电磁场刺激对骨髓间充质干细胞即刻早期基因表达的影响

Effects of electromagnetic field on the expression of immediate-early genes in bone marrow mesenchymal stem cells

背景:细胞受刺激后即刻早期基因在细胞核内快速表达,其编码的核蛋白作为反式作用因子,通过激活某些基因的转录而调控细胞的增殖、分化等生物学行为。目的:观察电磁场刺激对大鼠骨髓间充质干细胞c-fos、c-myc和c-jun即刻早期基因表达的影响。设计、时间及地点:电刺激干预的体外细胞学观察,于2006-10/2007-01在同济医院矫形外科实验室完成。材料:清洁级健康Sprague-Dawley大鼠6只,由华中科技大学同济医学院动物实验中心提供。电磁场发生器由海军工程大学电机系设计制造。方法:贴壁筛选法体外分离培养大鼠骨髓间充质干细胞,传至第4代按2×103个/孔接种,每孔均加入200μL含10%胎牛血清的DMEM-F12培养基,分为2组,对照组细胞置于普通培养箱中培养3d,暴磁组在培养箱中安装电磁场发生器,频率50Hz、强度1mT的正弦波,每次刺激30min,间隔11.5h,2次/d,共刺激5d。主要观察指标:MTT法检测细胞增殖活性,考马斯亮蓝法测定细胞碱性磷酸酶活性,RT-PCR检测即刻早期基因的表达。结果:电磁场刺激3d后,暴磁组MTT产物吸光度值明显高于对照组(P<0.01);电磁场刺激5d后,暴磁组细胞碱性磷酸酶活性明显高于对照组(P<0.01)。与对照组比较,暴磁组c-fos和c-myc基因表达均有不同水平的上调(P<0.01),c-jun基因无明显变化。结论:电磁场刺激可上调骨髓间充质干细胞中c-fos和c-myc即刻早期基因的表达,可能通过该途径来促进骨髓间充质干细胞的增殖活性,诱导其向成骨细胞分化。

BACKGROUND： The immediate-early genes may rapidly express in nucleus of the stimulated cells, its codogenic nucleoprotein serves as trans-acting factor to regulate the cells proliferation and differentiation through activating the transcription of some genes. OBJECTIVE： To study the effects of electromagnetic field on the expression of immediate-early genes, such as c-fos, c-myc and c-jun, in bone marrow mesenchymal stem cells. DESIGN, TIME AND SETTING： A cytological observation in vitro by electrostimulation was performed in the laboratory of Department of Orthopedics, Tongji Hospital from October 2006 to January 2007. MATERIALS： Six Sprague-Dawley rats of clean grade were offered by the Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology. Electromagnetic field generator was manufactured by Department of Electrical Engineering in Naval University of Engineering. METHODS： Adherence screening method was used to isolate and purify the rat bone marrow mesenchymal stem cells, and the fourth-passage cells were inoculated in 200 μL DMEM-F12 containing 10% fetal bovine serum at a density of 2×10^3 cells per well. Cells were divided into two groups. The exposure group was exposed to pulsed electromagnetic fields （50 Hz,1mT, sine wave form） for 5 days （30 minutes one time, twice a day, at an interval of 11.5 hours）, while the control group was cultured in the normal incubatoffor 3 days. MAIN OUTCOME MEASURES： The proliferation activity of cells was determined by methyl thiazolyl tetrazolium assay. The alkaline phosphatase activity of cells was determined according to the alkaline phosphatase kit. Semi-quantitative reverse transcription-polymerase chain reaction was used to detect the expression of immediate-early genes. RESULTS： Compared with control group, the expression of c-fos and c-myc were upregulated by exposure to electromagnetic field （P 〈 0.01） while c-jun had no significant change. The absorbance value of methyl thiazolyl tetrazolium product was promoted remarkably after exposure to electromagnetic field for 3 days （P 〈 0.01）. The activation of alkaline phosphatase in bone marrow mesenchymal stem ceils was promoted remarkably after exposure to electromagnetic field for 5 days （P 〈 0.01）. CONCLUSION： Electromagnetic field could upregulate the expression of immediate-early genes in bone marrow mesenchymal stem cells and this may play an important role in cell proliferation and differentiation.