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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: Pulsed electromagnetic fields as a non-invasive treatment of nonunion and other orthopedic diseases have been shown to have a clinically relevant effect. However, due to the limitations of understanding, the role of bone marrow mesenchymal stem cells in the treatment of fractures by pulsed electromagnetic fields has not been given enough attention. OBJECTIVE: To observe the proliferation of mouse bone marrow mesenchymal stem cells (BMSCs) under the condition of 50Hz, sinusoidal waveforms, different intensities and time-varying impulsive electromagnetic fields, as well as their cell cycle changes in order to find the best intervention window. Design: Single sample, block design, observation and comparison of in vitro cell culture experiments. Unit: Department of Orthopedics, Puai Hospital, Tongji Medical College, Huazhong University of Science and Technology. MATERIALS: Twenty BALB / C mice were selected, aged 4 to 5 weeks and body weight ranging from 18 to 22g. Both sexes were provided by Experimental Animal Center of Tongji Medical College. This animal experiment meets the ethical requirements of animals. Pulse electromagnetic field generator (Naval Engineering University Department of Electrical Design and Manufacturing). METHODS: The experiment was performed in the Department of Orthopedic Surgery, Puai Hospital, Tongji Medical College, Huazhong University of Science and Technology from February to December 2005. Mice bone marrow mesenchymal stem cells were separated by density gradient centrifugation, and then screened by adherence screening method. The third generation of mouse bone marrow mesenchymal stem cells were given 50Hz respectively with intensities of 4, 3, 2, 1mT sine wave pulse electromagnetic field, 2 times / d, each time 30min, interval 12h, with no electromagnetic field intervention stem cells as the control group. MAIN OUTCOME MEASURES: Bone marrow mesenchymal stem cell proliferation was measured by MTT assay at 24, 48 and 72 hours after irradiation. Cell cycle was analyzed by flow cytometry. Proliferation index (PI) Effect of Bone Marrow Mesenchymal Stem Cells on Cell Proliferation. Results: The effect of pulsed electromagnetic field on the proliferation of mouse bone marrow mesenchymal stem cells: there was no significant difference between the intervention group and the control group 24 and 48 hours after pulsed electromagnetic field irradiation (P> 0.05). The number of proliferation of bone marrow mesenchymal stem cells in each intervention group was significantly higher than that in control group at 72h after irradiation with pulsed electromagnetic field (P <0.05 ~ 0.01), of which 1mT intensity was the most obvious (P <0.01). Effects of pulsed electromagnetic fields on the cell cycle of mouse bone marrow mesenchymal stem cells: No diploid cells (diploid: 100%) were found in all the cell samples tested. The PI value of bone marrow mesenchymal stem cells after being exposed to pulsed electromagnetic fields with intensity of 1, 2, 3 and 4 mT at 72 h was significantly higher than that of the control group (P <0.05 ~ 0.01), especially 1 mT (P < 0.01). CONCLUSION: The irradiation of 50Hz, sinusoidal waveform and intensity 1mT pulsed electromagnetic field for 72h can significantly promote the proliferation of mouse bone marrow mesenchymal stem cells in vitro, which is the best intervention window.