타액산단백(BSP)、골교단백(OPN)기인표체량적변화；용천소홍염색방법비교각조개결절분포；용단백인적법(Western blot)검측비교각조RUNX2단백량적변화.결과 15Hz정현파전자장자격7d가이체외유도대서BMSCs향성골방향분화,이A2조적효응최위명현,병차이조기성골지표RUNX2적효응최위돌출；자장자격14d후,칙이B1조효용명현,병차이만기성골지표OPN적효응명현.Westem Blot비교각조RUNX2단백량적변화,추세상근.전자장자격14d화28d후,이B1조화C1조적개결절량최다.결론 15Hz정현파전자장유도BMSCs성골분화유명현적시간창구효응；수착폭자천수적증가,매천단시간(1h)폭자즉가체도교호적유도효과.
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,80-120g).The BMSCs (from passage 3) were exposed 0,1,4 or 8h/d for 7d,14,or 28d,respectively,to 15Hz sinusoidal electromagnetic fields with a maximum amplitude of lmT.Those exposed 0h/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 14d.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 the15 Hz sinusoidal electromagnetic fields,most obviously manifested by an outstanding increase of the early osteogenic index RUNX2 in those exposed 4h/d.After 14 days of intervention,the 1h/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 1h/d for 14 and 28days,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 (1h) every day.