不同处理时间50Hz 3.6mT正弦交变磁场对人脐带干细胞增殖与成骨性分化的研究

Effects of different durations of 50 Hz 3.6 mT sinusoidal electromagnetic field on proliferation and differentiation of human umbilical cord stem cells in vitro

目的：研究50 Hz 3.6 mT不同处理时间的正弦交变电磁场（Sinusoidal electromagnetic field,SEMFs）对体外培养人脐带干细胞（Human umbilical cord stem cells,HUCSC）增殖与成骨性分化的影响。方法：体外分离培养HUCSC,传代后随机分为6组。用频率50 Hz,3.6 mT的SEMFs分别每天处理HUCSC 0.0（对照组）、0.5、1.0、1.5、2.0 h和2.5 h,倒置相差显微镜观察细胞形态,MTT法测定细胞增殖,在磁场处理后的15 d和17 d分别用茜素红和vonkossa对钙化结节进行染色,在磁场处理后的第4天和第6天PCR检测胶原Ⅰ（Collagen-Ⅰ）和骨形态发生蛋白（Bone morphogenetic protein-2,BMP-2）mRNA表达量的变化,在磁场处理后的10、12、14 d和16 d测定ALP活性。结果：1.0、1.5、2.0 h和2.5 h组促进HUCSC增殖;磁场处理后的7~9 d细胞出现钙化结节;在SEMFs处理后的第14天和第16天0.5 h组和1.0 h组碱性磷酸酶（Alkaline phosphatase,ALP）活性显著高于对照;SEMFs处理组能增加HUCSC钙化面积,其中尤以0.5 h和1.0 h最为明显;SEMFs能增加Collagen-Ⅰ和BMP-2mRNA表达量,其中尤以0.5 h和1.0 h组作用最为明显。结论：50 Hz,3.6 mT 1.0、1.5、2.0 h和2.5 h促进HUCSC增殖,同时SEMFs组能促进体外培养HUCSC成骨性分化,尤以处理0.5 h和1.0 h促进成骨性分化最为明显。

Objective：To investigate the effects of different durations of 50 Hz 3.6 mT sinusoidal electromagnetic fields（SEMFs） on the proliferation and differentiation of human umbilical cord stem cells（HUCSCs） in vitro.Methods：The HUCSCs were isolated from the neonate umbilical cord and randomly divided into 6 groups after one passage.The treatment groups were under 50 Hz 3.6 mT SEMFs respectively for 0.5,1.0,1.5,2.0 h,and 2.5 h each day,while the control group underwent no SEMFs treatment.All groups were observed under the contrast phase microscope each day.The proliferation was tested by MTT.At 15 d and 17 d after the SEMFs treatment the calcified nodules were stained by alizarin red and vonkossa respectively.At 4 d and 6 d after the SEMFs treatment the Collagen-Ⅰand BMP-2 mRNA were tested by real time PCR.At 10,12,14 d,and 16 d after the SEMFs treatment the ALP activity was tested.Results：the SEMFs were arranged in spiral appearance 7~9 d after the treatment.The SEMFs promoted the proliferation of HUCSCs,except in 0.5 h group.The ALP activity in 0.5 h and 1.0 h groups significantly increased at 14 d and 16 d after the SEMFs treatment.The calcified nodules number in 0.5 h and 1.0 h groups significantly increased after the SEMFs treatment.The Collagen-Ⅰand BMP-2 mRNA expression in 0.5 h and 1.0 h groups significantly increased after the SEMFs treatment.Conclusion：1.0,1.5,2.0 h,and 2.5 h treatment of 50 Hz 3.6 mT SEMFs promoted the proliferation of HUCSC,0.5 h and 1.0 h treatment of 50 Hz 3.6 mT SEMFs significantly enhanced the osteogenesis differentiation of human umbilical cord.