Sugimoto M, Maeshige N, Honda H, Yoshikawa Y, Uemura M, Yamamoto M, Terashi H
Kobe University Graduate School of Health Sciences, Kobe, Japan.
J Wound Care. 2012 Jan;21(1):5-6, 8,10; discussion 10-1. doi: 10.12968/jowc.2012.21.Sup9.S5.
In this study, we develop methods to measure galvanotaxis of fibroblasts and determined the optimum conditions of electrical stimulation.
An inverted 35mm dish containing cell suspensions (3×105 primary human skin fibroblasts, DMEM, and 10% FBS) was placed on the centre of a 100mm dish. The 35mm dish was removed 24 hours later, and culture medium was added to the 100mm dish. Fibroblasts were randomised (double-blind) into three groups, where electrical stimulation was given at varying intensities: 0UA (control), 50UA, and 100UA. Electrical stimulation (frequency=0.3Hz) was conducted, for a duration of 4 hours, with platinum electrodes in a CO2 incubator. We took pictures immediately before and 20 hours after stimulation. We calculated the migration ratio to the negative pole by dividing the area of attached fibroblasts after stimulation with that before stimulation.
The migration ratio to the negative pole was significantly higher in the 100UA group than in the control group (p<0.05). The ratios were 0.902±0.292 in the control group, 1.128±0.253 in the 50UA group, and 1.24±0.300 in the 100UA group.
This study observed the change in cell proliferation during the initial 24-hour period after plating and was thus able to quantitatively evaluate the migration. The results suggest that a low-intensity direct current promotes migration to the negative pole of human dermal fibroblasts, which is charged with positive electricity. Several clinical reports using the methods in this study showed the microcurrent efficacy for pressure ulcer healing. Electrical stimulation based on our in vitro experiment might be important for the development of physical therapy for pressure ulcers.
在本研究中，我们开发了测量成纤维细胞电趋化性的方法，并确定了电刺激的最佳条件。
将一个装有细胞悬液（3×10⁵原代人皮肤成纤维细胞、DMEM和10%胎牛血清）的35mm倒置培养皿置于一个100mm培养皿的中心。24小时后移除35mm培养皿，并向100mm培养皿中加入培养基。将成纤维细胞随机（双盲）分为三组，分别给予不同强度的电刺激：0μA（对照组）、50μA和100μA。在二氧化碳培养箱中使用铂电极进行电刺激（频率 = 0.3Hz），持续4小时。在刺激前和刺激后20小时立即拍照。通过将刺激后附着的成纤维细胞面积除以刺激前的面积来计算向负极的迁移率。
100μA组向负极的迁移率显著高于对照组（p<0.05）。对照组的迁移率为0.902±0.292，50μA组为1.128±0.253，100μA组为1.24±0.300。
本研究观察了接种后最初24小时内细胞增殖的变化，从而能够定量评估迁移情况。结果表明，低强度直流电促进带正电的人真皮成纤维细胞向负极迁移。几项使用本研究方法的临床报告显示了微电流对压疮愈合的疗效。基于我们体外实验的电刺激可能对压疮物理治疗的发展具有重要意义。
