脉冲电磁场对小鼠骨髓间充质干细胞体外成骨分化的影响

Effects of pulsed electromagnetic fields on the differentiation of mouse bone marrow mesenchymal stem cells into osteoblasts in vitro

背景:间充质干细胞的增殖分化缺乏可调控性,结合适当载体后不能高效增殖分化是阻碍其进入临床的瓶颈。脉冲电磁场若能有效调控干细胞向骨源细胞分化将具有重要的临床价值。目的:观察脉冲电磁场刺激后,小鼠骨髓间充质干细胞体外成骨分化情况。设计、时间及地点:细胞学体外对照观察,于2004-05/2007-10在华中科技大学同济医学院附属普爱医院骨科实验室完成。材料:BALB/C小鼠20只,由同济医学院实验动物中心提供。脉冲电磁场发生器由海军工程大学电机系设计与制造。方法:无菌分离小鼠双侧股骨,Percoll密度梯度离心法分离骨髓间充质干细胞,再用贴壁筛选法纯化扩增。取生长良好的第3代细胞,调整细胞密度为2×107L-1接种于6孔板,分成4组:空白对照组加入完全培养基组;脉冲电磁场组给予50Hz、正弦波形、强度1mT的脉冲电磁场辐射,2次/d,30min/次,间隔12h,共10d;成骨诱导组加入含地塞米松、甘油磷酸钠、VitC的完全诱导培养基;脉冲电磁场+成骨诱导组给予相同的脉冲电磁场辐射后加入完全诱导培养基。主要观察指标:骨髓间充质干细胞的分化情况。结果:碱性磷酸酶染色结果显示,干预10d后,空白对照组为阴性;脉冲电磁场组呈弱阳性;成骨诱导组呈阳性;脉冲电磁场+成骨诱导组呈强阳性。与空白对照组比较,成骨诱导组、脉冲电磁场+成骨诱导组Ⅰ型胶原免疫组化染色吸光度值均明显升高(P<0.05,P<0.01)。结论:50Hz、正弦波形、强度1mT脉冲电磁场干预一定时间后,能够增强小鼠骨髓间充质干细胞碱性磷酸酶及Ⅰ型胶原的表达,促进其分化成骨。

BACKGROUND：The proliferation and differentiation of mesenchymal stem cells （MSCs） lack of regulatory functions.Following combining with suitable vectors,MSCs cannot highly effectively proliferate and differentiate,which are keys to prevent MSCs entering the clinic.It is of great importance to effectively regulate the differentiation of stem cells into osteoblasts using pulse electromagnetic field.OBJECTIVE：To investigate the differentiation of mouse MSCs into osteoblasts in vitro following stimulation of pulse electromagnetic field.DESIGN,TIME AND SETTING：The cytological in vitro controlled study was conducted at the Laboratory of Department of Orthopaedics,Puai Hospital of Tongji Medical College,Huazhong University of Science and Technology from May 2004 to October 2007.MATEIRALS：Totally 20 BALB/C mice were supplied by the Experimental Animal Center of Tongji Medical College.Pulse electromagnetic field deviser was designed and made by the Department of Electric Machine,Naval University of Engineering.METHODS：Mouse bilateral femur was sterilely isolated.BMSCs were harvested by the Percoll density gradient centrifugation,and purified and proliferated by the adherent method.Cells at the third passage （2×10^7/L） were incubated in a 6-well plate,and then divided into 4 groups.Cells in the blank control group were incubated in the complete medium.Cells in the pulse electromagnetic field underwent pulse electromagnetic field radiation of 50 Hz,sinusoidal wave,and 1 mT,twice a day,once 30 minutes,with an interval of 12 hours,totally 10 days.Cells in the osteogenic induction group were incubated in the complete medium,supplemented with dexamethasone,sodium glycerophosphate and VitC.Cells in the pulse electromagnetic field ＋ osteogenic induction group were subjected to the same pulse electromagnetic field radiation and then incubated in the complete medium.MAIN OUTCOME MEASURES：The differentiation of BMSCs was measured.RESULTS：Results of alkaline phosphatase staining showed that cells were negative in the blank control group,but weakly positive in the pulse electromagnetic field group,positive in the osteogenic induction group,and strongly positive in the pulse electromagnetic field ＋ osteogenic induction group 10 days following intervention.Compared with the blank control group,absorbance value of type I collagen immunohistochemistry was significantly greater in the osteogenic induction group,pulse electromagnetic field ＋ osteogenic induction group （P〈0.05,P 〈0.01）.CONCLUSION：Pulsed electromagnetism fields of 50 Hz,waves of sine,with the intensity of 1 mT could promote alkaline phosphatase and type I collagen expression and enhance the differentiation of mouse BMSCs into osteoblasts in vitro.