Regulatory effects of bio-intensity electric field on transformation of human skin fibroblasts
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摘要:
目的 探讨生物强度电场对人皮肤成纤维细胞（HSF）转化的调节作用。 方法 采用实验研究方法。取HSF，分为经200 mV/mm电场处理6 h的200 mV/mm电场组和置于电场装置中不通电处理6 h的模拟电场组，在活细胞工作站中观察细胞形态和排列变化；记录处理0、6 h细胞数，并计算细胞数变化率；观察并计算3 h内细胞运动方向、位移速度、轨迹速度（以上实验模拟电场组样本数为34、200 mV/mm电场组样本数为30）；采用免疫荧光法检测处理3 h细胞α平滑肌肌动蛋白（α-SMA）的蛋白表达（样本数为3）。取HSF分为置于电场装置中不通电处理3 h的模拟电场组和经相应强度电场处理3 h的100 mV/mm电场组、200 mV/mm电场组、400 mV/mm电场组，另取HSF分为置于电场装置中不通电处理6 h的模拟电场组和经200 mV/mm电场处理相应时间的电场处理1 h组、电场处理3 h组、电场处理6 h组，采用蛋白质印迹法检测α-SMA、增殖细胞核抗原（PCNA）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6 h，与模拟电场组相比，200 mV/mm电场组细胞形态拉长，并产生局部粘连；模拟电场组细胞任意排列，200 mV/mm电场组细胞呈有规律的纵向排列；2组细胞数变化率相近（P>0.05）。处理3 h内，200 mV/mm电场组细胞有明显的向正极运动趋势，模拟电场组细胞绕原点运动；与模拟电场组比较，200 mV/mm电场组细胞位移速度和轨迹速度均明显加快（Z值分别为-5.33、-5.41，P<0.01），方向性显著增强（Z=-4.39，P<0.01）。处理3 h，200 mV/mm电场组细胞α-SMA蛋白表达较模拟电场组明显增加（t=-9.81，P<0.01）。处理3 h，100 mV/mm电场组、200 mV/mm电场组、400 mV/mm电场组细胞α-SMA蛋白表达分别为1.195±0.057、1.606±0.041、1.616±0.039，均明显多于模拟电场组的0.649±0.028（P<0.01）。与100 mV/mm电场组比较，200 mV/mm电场组、400 mV/mm电场组细胞α-SMA蛋白表达均明显增加（P<0.01）。电场处理1 h组、电场处理3 h组、电场处理6 h组细胞α-SMA蛋白表达分别为0.730±0.032、1.561±0.031、1.553±0.045，均明显多于模拟电场组的0.464±0.020（P<0.01）；与电场处理1 h组比较，电场处理3 h组、电场处理6 h组细胞α-SMA蛋白表达均明显增加（P<0.01）。处理3 h，与模拟电场组比较，100 mV/mm电场组、200 mV/mm电场组、400 mV/mm电场组细胞PCNA蛋白表达均明显减少（P<0.05或P<0.01）；与100 mV/mm电场组比较，200 mV/mm电场组、400 mV/mm电场组细胞PCNA蛋白表达均明显减少（P<0.05或P<0.01）；与200 mV/mm电场组比较，400 mV/mm电场组细胞PCNA蛋白表达明显减少（P<0.01）。与模拟电场组比较，电场处理1 h组、电场处理3 h组、电场处理6 h组细胞PCNA蛋白表达均明显减少（P<0.01）；与电场处理1 h组比较，电场处理3 h组、电场处理6 h组细胞PCNA蛋白表达均明显减少（P<0.05或P<0.01）；与电场处理3 h组比较，电场处理6 h组细胞PCNA蛋白表达明显减少（P<0.01）。 结论 生物强度电场可诱导HSF迁移、促进Fb向肌Fb转化，且转化有一定的时间及电场强度依赖性。 Abstract:Objective To investigate the regulatory effects of bio-intensity electric field on the transformation of human skin fibroblasts (HSFs). Methods The experimental research methods were used. HSFs were collected and divided into 200 mV/mm electric field group treated with 200 mV/mm electric field for 6 h and simulated electric field group placed in the electric field device without electricity for 6 h. Changes in morphology and arrangement of cells were observed in the living cell workstation; the number of cells at 0 and 6 h of treatment was recorded, and the rate of change in cell number was calculated; the direction of cell movement, movement velocity, and trajectory velocity within 3 h were observed and calculated (the number of samples was 34 in the simulated electric field group and 30 in 200 mV/mm electric field group in the aforementioned experiments); the protein expression of α-smooth muscle actin (α-SMA) in cells after 3 h of treatment was detected by immunofluorescence method (the number of sample was 3). HSFs were collected and divided into simulated electric field group placed in the electric field device without electricity for 3 h, and 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group which were treated with electric fields of corresponding intensities for 3 h. Besides, HSFs were divided into simulated electric field group placed in the electric field device without electricity for 6 h, and electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group treated with 200 mV/mm electric field for corresponding time. The protein expressions of α-SMA and proliferating cell nuclear antigen (PCNA) were detected by Western blotting (the number of sample was 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 After 6 h of treatment, compared with that in simulated electric field group, the cells in 200 mV/mm electric field group were elongated in shape and locally adhered; the cells in simulated electric field group were randomly arranged, while the cells in 200 mV/mm electric field group were arranged in a regular longitudinal direction; the change rates in the number of cells in the two groups were similar (P>0.05). Within 3 h of treatment, the cells in 200 mV/mm electric field group had an obvious tendency to move toward the positive electrode, and the cells in simulated electric field group moved around the origin; compared with those in simulated electric field group, the movement velocity and trajectory velocity of the cells in 200 mV/mm electric field group were increased significantly (with Z values of -5.33 and -5.41, respectively, P<0.01), and the directionality was significantly enhanced (Z=-4.39, P<0.01). After 3 h of treatment, the protein expression of α-SMA of cells in 200 mV/mm electric field group was significantly higher than that in simulated electric field group (t=-9.81, P<0.01). After 3 h of treatment, the protein expressions of α-SMA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were 1.195±0.057, 1.606±0.041, and 1.616±0.039, respectively, which were significantly more than 0.649±0.028 in simulated electric field group (P<0.01). Compared with that in 100 mV/mm electric field group, the protein expressions of α-SMA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly increased (P<0.01). The protein expressions of α-SMA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were 0.730±0.032, 1.561±0.031, and 1.553±0.045, respectively, significantly more than 0.464±0.020 in simulated electric field group (P<0.01). Compared with that in electric field treatment 1 h group, the protein expressions of α-SMA in electric field treatment 3 h group and electric field treatment 6 h group were significantly increased (P<0.01). After 3 h of treatment, compared with that in simulated electric field group, the protein expressions of PCNA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were significantly decreased (P<0.05 or P<0.01); compared with that in 100 mV/mm electric field group, the protein expressions of PCNA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly decreased (P<0.05 or P<0.01); compared with that in 200 mV/mm electric field group, the protein expression of PCNA of cells in 400 mV/mm electric field group was significantly decreased (P<0.01). Compared with that in simulated electric field group, the protein expressions of PCNA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were significantly decreased (P<0.01); compared with that in electric field treatment 1 h group, the protein expressions of PCNA of cells in electric field treatment 3 h group and electric field treatment 6 h group were significantly decreased (P<0.05 or P<0.01); compared with that in electric field treatment 3 h group, the protein expression of PCNA of cells in electric field treatment 6 h group was significantly decreased (P<0.01). Conclusions The bio-intensity electric field can induce the migration of HSFs and promote the transformation of fibroblasts to myofibroblasts, and the transformation displays certain dependence on the time and intensity of electric field. -
Key words:
- Skin /
- Fibroblasts /
- Myofibroblasts /
- Cell proliferation /
- Cell transformation /
- Bio-intensity electric fields
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1 采用活细胞工作站观察电场刺激各时间点2组人皮肤成纤维细胞的形态和排列 倒置相差显微镜×100，图中标尺为100 μm。 1A、1B.分别为模拟电场组处理0（即刻）、6 h，细胞均呈任意方向排列；1C、1D.分别为200 mV/mm电场组处理0、6 h，图1C细胞呈任意方向排列，图1D细胞形态拉长，呈纵向排列，细胞长轴与电场方向垂直
注：图1D下方箭头为电场方向，“+” 为正极、“-” 为负极
2 采用活细胞工作站观察2组人皮肤成纤维细胞经电场处理3 h内运动轨迹 倒置相差显微镜×100。2A.模拟电场组细胞绕原点运动；2B.200 mV/mm电场组细胞趋向于正极运动
注：细胞运动起点为坐标（0，0），运动终点为4个象限中的圆点，连接前述2个点之间的曲线为细胞运动轨迹，圆点位于左上、左下象限代表细胞向负极方向迁移，圆点位于右上、右下象限代表细胞向正极方向迁移；图2B下方箭头为电场方向，“ + ” 为正极、“-”为负极
3 免疫荧光法检测电场处理3 h后2组人皮肤成纤维细胞α-SMA的表达 Alexa Fluor 488-4′,6-二脒基-2-苯基吲哚-罗丹明标记的鬼笔环肽×100，图中标尺为100 μm。3A、3B、3C.分别为模拟电场组细胞α-SMA蛋白表达、纤维状肌动蛋白排列、细胞核形态，α-SMA蛋白表达较低，纤维状肌动蛋白无序排列，细胞核完整；3D、3E、3F.分别为200 mV/mm电场组细胞α-SMA蛋白表达、纤维肌动蛋白排列、细胞核形态，图3D中α-SMA蛋白表达较图3A增加，纤维状肌动蛋白纵向排列，细胞核完整
注：绿色荧光标记α平滑肌肌动蛋白（α-SMA），红色荧光标记纤维状肌动蛋白，蓝色荧光标记细胞核
4 蛋白质印迹法检测不同强度电场处理3 h后4组人皮肤成纤维细胞α-SMA蛋白表达
注：α-SMA为α平滑肌肌动蛋白，GAPDH为3-磷酸甘油醛脱氢酶；条带上方1、2、3、4分别指模拟电场组、100 mV/mm电场组、200 mV/mm电场组、400 mV/mm电场组
5 蛋白质印迹法检测电场处理不同时间4组人皮肤成纤维细胞α-SMA蛋白表达
注：α-SMA为α平滑肌肌动蛋白，GAPDH为3-磷酸甘油醛脱氢酶；条带上方1、2、3、4分别指模拟电场组（处理6 h）、电场处理1 h组、电场处理3 h组、电场处理6 h组
6 蛋白质印迹法检测不同强度电场处理3 h后4组人皮肤成纤维细胞增殖细胞核抗原（PCNA）蛋白表达
注：条带上方1、2、3、4分别指模拟电场组、100 mV/mm电场组、200 mV/mm电场组、400 mV/mm电场组
7 蛋白质印迹法检测电场处理不同时间4组人皮肤成纤维细胞增殖细胞核抗原（PCNA）蛋白表达
注：条带上方1、2、3、4分别指模拟电场组（处理6 h）、电场处理1 h组、电场处理3 h组、电场处理6 h组
表1 2组人皮肤成纤维细胞经电场处理3 h内cosθ及位移速度和轨迹速度比较[M(Q1，Q3)]
组别 样本数 cosθ 位移速度（μm/min） 轨迹速度（μm/min） 模拟电场组 34 -0.184（-0.336，0.371） 0.207（0.160，0.261） 0.165（0.126，0.204） 200 mV/mm电场组 30 0.833（0.694，0.925） 0.470（0.419，0.523） 0.383（0.347，0.438） Z值 -4.39 -5.33 -5.41 P值 <0.001 <0.001 <0.001 
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