Objective:To investigate the regulation effect of biological intensity 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 epidermal cell line HaCaT cells in logarithmic growth phase and primary mouse epidermal cells isolated from 16 BALB/c mice aged 1-3 days were used for the experiment. HaCaT cells were divided into EF group treated with EF in the intensity of 200 mV/mm and sham EF group treated with simulated operation. The cell migration (displacement velocity, trajectory velocity, and direction, 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 operation) and 50 mV/mm group, 100 mV/mm group, 200 mV/mm group and 400 mV/mm group treated with EF in the corresponding intensity respectively. 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 in 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, 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 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 biological 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.

The referential significance of standardized training for residents in clinical practice teaching was explored by analyzing current teaching situation. We analyzed some plans for improvement in management construction,talent training, teaching content construction and so on, in order to standardize medical teaching management and improve teaching quality, so as to link up clinical practice teaching to standardized residency training tightly.

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.

OBJECTIVETo study the effects of hypoxia of different duration on movement and proliferation of human epidermal cell line HaCaT.

METHODS(1) HaCaT cells in logarithmic phase were cultured in RPMI 1640 medium containing 10% FBS (the same culture method below). Cells were divided into control group (routine culture) and hypoxia for 1, 3, 6 h groups according to the random number table (the same grouping method below), with 6 wells in each group. Cells in the 3 hypoxia groups were cultured in incubator containing 5% CO2, 2% O2, and 93% N2 (the same hypoxic condition below) for corresponding duration. Range of movement of cells in 3 hours was observed under live cell imaging workstation, and their curvilinear and rectilinear movement speeds were calculated at post observation hour (POH) 1, 2, 3. (2) HaCaT cells in logarithmic phase were divided into control group (routine culture) and hypoxia for 1, 3, 6, 9, 12, 24 h groups, with 20 wells in each group. Cells in the 6 hypoxia groups were cultured under hypoxic condition for corresponding duration. Proliferation of cells was examined with cell counting kit and microplate reader (denoted as absorbance value). (3) HaCaT cells in logarithmic phase were divided into control group (routine culture) and hypoxia for 1, 3, 6, 24 h groups, with 5 wells in each group. Cells in the 4 hypoxia groups were cultured under hypoxic condition for corresponding duration. Protein expression of proliferating cell nuclear antigen (PCNA) was determined with Western blotting. Data were processed with one-way analysis of variance and Dunnett- t test.

RESULTS(1) Compared with that of control group, the movement area of cells was obviously expanded in hypoxia for 1, 3, 6 h groups. The longer the hypoxic treatment, the greater the increase was. At POH 1, 2, 3, the curvilinear movement speeds of cells in hypoxia for 1, 3, 6 h groups were respectively (43 ± 18), (44 ± 17), (43 ± 16) µm/h; (44 ± 16), (44 ± 14), (45 ± 14) µm/h; (55 ± 19), (54 ± 17), (56 ± 18) µm/h. They were significantly higher than those of control group [(33 ± 13), (33 ± 12), (33 ± 10) µm/h, with t values from 2.840 to 9.330, P < 0.05 or P < 0.01]. The curvilinear movement speed of cells was significantly higher in hypoxia for 6 h group than in hypoxia for 1 or 3 h group (with t values from 3.474 to 4.545, P < 0.05 or P < 0.01). There was no significant difference in the curvilinear movement speed among the observation time points within each group (with F values from 0.012 to 0.195, P values above 0.05). At POH 1, the rectilinear movement speed of cells in hypoxia for 1 h group was (22 ± 11) µm/h, which was obviously higher than that of control group [(15 ± 10) µm/h, t = 2.697, P < 0.01]. At POH 1, 2, 3, rectilinear movement speeds of cells in hypoxia for 3 and 6 h groups were respectively (19 ± 14), (12 ± 8), (10 ± 6) µm/h; (32 ± 19), (21 ± 13), (17 ± 12) µm/h. They were significantly higher than those of control group [(9 ± 7) and (6 ± 5) µm/h at POH 2 and 3, with t values from 1.990 to 8.231, P < 0.05 or P < 0.01]. The rectilinear movement speed of cells in hypoxia for 6 h group was obviously higher than that of hypoxia for 1 or 3 h group (with t values from 3.394 to 6.008, P < 0.05 or P < 0.01). The rectilinear movement speed of cells in each group decreased at POH 2 or 3 in comparison with POH 1 (with t values from -8.208 to -4.232, P values below 0.01). The rectilinear movement speed of cells in control group at POH 3 was significantly different from that at POH 2 (t = -1.967, P < 0.05). (2) The proliferation levels of cells in control group and hypoxia for 1, 3, 6, 9, 12, 24 h groups were respectively 1.11 ± 0.08, 1.36 ± 0.10, 1.39 ± 0.05, 1.38 ± 0.05, 1.10 ± 0.14, 1.06 ± 0.09, 0.99 ± 0.06 (F = 39.19, P < 0.01). Compared with that of control group, the rate of proliferation of cells was obviously increased in hypoxia for 1, 3, 6 h groups (with t values respectively 6.639, 7.403, 7.195, P values below 0.01), but obviously decreased in hypoxia for 24 h group (t = -3.136, P < 0.05). The proliferation of cells decreased in hypoxia for 9, 12, 24 h groups in comparison with hypoxia for 1, 3, 6 h groups (with t values from -10.538 to -6.775, P values below 0.01). (3) The protein expressions of PCNA of cells in control group and hypoxia for 1, 3, 6, 24 h groups were respectively 0.93 ± 0.12, 0.97 ± 0.14, 1.62 ± 0.18, 0.95 ± 0.09, 0.66 ± 0.21 (F = 20.11, P < 0.01). Compared with that of control group, the expression of PCNA was obviously increased in hypoxia for 1, 3, 6 h groups (with t values respectively 2.339, 5.783, 2.235, P < 0.05 or P < 0.01), but obviously decreased in hypoxia for 24 h group (t = -1.998, P < 0.05). The protein expression of PCNA was higher in hypoxia for 3 h group than in hypoxia for 1 or 6 h group (with t values respectively 4.312 and 3.947, P values below 0.01), and it was increased in the 3 groups in comparison with that of hypoxia for 24 h group (with t values respectively 2.011, 6.193, 3.287, P < 0.05 or P < 0.01).

CONCLUSIONSShort-time hypoxia (1, 3, 6 h) treatment can promote the movement and proliferation of HaCaT cells. Hypoxia for 6 h is the best condition to promote their movement, while hypoxia for 3 or 6 h is better for their proliferation.

Carbon Dioxide ; pharmacology ; Cell Cycle ; drug effects ; Cell Line ; Cell Movement ; physiology ; Cell Proliferation ; drug effects ; physiology ; Cells, Cultured ; Epithelial Cells ; cytology ; drug effects ; Humans ; Hypoxia ; physiopathology ; Nitric Oxide ; pharmacology ; Oxygen ; pharmacology ; Phosphorylation ; Proliferating Cell Nuclear Antigen ; Signal Transduction