Effects of direct current electric field on directional migration and arrangement of dermal fibroblasts in neonatal BALB/c mice and the mechanisms
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摘要: 目的 明确外源性直流电场对BALB/c小鼠乳鼠真皮Fb定向迁移与排列的作用,初步探讨其相关机制。 方法 取12只BALB/c小鼠乳鼠,分4批次,每批次取3只乳鼠背部皮肤培养真皮Fb,将第2代Fb以5×104个/mL接种于27块方形盖玻片。(1)实验1。取6块接种了第2代Fb的盖玻片(下同)分为加电组和假电组,各3块。于活细胞工作站内,加电组加电处理,电场强度为200 mV/mm,假电组模拟操作但不通电(下同),持续作用6 h,计算细胞增殖率。(2)实验2。取6块盖玻片同实验1分组并处理,活细胞工作站下观察并分析电场作用6 h内细胞的运动轨迹;电场作用0(即刻)、1、2、3、4、5、6 h细胞迁移方向性变化,以cos(α)表示;电场作用6 h内细胞的迁移速度;电场作用6 h内细胞长轴方向变化;电场作用0、1、2、3、4、5、6 h细胞排列的方向性变化,以极向值cos[2(θ-90)]表示。电场作用6 h后,采用免疫荧光染色法观察细胞微丝、微管形态变化。(3)实验3。取6块盖玻片,分为细胞松弛素D组(加入1 μmol/L的细胞松弛素D处理10 min)和秋水仙素组(加入5 μmol/L的秋水仙素处理10 min),各3块。同实验2分别观察2组细胞微丝、微管形态变化。(4)实验4。取9块盖玻片分为对照组、细胞松弛素D组、秋水仙素组,各3块。对照组不进行任何处理,细胞松弛素D组、秋水仙素组处理同实验3。均加电处理6 h,电场强度200 mV/mm。分析电场作用6 h内细胞运动轨迹,并计算电场作用6 h内细胞迁移速度;计算电场作用0、3、6 h细胞极向值;记录电场作用0、6 h细胞形态变化。对数据行独立样本
t 检验、单因素方差分析、LSD检验。 结果 (1)加电组、假电组细胞增殖率比较,差异无统计学意义(t =-0.24,P >0.05)。(2)加电组细胞电场作用6 h内整体趋向于朝电场阳极方向迁移,假电组细胞6 h内呈任意方向运动。电场作用0 h,2组细胞的cos(α)均为0;电场作用1 h,加电组细胞的cos(α)达-0.57±0.06,绝对值显著高于假电组的0.13±0.09(t =6.68,P <0.01);此后2~6 h一直明显高于假电组(t 值为5.33~6.83,P 值均小于0.01)。电场作用6 h内,加电组细胞的迁移速度为(0.308±0.019)μm/min,显著高于假电组的(0.228±0.021)μm/min(t =-2.76,P <0.01)。加电组细胞电场作用6 h内细胞长轴趋向于与电场方向垂直,假电组细胞6 h内呈任意取向排列。电场作用2~6 h,加电组细胞极向值均显著高于假电组(t 值为-7.52~-0.90,P 值均小于0.01)。电场作用6 h,加电组细胞的微丝和微管形态与假电组相似。(3)细胞松弛素D组细胞的微丝荧光强度显著减弱,束状结构模糊;秋水仙素组细胞的微管呈弱荧光强度的弥散模糊结构。(4)电场作用6 h内,对照组细胞整体趋向于朝电场阳极方向迁移,而细胞松弛素D组和秋水仙素组细胞均呈任意方向运动。电场作用6 h内,3组细胞的迁移速度差异明显(F =6.36,P <0.01)。与对照组相比,细胞松弛素D组、秋水仙素组细胞迁移速度显著降低(P <0.05或P <0.01)。电场作用0、3、6 h,3组细胞极向值相近(F 值为0.99~1.51,P 值均大于0.05)。电场作用0 h,对照组细胞为梭形,细胞松弛素D组细胞呈多角形或不规则形,秋水仙素组细胞呈锯齿状圆形或椭圆形;电场作用6 h,对照组细胞形态无明显变化,细胞松弛素D组细胞呈梭形且两端呈多分叉形态,秋水仙素组细胞呈锯齿状椭圆形。 结论 生理强度外源性直流电场可诱导BALB/c小鼠乳鼠真皮Fb发生定向迁移和排列。微丝、微管是电场诱导Fb定向迁移所必需的骨架结构,而非电场诱导Fb定向排列所必需。Abstract: Objective To explore the effects of direct current electric fields on directional migration and arrangement of dermal fibroblasts in neonatal BALB/c mice and the related mechanisms. Methods Twelve neonatal BALB/c mice were divided into 4 batches. The skin on the back of 3 neonatal mice in each batch was obtained to culture fibroblasts. Fibroblasts of the second passage were inoculated in 27 square cover slips with the concentration of 5×104 cells per mL. (1) Experiment 1. Six square cover slips inoculated with fibroblasts of the second passage were divided into electric field group (EF) and sham electric field group (SEF), with 3 cover slips in each group. The cover slips were put in live cell imaging workstation. The cells in group EF was treated with electric power with EF intensity of 200 mV/mm, while simulating process without actual power was given to SEF group (the same below) for 6 h. Cell proliferation rate was subsequently counted. (2) Experiment 2. Six cover slips were divided and underwent the same processes as in experiment 1. Cell movement locus within EF hour (EFH) 6, direction change of cell migration at EFH 0 (immediately), 1, 2, 3, 4, 5, and 6 which was denoted as cos(α), cell migration velocity within EFH 6, direction change of long axis of cell within EFH 6, and direction change of cell arrangement at EFH 0, 1, 2, 3, 4, 5, and 6 which was denoted as polarity value cos[2(θ-90)] were observed under live cell imaging workstation. After EFH 6, the morphological changes in microtubules and microfilaments were observed with immunofluorescent staining. (3) Experiment 3. Six cover slips were divided into cytochalasin D group (treated with 1 μmol/L cytochalasin D for 10 min) and colchicine group (treated with 5 μmol/L colchicine for 10 min), with 3 cover slips in each group. The morphological changes in microfilaments and microtubules were observed with the same method as in experiment 2. (4) Experiment 4. Nine cover slips were divided into control group (no reagent was added), cytochalasin D group and colchicine group (added with the same reagents as in experiment 3), with 3 cover slips in each group. Cells in the 3 groups were exposed to an EF of 200 mV/mm for 6 h. Cell movement locus within EFH 6, cell migration velocity within EFH 6, cell polarity values at EFH 0, 3, and 6, and morphological changes of cells at EFH 0 and 6 were observed. Data were processed with independent samplest -test, one-way analysis of variance, and LSD test. Results (1) There was no statistically significant difference in cell proliferation rate in group EF and group SEF (t =-0.24,P ﹥0.05). (2) Within EFH 6, cells in group EF migrated towards the anode of EF, while cells in group SEF moved randomly. At EFH 0, the values of cos(α) of cells in the 2 groups were both 0. The absolute value of cos(α) of cells in group EF (-0.57±0.06) was significantly higher than that in group SEF (0.13±0.09,t =6.68,P <0.01) at EFH 1, and it was still higher than that in group SEF from EFH 2 to 6 (witht values from 5.33 to 6.83,P values below 0.01). Within EFH 6, migration velocity of cells in group EF was (0.308±0.019) μm/min, which was significantly higher than that in group SEF [(0.228±0.021) μm/min,t =-2.76,P <0.01]. Within EFH 6, long axis of cells in group EF was perpendicular to the direction of EF, while arrangement of cells in group SEF was irregular. Cell polarity values in group EF were significantly higher than that in group SEF from EFH 2 to 6 (witht values from -7.52 to -0.90,P values below 0.01). At EFH 6, the morphology of microfilaments and microtubules of cells in EF group was similar to that in SEF group. (3) The fluorescent intensity of microfilaments of cells in cytochalasin D group became weakened, and the filamentary structure became fuzzy. The microtubules of cells in colchicine group became fuzzy with low fluorescent intensity. (4) Within EFH 6, cells in control group migrated towards the anode of EF, while cells in cytochalasin D group and colchicine group moved randomly. Within EFH 6, there was statistically significant difference in migration velocity of cells in the 3 groups (F =6.36,P <0.01). Migration velocity of cells in cytochalasin D group and colchicine group was significantly slower than that in control group (P <0.05 orP <0.01). At EFH 0, 3, and 6, cell polarity values in the 3 groups were close (withF values from 0.99 to 1.51,P values above 0.05). At EFH 0, cells in control group were spindle; cells in cytochalasin D group were polygonal or in irregular shapes; cells in colchicine group were serrated circle or oval. At EFH 6, no morphological change was observed in cells in control group; cells in cytochalasin D group were spindle with split ends on both ends; cells in colchicine group were serrated oval. Conclusions The physiologic strength of exogenous direct current EF can induce directional migration and alignment of dermal fibroblasts in neonatal BALB/c mice. Microfilaments and microtubules are necessary skeleton structure for cell directional migration induced by EF, while they are not necessary for cell directional arrangement induced by EF.-
Key words:
- Fibroblasts /
- Cell Movement /
- Microfilaments /
- Microtubules /
- Electric Fields
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