不同时间低氧处理对人表皮细胞株HaCaT运动和增殖的影响
불동시간저양처리대인표피세포주HaCaT운동화증식적영향
Effects of hypoxia of different duration on movement and proliferation of human epidermal cell line HaCaT
  • 万方数据
    中华烧伤杂志 중화소상잡지
    16
    2014年 3期 231-236 ,共6页

    闫甜甜%张东霞%江旭品%张琼%黄跃生

    염첨첨%장동하%강욱품%장경%황약생

    缺氧%细胞运动%细胞增殖%HaCaT细胞 缺氧%細胞運動%細胞增殖%HaCaT細胞
    결양%세포운동%세포증식%HaCaT세포
    Anoxia%Cell movement%Cell proliferation%HaCaT cells
    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.
    목적 탐토저양처리인표피세포주HaCaT불동시간대기운동화증식적영향. 방법 (1)취대수생장기HaCaT세포,용함체적분수10％ FBS적RPMI 1640배양액(배양방법하동)배양,안수궤수자표법(분조방법하동)분위정상대조조(상규배양,하동)이급저양1、3、6h조,매조6공,후3조세포우함체적분수5％이양화탄、2％양기、93％담기적배경중(저양배양조건하동)분별배양상응시간.활세포공작참하관찰각조세포재3h내적운동범위,계산세포곡선운동속도급직선운동속도.(2)취대수생장기HaCaT세포분위정상대조조이급저양1、3、6、9、12、24 h조,매조20공,후6조세포분별행상응시간저양배양.채용세포계수시제합여매표의검측세포증식정황(이흡광도치표시).(3)취대수생장기HaCaT세포분위정상대조조이급저양1、3、6、24 h조,매조5공,후4조세포분별행상응시간저양배양.단백질인적법검측증식세포핵항원(PCNA)단백수평.대수거행단인소방차분석、Dunnett-t검험. 결과 (1)여정상대조조비교,저양1、3、6h조세포적운동범위명현증대,저양배양시간월장,증가폭도월대.저양1、3、6h조세포재관찰1、2、3h적곡선운동속도분별위(43±18)、(44±17)、(43±16) μm/h,(44±16)、(44±14)、(45±14) μm/h,(55±19)、(54±17)、(56±18) μm/h,현저고우정상대조조적(33±13)、(33±12)、(33±10) μm/h(t치위2.840～9.330,P＜0.05혹P＜0.01);저양6h조세포곡선운동속도현저고우저양1、3 h조(t치위3.474～ 4.545,P＜0.05혹P＜0.01).각조내각관찰시상점간세포곡선운동속도상근(F치위0.012～0.195,P치대우0.05).저양1h조관찰1h세포직선운동속도위(22±11) μm/h,현저고우정상대조조적(15±10)μm/h(t=2.697,P＜0.01);관찰1、2、3h,저양3、6 h조세포직선운동속도분별위(19±14)、(12±8)、(10±6)μm/h,(32±19)、(21±13)、(17±12) μm/h,현저고우정상대조조[관찰2、3h분별위(9±7)、(6±5) μm/h,t치위1.990 ～8.231,P＜0.05혹P＜0.01];저양6h조세포직선운동속도현저고우저양1、3h조(t치위3.394～6.008,P＜0.05혹P＜0.01).여관찰1h비교,각조관찰2、3h세포직선운동속도균현저강저(t치위-8.208 ～-4.232,P치균소우0.01);정상대조조관찰2、3h세포직선운동속도차이명현(t=-1.967,P＜0.05).(2)정상대조조이급저양1、3、6、9、12、24 h조세포증식수평분별위1.11±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).여정상대조조비교,저양1、3、6h조세포증식수평현저승고(t치분별위6.639、7.403、7.195,P치균소우0.01),저양24 h조세포적증식수평현저강저(t=-3.136,P＜0.05);여저양1、3、6h조비교,저양9、12、24 h조세포적증식수평균현저강저(t치위-10.538～-6.775,P치균소우0.01).(3)정상대조조이급저양1、3、6、24 h조세포PCNA단백수평분별위0.93±0.12、0.97±0.14、1.62±0.18、0.95±0.09、0.66±0.21(F=20.11,P＜0.01).여정상대조조비교,저양1、3、6h조세포PCNA단백수평현저승고(t치분별위2.339、5.783、2.235,P＜0.05혹P＜0.01),저양24 h조세포PCNA단백수평현저강저(t=-1.998,P＜0.05).저양3h조세포PCNA단백수평현저고우저양1、6h조(￡치분별위4.312、3.947,P치균소우0.01),해3조세포PCNA단백수평균현저고우저양24 h조(￡치분별위2.011、6.193、3.287,P＜0.05혹P＜0.01).결론 단시(1、3、6h)저양처리가대HaCaT세포적운동급증식발휘촉진작용,처리6h후세포운동능력제고폭도최대,처리3、6 h대세포증식능력적촉진작용경명습.
    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.
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