Yan Tiantian, Zhang Dongxia, Jiang Xupin, Zhang Qiong, Huang Yuesheng
Institute of Burn Research, Southwest Hospital, State Key Laboratory of Trauma, Burns and Combined Injury, the Third Military Medical University, Chongqing 400038, China.
Institute of Burn Research, Southwest Hospital, State Key Laboratory of Trauma, Burns and Combined Injury, the Third Military Medical University, Chongqing 400038, China. Email:
Zhonghua Shao Shang Za Zhi. 2014 Jun;30(3):231-6.
To study the effects of hypoxia of different duration on movement and proliferation of human epidermal cell line HaCaT.
(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.
(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).
Short-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.
研究不同时长缺氧对人表皮细胞系HaCaT运动及增殖的影响。
（1）将对数期HaCaT细胞培养于含10%胎牛血清的RPMI 1640培养基中（以下同此培养方法）。按随机数字表将细胞分为对照组（常规培养）及缺氧1、3、6小时组（以下同此分组方法），每组6孔。3个缺氧组细胞于含5%二氧化碳、2%氧气和93%氮气的培养箱中（以下同此缺氧条件）培养相应时长。在活细胞成像工作站下观察细胞3小时内的运动范围，并于观察后第1、2、3小时计算其曲线运动速度和直线运动速度。（2）将对数期HaCaT细胞分为对照组（常规培养）及缺氧1、3、6、9、12、24小时组，每组20孔。6个缺氧组细胞于缺氧条件下培养相应时长。用细胞计数试剂盒及酶标仪检测细胞增殖情况（以吸光度值表示）。（3）将对数期HaCaT细胞分为对照组（常规培养）及缺氧1、3、6、24小时组，每组5孔。4个缺氧组细胞于缺氧条件下培养相应时长。采用蛋白质印迹法检测增殖细胞核抗原（PCNA）的蛋白表达。数据采用单因素方差分析及Dunnett - t检验进行处理。
（1）与对照组相比，缺氧1、3、6小时组细胞的运动面积明显扩大。缺氧处理时间越长，增加越明显。在观察后第1、2、3小时，缺氧1、3、6小时组细胞的曲线运动速度分别为（43±18）、（44±17）、（43±16）μm/h；（44±16）、（44±14）、（45±14）μm/h；（55±19）、（54±17）、（56±18）μm/h。显著高于对照组[（33±13）、（33±12）、（33±10）μm/h，t值为2.840至9.330，P<0.05或P<0.01]。缺氧6小时组细胞的曲线运动速度显著高于缺氧1或3小时组（t值为3.474至4.545，P<0.05或P<0.01）。每组观察时间点间曲线运动速度差异无统计学意义（F值为0.012至0.195，P值>0.05）。在观察后第1小时，缺氧1小时组细胞的直线运动速度为（22±11）μm/h，明显高于对照组[（15±10）μm/h，t = 2.697，P<0.01]。在观察后第1、2、3小时，缺氧3和6小时组细胞的直线运动速度分别为（19±14）、（12±8）、（10±6）μm/h；（32±19）、（21±13）、（17±12）μm/h。显著高于对照组[在观察后第2和3小时分别为（9±7）和（6±5）μm/h，t值为1.990至8.231，P<0.05或P<0.01]。缺氧6小时组细胞的直线运动速度明显高于缺氧1或3小时组（t值为3.394至6.008，P<0.05或P<0.01）。与观察后第1小时相比，每组细胞在观察后第2或3小时的直线运动速度均降低（t值为 - 8.208至 - 4.232，P值<0.01）。对照组细胞在观察后第3小时的直线运动速度与第2小时有显著差异（t = - 1.967，P<0.05）。（2）对照组及缺氧1、3、6、9、12、24小时组细胞的增殖水平分别为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）。与对照组相比，缺氧1、3、6小时组细胞的增殖率明显升高（t值分别为6.639、7.403、7.195，P值<0.01），但缺氧24小时组明显降低（t = - 3.136，P<0.05）。与缺氧1、3、6小时组相比，缺氧9、12、24小时组细胞的增殖降低（t值为 - 10.538至 - 6.775，P值<0.01）。（3）对照组及缺氧1、3、6、24小时组细胞PCNA的蛋白表达分别为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）。与对照组相比，缺氧1、3、6小时组PCNA的表达明显升高（t值分别为2.339、5.783、2.235，P<0.05或P<0.01），但缺氧24小时组明显降低（t = - 1.998，P<0.05）。缺氧3小时组PCNA的蛋白表达高于缺氧1或6小时组（t值分别为4.312和3.947，P值<0.01），且这3组与缺氧24小时组相比均升高（t值分别为2.011、6.193、3.287，P<0.05或P<0.01）。
短时缺氧（1、3、6小时）处理可促进HaCaT细胞的运动及增殖。缺氧6小时是促进其运动的最佳条件，而缺氧3或6小时对其增殖更有利。
