低强度脉冲超声促进MC3T3-E1成骨分化的机制研究  

Mechanism of low intensity pulsed ultrasound promoting osteogenic differentiation of MC3T3-E1 cells

作　　者：易雪婷 张俊[1] YI Xueting;ZHANG Jun(Department of Ultrasound,the Affiliated Suzhou Hospital of Nanjing Medical University,Suzhou Municipal Hospital,Gusu School,Nanjing Medical University,Nanjing 215008,Jiangsu,China)

机构地区：[1]南京医科大学姑苏学院,苏州市立医院,南京医科大学附属苏州医院超声科,江苏苏州215008

出　　处：《生物医学工程与临床》2021年第6期663-668,共6页Biomedical Engineering and Clinical Medicine

基　　金：苏州市“科教兴卫”项目(KJXW2018034);南京医科大学校级课题(NMUB2018228)。

摘　　要：目的探讨低强度脉冲超声(LIPUS)促进成骨细胞MC3T3-E1成骨分化的机制研究。方法采用LIPUS辐照小鼠MC3T3-E1成骨细胞系(30 mW/cm^(2)、1.5 MHz、25 min/d)。在光学显微镜下观察24 h后成骨细胞形态,通过镁离子探针Mag-Fluo-4 AM染色,实时观察LIPUS干预对成骨细胞镁离子内流的影响。通过碱性磷酸酶(ALP)染色及茜素红染色检测LIPUS对成骨细胞矿化能力的影响。通过实时定量-聚合酶链式反应(PCR)检测LIPUS对ALP和RUNT相关转录因子2(RUNX2)相对表达量的影响。结果经过LIPUS辐照25 min/d后,小鼠成骨细胞胞浆饱满,细胞间彼此连接增多,细胞密度相比对照组提高。相比对照组,LIPUS处理后小鼠成骨细胞中镁离子探针阳性细胞面积增加[(16.81±3.70)%vs(4.94±1.85)%],差异具有显著统计学意义(P<0.01)。相比只使用成骨诱导液的对照组,连续LIPUS刺激的实验组显著增加了ALP阳性细胞面积[(51.80±3.60)%vs(11.38±2.33)%],差异具有显著统计学意义(P<0.01)。此外,与对照组相比,LIPUS刺激显著增加了矿化结节阳性的细胞面积[(39.24±7.57)%vs(1.54±0.41)%],差异具有显著统计学意义(P<0.01)。表明LIPUS干预可以显著增强成骨细胞ALP表达及矿化能力。实时定量-PCR结果显示,LIPUS刺激后,成骨细胞ALP表达增加(2.79±0.48)倍,相比阴性对照组显著增加(P<0.01);LIPUS组RUNX2表达增加(18.22±1.35)倍,相比阴性对照组显著增加(P<0.01)。结论LIPUS促进镁离子内流,上调成骨相关标志物ALP、RUNX2表达量,从而促进成骨细胞矿化及钙沉积。Objective To explore promotion mechanism of low intensity pulsed ultrasound(LIPUS)in osteogenic differentiation of osteoblast MC3T3-E1.Methods The murine osteoblast MC3T3-E1 cells were exposed to LIPUS with frequency of 1.5 MHz,power intensity of 30 mW/cm^(2)for 25 minutes per day.The morphology and proliferation of osteoblasts was detected using light microscope after 24 hours,and the effect of LIPUS treatment on magnesium influx of osteoblasts was observed by Mag-Fluo-4 AM staining.The alkaline phosphatase(ALP)staining and alizarin red staining were employed to evaluate biomineralization.The expression level of ALP and RUNT-related transcription factor 2(RUNX2)were examined by quantitative real-time polymerase chain reaction(qRT-PCR).Results After 25 min/d treatment of LIPUS,the cytoplasm of osteoblasts was plump,the interaction between cells was increased,and cell density was higher than that of control group.Compared with control group,the area of magnesium probe-positive cells in mouse osteoblasts after LIPUS treatment was increased[(16.81±3.70)%vs(4.94±1.85)%],and the difference was statistically significant(P<0.01).Compared with control group using only osteoinductive fluid,the area of ALP-positive cells in experimental group which stimulated by continuous LIPUS significantly increased[(51.80±3.60)%vs(11.38±2.33)%],and the difference was statistically significant(P<0.01).Compared with the control group,LIPUS stimulation significantly increased area of positive calcium nodules[(39.24±7.57)%vs(1.54±0.41)%],and the difference was statistically significant(P<0.01),which showed that LIPUS intervention significantly enhance the ALP expression and mineralization ability of osteoblast.qRT-PCR results showed that after LIPUS stimulation,the expression of ALP in osteoblasts increased(2.79±0.48)times,which was significantly higher than that of control group(P<0.01).Expression of RUNX2 in LIPUS group increased(18.22±1.35)times,which was significantly higher than that of control group(P<0.01).Conclusion It is d

关 键 词：低强度脉冲超声(LIPUS) 镁离子(Mg^(2+)) RUNT相关转录因子2(RUNX2) 碱性磷酸酶(ALP) 

分 类 号：R318.03[医药卫生—生物医学工程]