不同时间低氧处理对人表皮细胞株HaCaT运动和增殖的影响

Effects of hypoxia of different duration on movement and proliferation of human epidermal cell lineHaCaT

目的 探讨低氧处理人表皮细胞株HaCaT不同时间对其运动和增殖的影响. 方法 （1）取对数生长期HaCaT细胞,用含体积分数10％ FBS的RPMI 1640培养液（培养方法下同）培养,按随机数字表法（分组方法下同）分为正常对照组（常规培养,下同）以及低氧1、3、6h组,每组6孔,后3组细胞于含体积分数5％二氧化碳、2％氧气、93％氮气的环境中（低氧培养条件下同）分别培养相应时间.活细胞工作站下观察各组细胞在3h内的运动范围,计算细胞曲线运动速度及直线运动速度.（2）取对数生长期HaCaT细胞分为正常对照组以及低氧1、3、6、9、12、24 h组,每组20孔,后6组细胞分别行相应时间低氧培养.采用细胞计数试剂盒与酶标仪检测细胞增殖情况（以吸光度值表示）.（3）取对数生长期HaCaT细胞分为正常对照组以及低氧1、3、6、24 h组,每组5孔,后4组细胞分别行相应时间低氧培养.蛋白质印迹法检测增殖细胞核抗原（PCNA）蛋白水平.对数据行单因素方差分析、Dunnett-t检验. 结果 （1）与正常对照组比较,低氧1、3、6h组细胞的运动范围明显增大,低氧培养时间越长,增加幅度越大.低氧1、3、6h组细胞在观察1、2、3h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,显著高于正常对照组的（33±13）、（33±12）、（33±10） μm/h（t值为2.840～9.330,P＜0.05或P＜0.01）;低氧6h组细胞曲线运动速度显著高于低氧1、3 h组（t值为3.474～ 4.545,P＜0.05或P＜0.01）.各组内各观察时相点间细胞曲线运动速度相近（F值为0.012～0.195,P值大于0.05）.低氧1h组观察1h细胞直线运动速度为（22±11） μm/h,显著高于正常对照组的（15±10）μm/h（t=2.697,P＜0.01）;观察1、2、3h,低氧3、6 h组细胞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,显著高于正常对照组[观察2、3h分别为（9±7）、（6±5） μm/h,t值为1.990 ～8.231,P＜0.05或P＜0.01];低氧6h组细胞直线运动速度显著高于低氧1、3h组（t值为3.394～6.008,P＜0.05或P＜0.01）.与观察1h比较,各组观察2、3h细胞直线运动速度均显著降低（t值为-8.208 ～-4.232,P值均小于0.01）;正常对照组观察2、3h细胞直线运动速度差异明显（t=-1.967,P＜0.05）.（2）正常对照组以及低氧1、3、6、9、12、24 h组细胞增殖水平分别为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）.与正常对照组比较,低氧1、3、6h组细胞增殖水平显著升高（t值分别为6.639、7.403、7.195,P值均小于0.01）,低氧24 h组细胞的增殖水平显著降低（t=-3.136,P＜0.05）;与低氧1、3、6h组比较,低氧9、12、24 h组细胞的增殖水平均显著降低（t值为-10.538～-6.775,P值均小于0.01）.（3）正常对照组以及低氧1、3、6、24 h组细胞PCNA蛋白水平分别为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）.与正常对照组比较,低氧1、3、6h组细胞PCNA蛋白水平显著升高（t值分别为2.339、5.783、2.235,P＜0.05或P＜0.01）,低氧24 h组细胞PCNA蛋白水平显著降低（t=-1.998,P＜0.05）.低氧3h组细胞PCNA蛋白水平显著高于低氧1、6h组（￡值分别为4.312、3.947,P值均小于0.01）,该3组细胞PCNA蛋白水平均显著高于低氧24 h组（￡值分别为2.011、6.193、3.287,P＜0.05或P＜0.01）.结论 短时（1、3、6h）低氧处理可对HaCaT细胞的运动及增殖发挥促进作用,处理6h后细胞运动能力提高幅度最大,处理3、6 h对细胞增殖能力的促进作用更明湿.

Objective To 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 （withtvalues from 3.474 to 4.545,P 〈0.05 orP 〈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 and6 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 or3 in comparison with POH 1 （withtvalues from-8.208 to-4.232,Pvalues 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 hypoxiafor 1,3,6 h groups （withtvalues from-10.538 to-6.775,Pvalues 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 （withtvalues respectively 2.339,5.783,2.235,P 〈0.05 orP 〈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 orP 〈0.01）.Conclusions Short-timehypoxia （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.