Ji R, Zhang Z, Wang W P, Zhang Q, Lyu Y L, Jiang X P, Teng M
Department of Plastic and Burn Surgery, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China.
State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Burn Research, the First Affiliated Hospital of Army Medical University (the Third Military Medical University), Chongqing 400038, China.
Zhonghua Shao Shang Za Zhi. 2021 Jan 20;37(1):34-41. doi: 10.3760/cma.j.cn501120-20200115-00023.
To investigate the regulatory effect of bio-strength electric field (EF) on the motility and CD9 expression of human epidermal cell line HaCaT and mouse epidermal cells. The experimental research method was used. Human immortal epidermal cell line HaCaT cells in logarithmic growth phase and primary epidermal cells isolated from 16 BALB/c mice (no matter male or female) aged 1-3 days were used for experiments. HaCaT cells were divided into EF group treated for 3 h at the EF intensity of 200 mV/mm and sham EF group with simulated treatment. The cell migration (direction, displacement velocity, and trajectory velocity, with 46 samples in EF group and 34 samples in sham EF group) and arrangement were observed in the living cell workstation, and the distribution and expression of CD9 protein were detected by immunofluorescence method. Both HaCaT cells and mouse epidermal cells were divided into sham EF group (simulated treatment) and EF groups treated respectively for 3 h at the corresponding EF intensity of 50, 100, 200, and 400 mV/mm. Both HaCaT cells and mouse epidermal cells were divided into blank control group without any treatment, and 1 h group, 3 h group, and 6 h group treated with EF at the intensity of 200 mV/mm for corresponding time respectively. The expression of CD9 protein was detected by Western blotting (=3). Data were statistically analyzed with Mann-Whitney test, one-way analysis of variance, independent sample test and least significant difference test. Within 3 hours of treatment, HaCaT cells in EF group tended to move towards the negative electrode obviously, while HaCaT cells in sham EF group moved randomly around the origin; compared with those of sham EF group, the directivity of HaCaT cells in EF group was significantly enhanced, and the displacement velocity and trajectory velocity were significantly increased (=-3.975, -6.052, -6.299, <0.01). After 3 hours of treatment, the long axis of HaCaT cells in EF group was perpendicular to the direction of EF, while HaCaT cells in sham EF group arranged randomly. After 3 hours of treatment, the expression of CD9 protein in HaCaT cells in EF group was significantly down-regulated compared with that of sham EF group (=4.527, <0.01), although both expressed on cytomembrane. After 3 hours of treatment, the expression of CD9 protein in HaCaT cells and mouse epidermal cells in sham EF group, 50 mV/mm group, 100 mV/mm group, 200 mV/mm group, and 400 mV/mm group were 0.332±0.021, 0.283±0.032, 0.254±0.020, 0.231±0.041, 0.212±0.031 and 0.565±0.021, 0.453±0.022, 0.389±0.020, 0.338±0.021, 0.233±0.011, respectively. For both types of cells, compared with that of sham EF group, the expression of CD9 protein in cells was significantly decreased in the four groups of EF treatment (<0.01); compared with that of 50 mV/mm group, the expression of CD9 protein in cells was significantly decreased in the other three groups of EF treatment (<0.01); compared with that of 100 mV/mm group, the expression of CD9 protein in cells was significantly decreased in 200 mV/mm group and 400 mV/mm group (<0.01); compared with that of 200 mV/mm group, the expression of CD9 protein in cells was significantly decreased in 400 mV/mm group (<0.01). The expression levels of CD9 protein in HaCaT cells and mouse epidermal cells in blank control group, 1 h group, 3 h group, and 6 h group were 0.962±0.031, 0.784±0.020, 0.531±0.021, 0.409±0.011 and 0.963±0.031, 0.872±0.031, 0.778±0.040, 0.591±0.041, respectively. For both types of cells, compared with that of blank control group, the expression of CD9 protein in cells was significantly decreased in 1 h group, 3 h group, and 6 h group (<0.01); compared with that of 1 h group, the expression of CD9 protein in cells was significantly decreased in 3 h group and 6 h group (<0.05 or <0.01); compared with that of 3 h group, the expression of CD9 protein in cells was significantly decreased in 6 h group (<0.01). The bio-strength intensity EF can induce the directional migration and arrangement of HaCaT cells and down-regulate the expression of CD9 in HaCaT cells and mouse epidermal cells in a time-dependent and intensity-dependent manner.
探讨生物强度电场（EF）对人表皮细胞系HaCaT及小鼠表皮细胞运动性和CD9表达的调控作用。采用实验研究方法。选用处于对数生长期的人永生化表皮细胞系HaCaT细胞以及从16只1 - 3日龄BALB/c小鼠（雌雄不限）分离的原代表皮细胞进行实验。将HaCaT细胞分为在200 mV/mm的EF强度下处理3小时的EF组和模拟处理的假EF组。在活细胞工作站中观察细胞迁移（方向、位移速度和轨迹速度，EF组46个样本，假EF组34个样本）和排列情况，并用免疫荧光法检测CD9蛋白的分布及表达。将HaCaT细胞和小鼠表皮细胞均分为假EF组（模拟处理）以及分别在50、100、200和400 mV/mm相应EF强度下处理3小时的EF组。将HaCaT细胞和小鼠表皮细胞均分为未作任何处理的空白对照组以及分别在200 mV/mm强度的EF下处理相应时间的1小时组、3小时组和6小时组。采用蛋白质免疫印迹法检测CD9蛋白的表达（n = 3）。数据采用曼 - 惠特尼检验、单因素方差分析、独立样本t检验和最小显著差检验进行统计学分析。处理3小时内，EF组的HaCaT细胞明显倾向于向负极移动，而假EF组的HaCaT细胞在原点周围随机移动；与假EF组相比，EF组HaCaT细胞的方向性显著增强，位移速度和轨迹速度显著增加（Z = -3.975、-6.052、-6.299，P < 0.01）。处理3小时后，EF组HaCaT细胞的长轴垂直于EF方向，而假EF组HaCaT细胞随机排列。处理3小时后，EF组HaCaT细胞中CD9蛋白的表达与假EF组相比显著下调（t = 4.527，P < 0.01），尽管两者均表达于细胞膜上。处理3小时后，假EF组、50 mV/mm组、100 mV/mm组、200 mV/mm组和400 mV/mm组的HaCaT细胞及小鼠表皮细胞中CD9蛋白的表达分别为0.332±0.021、0.283±0.032、0.254±0.020、0.231±0.041、0.212±0.031和0.565±0.021、0.453±0.022、0.389±0.020、0.338±0.021、0.233±0.011。对于两种细胞类型，与假EF组相比，EF处理的四组细胞中CD9蛋白的表达均显著降低（P < 0.01）；与50 mV/mm组相比，EF处理的其他三组细胞中CD9蛋白的表达均显著降低（P < 0.01）；与100 mV/mm组相比，200 mV/mm组和400 mV/mm组细胞中CD9蛋白的表达显著降低（P < 0.01）；与200 mV/mm组相比，400 mV/mm组细胞中CD9蛋白的表达显著降低（P < 0.01）。空白对照组、1小时组、3小时组和6小时组的HaCaT细胞及小鼠表皮细胞中CD9蛋白的表达水平分别为0.962±0.031、0.784±0.020、0.531±0.021、0.409±0.011和0.963±0.031、0.872±0.031、0.778±0.040、0.591±0.041。对于两种细胞类型，与空白对照组相比，1小时组、3小时组和6小时组细胞中CD9蛋白的表达均显著降低（P < 0.01）；与1小时组相比，3小时组和6小时组细胞中CD9蛋白的表达显著降低（P < 0.05或P < 所提供的生物强度EF可诱导HaCaT细胞的定向迁移和排列，并以时间和强度依赖的方式下调HaCaT细胞及小鼠表皮细胞中CD9的表达。 0.01）；与
