生物强度电场对人表皮细胞株HaCaT和小鼠表皮细胞运动性及CD9表达的影响

Effects of bio-strength electric field on the motility and CD9 expression of human epidermal cell line HaCaT and mouse epidermal cells

目的探讨生物强度电场对人表皮细胞株HaCaT和小鼠表皮细胞运动性及CD9表达的调节作用。方法采用实验研究方法。取对数生长期人永生化表皮细胞株HaCaT细胞及分离自16只1~3 d龄雌雄不拘BALB/c小鼠的原代表皮细胞进行实验。将HaCaT细胞分为200 mV/mm电场强度处理3 h的加电组和模拟处理的假电组,在活细胞工作站中观察细胞迁移(运动方向、位移速度、轨迹速度,加电组样本数为46、假电组样本数为34)及排列,免疫荧光法检测CD9蛋白的分布及表达。将HaCaT细胞与小鼠表皮细胞均分为假电组(模拟处理)和进行相应电场强度处理3 h的50 mV/mm组、100 mV/mm组、200 mV/mm组、400 mV/mm组,将HaCaT细胞与小鼠表皮细胞均分为未行处理的空白对照组和用200 mV/mm电场强度分别处理相应时间点的1 h组、3 h组、6 h组,蛋白质印迹法检测CD9的蛋白表达(样本数为3)。对数据行Mann-Whitney U检验、单因素方差分析、独立样本t检验及LSD检验。结果处理3 h内,加电组HaCaT细胞明显趋向负极移动,假电组HaCaT细胞围绕原点随机运动;与假电组比较,加电组HaCaT细胞方向性显著增强,位移速度、轨迹速度显著加快(Z=-3.975、-6.052、-6.299,P<0.01)。处理3 h后,加电组HaCaT细胞长轴与电场方向垂直,假电组HaCaT细胞呈任意取向排列。处理3 h后,加电组HaCaT细胞CD9蛋白(均位于细胞膜上)相对表达量明显低于假电组(t=4.527,P<0.01)。处理3 h后,假电组、50 mV/mm组、100 mV/mm组、200 mV/mm组、400 mV/mm组HaCaT细胞、小鼠表皮细胞CD9蛋白表达量分别为0.332±0.021、0.283±0.032、0.254±0.020、0.231±0.041、0.212±0.031与0.565±0.021、0.453±0.022、0.389±0.020、0.338±0.021、0.233±0.011。对于2种细胞而言,与假电组比较,电场处理4组细胞CD9蛋白表达量均明显降低(P<0.01);与50 mV/mm组比较,另外3个电场强度处理组细胞CD9蛋白表达量均明显降低(P<0.01);与100 mV/mm组比较,200 mV/mm组、400 mV/mm组细胞CD9蛋白表达量均明显降低(P<0.01);与200 mV/mm组比较,400 mV/mm组细胞CD9蛋白表达量明显降低(P<0.01)。空白对照组、1 h组、3 h组、6 h组HaCaT细胞、小鼠表皮细胞CD9蛋白表达量分别为0.962±0.031、0.784±0.020、0.531±0.021、0.409±0.011与0.963±0.031、0.872±0.031、0.778±0.040、0.591±0.041。对于2种细胞而言,与空白对照组比较,1 h组、3 h组、6 h组细胞CD9蛋白表达量均明显降低(P<0.01);与1 h组比较,3 h组、6 h组细胞CD9蛋白表达量均明显降低(P<0.05或P<0.01);与3 h组比,6 h组细胞CD9蛋白表达量明显降低(P<0.01)。结论生物强度电场可使HaCaT细胞发生定向迁移和排列,可下调HaCaT细胞和小鼠表皮细胞中CD9的表达,且呈电场强度与处理时间依赖性。

Objective 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.Methods 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(n=3).Data were statistically analyzed with Mann-Whitney U test,one-way analysis of variance,independent sample t test and least significant difference test.Results 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(Z=-3.975,-6.052,-6.299,P<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(t=4.527,P<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(P<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(P<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(P<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(P<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(P<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(P<0.05 or P<0.01);compared with that of 3 h group,the expression of CD9 protein in cells was significantly decreased in 6 h group(P<0.01).Conclusions 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.