低幅高频机械振动波（LMHF）对人成纤维细胞影响的实验研究
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摘要
目的：为了探讨机械振动刺激对皮肤创面的影响，本研究欲建立正弦式低幅高频（Low-magnitude and high-frequency，LMHF）振动波体外研究的生物模型，研究机械振动波对人类皮肤成纤维细胞迁移、增殖、分化以及细胞外基质的影响，进而深入理解机械力对皮肤创面愈合影响的机制。
方法：①在以往的关于全身振动的研究基础上，利用特制的正弦式低幅高频振动器，对人类皮肤成纤维细胞进行体外振动，尝试不同的振动参数，观察振动后细胞的变化情况，分析不同条件的振动对成纤维细胞的不同影响，最终选择合适的振动参数作为后续研究的基础。根据前期振动研究的结果以及本研究观察到的现象，本研究最终选择的振动参数为：振动加速度a=0.4g，振动时间t=15分钟，振动频率分别为f=30、50、65、100Hz。②对人类成纤维细胞进行正弦式低幅高频振动处理后，利用细胞迁移划痕实验的方法，来检测振动处理后22小时细胞迁移的速率。③分别应用CellQuanti-Blue和BrdU细胞活性检测方法检测正弦式低幅高频振动波对人类成纤维细胞增殖能力的影响。④利用Real-time PCR技术检测经过正弦式低幅高频振动波长时间（3-5d）刺激之后的人类成纤维细胞内α-SMA,以及主要细胞外基质的mRNA基因表达水平，进而研究机械振动波对成纤维细胞的分化和细胞外基质分泌的影响。
结果：①在本实验的振动参数条件下，长时间（30min）的振动刺激会导致部分细胞发生坏死。镜下可见成纤维细胞突起数目减少，胞体由长梭形变为细长型或椭圆形，部分胞体内可以看到聚集的小空泡样改变，活细胞率下降。但是15min的振动刺激后，细胞的外形和活性跟对照组相比较，并没有明显的变化。②与对照组相比较，所有振动处理组内成纤维细胞的迁移能力均有明显降低（p<0.05)。并且这种降低的趋势与频率的增高成反比，即频率越高，细胞迁移的速度越慢，频率65Hz和100Hz的两组之间并没有差异性，但是从分析图表上我们可以发现仍呈下降趋势。同时我们还发现振动方向对细胞迁移的速率的影响不大。③与对照组相比，f=30Hz、50Hz、65Hz的振动刺激对细胞的增殖能力并没有显著的影响（p>0.05)，但是f=100Hz的振动会显著降低成纤维细胞的增殖能力。④对人类成纤维细胞进行长时间（3-5d）体外低幅高频振动刺激，成纤维细胞内的α-SMA有了增高的趋势，其中以f=50Hz振动组增高最明显，具有统计学意义。但是对细胞外基质蛋白的基因表达影响不明显。
结论：本研究在基于前人研究的基础上，首次建立了正弦式机械振动波对人类皮肤成纤维细胞影响的体外生物模型，为后续进一步开展广泛研究打下了基础。低幅高频的机械振动波会减缓皮肤成纤维细胞的迁移速率，这种降低的趋势与一定范围内频率的增高成反比（f=30-100Hz），但是不同方向的振动对细胞的迁移速度并没有明显的影响。正弦式机械振动波对人类成纤维细胞增值能力的影响与振动的条件有关，当振动频率小于100Hz时，机械振动波对细胞的增殖能力并没有明显的影响。但是在振动频率大于100Hz的时候，细胞的增殖能力会受到抑制。长时间低幅高频机械振动波对人类皮肤成纤维细胞向肌成纤维细胞的分化具有一定的促进作用，但是对细胞外基质蛋白的分泌则影响不明显。由于机械力振动对生物体的影响是一个非常复杂的过程，体内实验和体外实验之间存在非常大的差别，因此本研究的发现具有一定的局限性，需要更进一步的详细探索来逐渐认识机械振动波对皮肤创面的影响及其相关机制。
Objective: Low-Magnitude and High-Frequency (LMHF) vibration has becomeincreasingly popular over the last several years as a method of exercise or clinical treatment.However, the efficiency of LMHF applied on the cutaneous wound healing has not beenreported. The authors developed a novel in vitro model to investigate the effects of lowmagnitude and high frequency vibration on the migration, proliferation, differentiation andextracellular matrix of human cutaneous fibroblasts in order to understand the potentialapplication of mechanical vibration on wound healing.
Methods: The authors tested a vibration bioreactor customized by company to figure outthe suitable parameters of mechanical vibration for human fibroblasts in vitro study. Thevibration bioreactor could stimulate sinusoid mechanical vibration waves with lowmagnitude (a<1g) and high frequency (f=30-100Hz). Based on the previously study, humancutaneous fibroblasts were subjected to vibration treatment (f=30,50,65,100Hz; a=0.4×g;t=15minutes). The effect of low magnitude and high frequency on fibroblasts migrationwas investigated via in-vitro scratch assay which means a wound gap was created on themonolayer and healing percentage was measured after vibration. Cells proliferationchanging after vibration treatment was analyzed using CellQuanti-Blue Assay and BrdUassay. The mRNA level of α-SMA and several vital extracellular matrix protein weredetected by real-time polymerase chain reaction (PCR) after mechanical vibration.
Results: After exposure to sinusoid mechanical vibration for longer time (30min), part offibroblasts would be detached from bottom under microscope. Cellular morphologychanged from long spindle to short and round with the number of dendrites decreased. While, there were no significant difference of fibroblasts morphology and cellular viabilitybetween15min vibration group and control. Migration of human cutaneous fibroblast in allexperimental groups significantly (p<0.01) decreased compared with control group. Themigration speed tended to decrease with the frequency increasing but have no relationshipwith the direction of sinusoid vibration. The proliferation of fibroblasts stimulated afterexposure to low-magnitude and high frequency vibration (f=30Hz,50Hz,65Hz, a=0.4×g,t=15minutes) has no significant difference with control group, whereas f=100Hz vibrationconsiderably decreased cell proliferation (p<0.01). The mRNA expression of α-SMA hadbeen increased by3-5days vibration, which means mechanical vibration might bias thefibroblasts to differentiate into myofibroblasts. However, no significant differences ofextracellular matrix protein mRNA was observed between any experimental groups andcontrol.
Conclusions: We reported the effect of the LMHF vibration-wound healing in-vitro modelfor the first time. The present results showed that horizontal low magnitude and highfrequency vibration (f=30Hz,50Hz,65Hz, a=0.4×g, t=15min) could slow down the humancutaneous fibroblasts migration but haven’t significant effect on the cells proliferation.Long-term low magnitude and high frequency vibration tend to bias cutaneous fibroblastsdifferentiate into myofibroblasts while could not change the extracellular matrixarrangement. This study has certain limitation because the effects of mechanical vibrationon the biology field are so complicated and confusing. Further investigations should beperformed to discover the mechanism of the biological application of mechanical forces.
方法：①在以往的关于全身振动的研究基础上，利用特制的正弦式低幅高频振动器，对人类皮肤成纤维细胞进行体外振动，尝试不同的振动参数，观察振动后细胞的变化情况，分析不同条件的振动对成纤维细胞的不同影响，最终选择合适的振动参数作为后续研究的基础。根据前期振动研究的结果以及本研究观察到的现象，本研究最终选择的振动参数为：振动加速度a=0.4g，振动时间t=15分钟，振动频率分别为f=30、50、65、100Hz。②对人类成纤维细胞进行正弦式低幅高频振动处理后，利用细胞迁移划痕实验的方法，来检测振动处理后22小时细胞迁移的速率。③分别应用CellQuanti-Blue和BrdU细胞活性检测方法检测正弦式低幅高频振动波对人类成纤维细胞增殖能力的影响。④利用Real-time PCR技术检测经过正弦式低幅高频振动波长时间（3-5d）刺激之后的人类成纤维细胞内α-SMA,以及主要细胞外基质的mRNA基因表达水平，进而研究机械振动波对成纤维细胞的分化和细胞外基质分泌的影响。
结果：①在本实验的振动参数条件下，长时间（30min）的振动刺激会导致部分细胞发生坏死。镜下可见成纤维细胞突起数目减少，胞体由长梭形变为细长型或椭圆形，部分胞体内可以看到聚集的小空泡样改变，活细胞率下降。但是15min的振动刺激后，细胞的外形和活性跟对照组相比较，并没有明显的变化。②与对照组相比较，所有振动处理组内成纤维细胞的迁移能力均有明显降低（p<0.05)。并且这种降低的趋势与频率的增高成反比，即频率越高，细胞迁移的速度越慢，频率65Hz和100Hz的两组之间并没有差异性，但是从分析图表上我们可以发现仍呈下降趋势。同时我们还发现振动方向对细胞迁移的速率的影响不大。③与对照组相比，f=30Hz、50Hz、65Hz的振动刺激对细胞的增殖能力并没有显著的影响（p>0.05)，但是f=100Hz的振动会显著降低成纤维细胞的增殖能力。④对人类成纤维细胞进行长时间（3-5d）体外低幅高频振动刺激，成纤维细胞内的α-SMA有了增高的趋势，其中以f=50Hz振动组增高最明显，具有统计学意义。但是对细胞外基质蛋白的基因表达影响不明显。
结论：本研究在基于前人研究的基础上，首次建立了正弦式机械振动波对人类皮肤成纤维细胞影响的体外生物模型，为后续进一步开展广泛研究打下了基础。低幅高频的机械振动波会减缓皮肤成纤维细胞的迁移速率，这种降低的趋势与一定范围内频率的增高成反比（f=30-100Hz），但是不同方向的振动对细胞的迁移速度并没有明显的影响。正弦式机械振动波对人类成纤维细胞增值能力的影响与振动的条件有关，当振动频率小于100Hz时，机械振动波对细胞的增殖能力并没有明显的影响。但是在振动频率大于100Hz的时候，细胞的增殖能力会受到抑制。长时间低幅高频机械振动波对人类皮肤成纤维细胞向肌成纤维细胞的分化具有一定的促进作用，但是对细胞外基质蛋白的分泌则影响不明显。由于机械力振动对生物体的影响是一个非常复杂的过程，体内实验和体外实验之间存在非常大的差别，因此本研究的发现具有一定的局限性，需要更进一步的详细探索来逐渐认识机械振动波对皮肤创面的影响及其相关机制。
Objective: Low-Magnitude and High-Frequency (LMHF) vibration has becomeincreasingly popular over the last several years as a method of exercise or clinical treatment.However, the efficiency of LMHF applied on the cutaneous wound healing has not beenreported. The authors developed a novel in vitro model to investigate the effects of lowmagnitude and high frequency vibration on the migration, proliferation, differentiation andextracellular matrix of human cutaneous fibroblasts in order to understand the potentialapplication of mechanical vibration on wound healing.
Methods: The authors tested a vibration bioreactor customized by company to figure outthe suitable parameters of mechanical vibration for human fibroblasts in vitro study. Thevibration bioreactor could stimulate sinusoid mechanical vibration waves with lowmagnitude (a<1g) and high frequency (f=30-100Hz). Based on the previously study, humancutaneous fibroblasts were subjected to vibration treatment (f=30,50,65,100Hz; a=0.4×g;t=15minutes). The effect of low magnitude and high frequency on fibroblasts migrationwas investigated via in-vitro scratch assay which means a wound gap was created on themonolayer and healing percentage was measured after vibration. Cells proliferationchanging after vibration treatment was analyzed using CellQuanti-Blue Assay and BrdUassay. The mRNA level of α-SMA and several vital extracellular matrix protein weredetected by real-time polymerase chain reaction (PCR) after mechanical vibration.
Results: After exposure to sinusoid mechanical vibration for longer time (30min), part offibroblasts would be detached from bottom under microscope. Cellular morphologychanged from long spindle to short and round with the number of dendrites decreased. While, there were no significant difference of fibroblasts morphology and cellular viabilitybetween15min vibration group and control. Migration of human cutaneous fibroblast in allexperimental groups significantly (p<0.01) decreased compared with control group. Themigration speed tended to decrease with the frequency increasing but have no relationshipwith the direction of sinusoid vibration. The proliferation of fibroblasts stimulated afterexposure to low-magnitude and high frequency vibration (f=30Hz,50Hz,65Hz, a=0.4×g,t=15minutes) has no significant difference with control group, whereas f=100Hz vibrationconsiderably decreased cell proliferation (p<0.01). The mRNA expression of α-SMA hadbeen increased by3-5days vibration, which means mechanical vibration might bias thefibroblasts to differentiate into myofibroblasts. However, no significant differences ofextracellular matrix protein mRNA was observed between any experimental groups andcontrol.
Conclusions: We reported the effect of the LMHF vibration-wound healing in-vitro modelfor the first time. The present results showed that horizontal low magnitude and highfrequency vibration (f=30Hz,50Hz,65Hz, a=0.4×g, t=15min) could slow down the humancutaneous fibroblasts migration but haven’t significant effect on the cells proliferation.Long-term low magnitude and high frequency vibration tend to bias cutaneous fibroblastsdifferentiate into myofibroblasts while could not change the extracellular matrixarrangement. This study has certain limitation because the effects of mechanical vibrationon the biology field are so complicated and confusing. Further investigations should beperformed to discover the mechanism of the biological application of mechanical forces.
引文
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7. Rittweger, J. Vibration as an exercise modality: how it may work, and what itspotential might be. European journal of applied physiology108:877-904,2010.
8. Rittweger, J., Schiessl, H., Felsenberg, D. Oxygen uptake during whole-body vibrationexercise: comparison with squatting as a slow voluntary movement. European journalof applied physiology86:169-173,2001.
9. Xie, L., Jacobson, J. M., Choi, E. S., et al. Low-level mechanical vibrations caninfluence bone resorption and bone formation in the growing skeleton. Bone39:1059-1066,2006.
10.Rauch, F. Vibration therapy. Developmental medicine and child neurology51Suppl4:166-168,2009.
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24.Figueroa, A., Gil, R., Wong, A., et al. Whole-body vibration training reduces arterialstiffness, blood pressure and sympathovagal balance in young overweight/obesewomen. Hypertension research: official journal of the Japanese Society ofHypertension35:667-672,2012.
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26.Rittweger, J., Ehrig, J., Just, K., et al. Oxygen uptake in whole-body vibration exercise:influence of vibration frequency, amplitude, and external load. International journalof sports medicine23:428-432,2002.
27.Rubin, C. T., Capilla, E., Luu, Y. K., et al. Adipogenesis is inhibited by brief, dailyexposure to high-frequency, extremely low-magnitude mechanical signals.Proceedings of the National Academy of Sciences of the United States of America104:17879-17884,2007.
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32.van Nes, I. J., Latour, H., Schils, F., et al. Long-term effects of6-week whole-bodyvibration on balance recovery and activities of daily living in the postacute phase ofstroke: a randomized, controlled trial. Stroke; a journal of cerebral circulation37:2331-2335,2006.
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