脉冲电磁场对小鼠骨髓间充质干细胞增殖的影响(英文)

Effects of pulsed electromagnetic fields on the proliferation of bone marrow mesenchymal stem cells

背景:脉冲电磁场作为一种非侵入性疗法治疗骨不连及其他的骨科疾病已被证实有确切的临床疗效。但由于认识的局限性,骨髓间充质干细胞在脉冲电磁场治疗骨折中的作用未被充分重视。目的:观察小鼠骨髓间充质干细胞在50Hz、正弦波形、不同强度、不同作用时间脉冲电磁场干预下的增殖情况,以及其细胞周期变化,以寻求最佳干预窗口。设计:单一样本、区组设计,观察对比体外细胞培养实验。单位:华中科技大学同济医学院附属普爱医院骨科。材料:选用BALB/C小鼠20只,鼠龄4～5周,体质量18～22g,雌雄不拘,由同济医学院实验动物中心提供。此动物实验符合动物伦理学要求。脉冲电磁场发生器(海军工程大学电机系设计与制造)。方法:实验于2005-02/12在华中科技大学同济医学院附属普爱医院骨外科实验室完成。用密度梯度离心法分离小鼠骨髓间充质干细胞,再用贴壁筛选法筛选,对生长良好的第3代的小鼠骨髓间充质干细胞分别给予50Hz、强度分别为4,3,2,1mT的正弦波形脉冲电磁场,2次/d,每次30min,间隔12h,以不加电磁场干预的干细胞为对照组。主要观察指标:在照射第24,48,72h后采用MTT法测定骨髓间充质干细胞增殖水平;采用流式细胞仪分析细胞周期,以增殖指数(prolifera-tionindex,PI)表示脉冲电磁场对小鼠骨髓间充质干细胞分裂增殖的影响。结果:脉冲电磁场对小鼠骨髓间充质干细胞增殖的影响:脉冲电磁场照射24,48h后各干预组与对照组比较,差异无显著性意义(P>0.05)。脉冲电磁场照射72h后各干预组骨髓间充质干细胞增殖数目多于对照组,差异有显著性意义(P<0.05～0.01),其中以1mT强度照射最为明显(P<0.01)。脉冲电磁场对小鼠骨髓间充质干细胞细胞周期的影响:在检测的所有细胞样本中均未发现有DNA倍体异常的细胞(diploid:100%)。骨髓间充质干细胞经过72h的强度为1,2,3,4mT的脉冲电磁场干预后PI值高于对照组,差异有显著性意义(P<0.05～0.01),其中以1mT最为明显(P<0.01)。结论:50Hz、正弦波形、强度为1mT的脉冲电磁场照射72h能明显促进体外小鼠骨髓间充质干细胞增殖,是最佳干预窗口。

BACKGROUND： As a non-invasive therapy, pulsed electromagnetism fields （PEMFs） have been confirmed to have definite clinical efficacy on bone nonunion and other orthopaedic diseases. However, the action of bone marrow mesenchymal stem cells （MSCs） in the treatment of fracture by PEMFs is always neglected due to the restriction of previous knowledge. OB3ECTIVE： To observe the proliferation and changes of cell cycle of MSCs in mice under PEMFs of 50 Hz, sine waves, at different intensity and different time, so as to find out the optimal window for intervention. DESIGN ： A single-sample, grouped design, comparative observation of cell culture in vitro. SETTING： Department of Orthopaedics, Puai Hospital of Tongji Medical College, Huazhong University of Science and Technology. MATERIALS： Twenty BALB/C mice of 4-5 weeks old, weighing 18-22 g, either male or female, clean degree, were provided by the experimental animal center of Tongji Medical College [No. SYXK（E）2004-0028]. The animal experiment met the requirements of animal ethics. Pulse magnetic field generator was designed and manufactured by the Department of Electrical Engineering, Naval University of Engineering. METHODS： The experiments were carried out in the orthopaedic laboratory of Puai Hospital of Tongji Medical College, Huazhong University of Science and Technology from January to June in 2004. Mice MSCs were isolated using the density gradient centrifugation, then screened by means of adhesion, and the mice MSCs of the 3^rd passage that grew well were treated with 50 Hz sine-wave PEMFs of 4, 3, 2 and 1 mT respectively, twice a day, 30 minutes for each time, with an interval of 12 hours. The stem cells untreated with PEMFs were taken as the control group. MAIN OUTCOME MEASURES： At 24, 48 and 72 hours after irradiation, the level of MSCs proliferation was detected with methyl-thiazol-tetrazolium （Ml-r） assay, cell cycle was analyzed with flow cytometer, and the influence of PEMFs on the proliferation of mice MSCs was expressed as proliferation index （PI）. RESULTS： Effect of PEMFs on the proliferation of mice MSCs： There were no significant differences after irradiation for 24 and 48 hours between the PEMFs intervention groups and control group （P 〉 0.05）. The proliferated MSCs after irradiation for 72 hours in the PEMFs intervention groups were significantly more than those in the control group （P 〈 0.05-0.01）, and which was the most obvious by PEMFs of 1 mT （P 〈 0.01）. Effect of PEMFs on the cell cycle of mice MSCs： No cell with abnormal DNA ploidy （diploid： 100%） was observed in all the samples. The PI values after PEMFs intervention of 1, 2, 3 and 4 mT for 72 hours were significantly higher than that in the control group （P 〈 0.05-0.01）, which was the most obvious by PEMFs of 1 mT （P 〈 0.01）. CONCLUSION ： Pulsed electromagnetism fields of 50 Hz, sine waves and intensity of 1 mT for 72 hours could obviously promote the proliferation of mice MSCs in vitro, this being the optimal window for intervention.