Wu Y T, Zhang Z, Ji R, Zhang S H, Wang W P, Wu C, Zhang J P, Jiang X P, Zhang Hengshu
Department of Plastic and Burn Surgery, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China.
Department of Plastic Surgery, the First Affiliated Hospital of Army Medical University (the Third Military Medical University), Chongqing 400038, China.
Zhonghua Shao Shang Yu Chuang Mian Xiu Fu Za Zhi. 2022 Nov 20;38(11):1066-1072. doi: 10.3760/cma.j.cn501120-20211105-00377.
To investigate the regulatory effects of bio-intensity electric field on directional migration and microtubule acetylation in human epidermal cell line HaCaT, aiming to provide molecular theoretical basis for the clinical treatment of wound repair. The experimental research methods were used. HaCaT cells were collected and divided into simulated electric field group (=54) placed in the electric field device without electricity for 3 h and electric field treatment group (=52) treated with 200 mV/mm electric field for 3 h (the same treatment methods below). The cell movement direction was observed in the living cell workstation and the movement velocity, trajectory velocity, and direction of cosθ of cell movement within 3 h of treatment were calculated. HaCaT cells were divided into simulated electric field group and electric field treatment 1 h group, electric field treatment 2 h group, and electric field treatment 3 h group which were treated with 200 mV/mm electric field for corresponding time. HaCaT cells were divided into simulated electric field group and 100 mV/mm electric field group, 200 mV/mm electric field group, and 300 mV/mm electric field group treated with electric field of corresponding intensities for 3 h. The protein expression of acetylated α-tubulin was detected by Western blotting (3). HaCaT cells were divided into simulated electric field group and electric field treatment group, and the protein expression of acetylated α-tubulin was detected and located by immunofluorescence method (=3). Data were statistically analyzed with Kruskal-Wallis test,Mann-Whitney test, Bonferroni correction, one-way analysis of variance, least significant difference test, and independent sample test. Within 3 h of treatment, compared with that in simulated electric field group, the cells in electric field treatment group had obvious tendency to move directionally, the movement velocity and trajectory velocity were increased significantly (with values of -8.53 and -2.05, respectively, <0.05 or <0.01), and the directionality was significantly enhanced (=-8.65, <0.01). Compared with (0.80±0.14) in simulated electric field group, the protein expressions of acetylated α-tubulin in electric field treatment 1 h group (1.50±0.08) and electric field treatment 2 h group (1.89±0.06) were not changed obviously (>0.05), while the protein expression of acetylated α-tubulin of cells in electric field treatment 3 h group (3.37±0.36) was increased significantly (=-3.06, <0.05). After treatment for 3 h, the protein expressions of acetylated α-tubulin of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 300 mV/mm electric field group were 1.63±0.05, 2.24±0.08, and 2.00±0.13, respectively, which were significantly more than 0.95±0.27 in simulated electric field group (<0.01). Compared with that in 100 mV/mm electric field group, the protein expressions of acetylated α-tubulin in 200 mV/mm electric field group and 300 mV/mm electric field group were increased significantly (<0.01); the protein expression of acetylated α-tubulin of cells in 300 mV/mm electric field group was significantly lower than that in 200 mV/mm electric field group (<0.05). After treatment for 3 h, compared with that in simulated electric field group, the acetylated α-tubulin of cells had enhanced directional distribution and higher protein expression (=5.78, <0.01). Bio-intensity electric field can induce the directional migration of HaCaT cells and obviously up-regulate the level of α-ubulin acetylation after treatment at 200 mV/mm bio-intensity electric field for 3 h.
为研究生物强度电场对人表皮细胞系HaCaT定向迁移及微管乙酰化的调控作用,旨在为伤口修复的临床治疗提供分子理论依据。采用实验研究方法。收集HaCaT细胞,分为置于无电电场装置中3小时的模拟电场组(n = 54)和用200 mV/mm电场处理3小时的电场处理组(n = 52)(以下处理方法相同)。在活细胞工作站中观察细胞运动方向,并计算处理3小时内细胞的运动速度、轨迹速度及运动方向的cosθ值。将HaCaT细胞分为模拟电场组和分别用200 mV/mm电场处理相应时间的电场处理1小时组、电场处理2小时组和电场处理3小时组。将HaCaT细胞分为模拟电场组和分别用相应强度电场处理3小时的100 mV/mm电场组、200 mV/mm电场组和300 mV/mm电场组。通过蛋白质免疫印迹法检测乙酰化α-微管蛋白的蛋白表达(n = 3)。将HaCaT细胞分为模拟电场组和电场处理组,采用免疫荧光法检测并定位乙酰化α-微管蛋白的蛋白表达(n = 3)。数据采用Kruskal-Wallis检验、Mann-Whitney检验、Bonferroni校正、单因素方差分析、最小显著差异检验及独立样本t检验进行统计学分析。处理3小时内,与模拟电场组相比,电场处理组细胞有明显的定向移动趋势,运动速度和轨迹速度显著增加(z值分别为 - 8.53和 - 2.05,P < 0.05或P < 0.01),方向性显著增强(z = - 8.65,P < 0.01)。与模拟电场组的(0.80±0.14)相比,电场处理1小时组(1.50±0.08)和电场处理2小时组(1.89±0.06)乙酰化α-微管蛋白的蛋白表达无明显变化(P > 0.05),而电场处理3小时组细胞乙酰化α-微管蛋白的蛋白表达(3.37±0.36)显著增加(t = - 3.06,P < 0.05)。处理3小时后,100 mV/mm电场组、200 mV/mm电场组和300 mV/mm电场组细胞乙酰化α-微管蛋白的蛋白表达分别为1.63±0.05、2.24±0.08和2.00±0.13,均显著高于模拟电场组的0.95±0.27(P < 0.01)。与100 mV/mm电场组相比,200 mV/mm电场组和300 mV/mm电场组乙酰化α-微管蛋白的蛋白表达显著增加(P < 0.01);与200 mV/mm电场组相比,300 mV/mm电场组细胞乙酰化α-微管蛋白的蛋白表达显著降低(P < 0.05)。处理3小时后,与模拟电场组相比,细胞乙酰化α-微管蛋白的定向分布增强,蛋白表达升高(t = 5.78,P < 0.01)。生物强度电场可诱导HaCaT细胞定向迁移,并在生物强度为200 mV/mm的电场处理3小时后明显上调α-微管蛋白乙酰化水平。