低能量633纳米红光刺激对人表皮细胞株HaCaT增殖及活性氧水平的影响

Effect of low-energy 633 nm red light stimulation on proliferation and reactive oxygen species level of human epidermal cell line HaCaT

目的 观察低能量633 nm红光照射人表皮细胞株HaCaT后，细胞增殖活性及细胞内活性氧水平的变化。方法 预实验选择照射距离。采用含体积分数10% FBS、100 U/mL青霉素、100 μg/mL链霉素的RPMI 1640培养液常规传代培养HaCaT细胞。采用第3代细胞进行以下实验。（1）取细胞，按随机数字表法分为空白对照组和0.082、0.164、0.245、0.491、1.472、2.453、4.910、9.810 J/cm^2照射组，每组3孔。空白对照组细胞不照光，后8个照射组细胞依次照633 nm红光10、20、30、60、180、300、600、1 200 s，每8小时重复照射1次。照射组照射48 h（6次）后，9组均用细胞计数试剂盒8和酶标仪检测细胞增殖活性（以吸光度值表示）。（2）另取细胞，分组及照光方法同实验（1），照射组照光1次后9组均加入活性氧检测试剂，常规培养60 min。分别于培养后0（即刻）、30、60、120 min，采用酶标仪检测细胞内活性氧水平（以吸光度值表示）。（3）另取细胞，按随机数字表法分为空白对照组，0.082、0.491、2.453、9.810 J/cm^2照射组及阳性对照组。空白对照组和阳性对照组细胞不照光（阳性对照组另加入活性氧阳性对照试剂），照射组细胞按实验（1）方法照光1次。激光扫描共聚焦显微镜观察各组细胞内活性氧表达。对数据行单因素方差分析、重复测量方差分析、t检验。结果 （1）照射距离为10 cm。空白对照组以及0.082、0.164、0.245、0.491、1.472、2.453、4.910、9.810 J/cm^2照射组细胞增殖活性分别为1.000、1.116±0.031、1.146±0.016、1.162±0.041、1.179±0.016、1.207±0.016、1.247±0.040、1.097±0.059、0.951±0.118。与空白对照组比较，0.082～2.453 J/cm^2照射组细胞增殖活性明显增强（t值为－22.803～－6.779，P值均小于0.05）。4.910、9.810 J/cm^2照射组细胞增殖活性与空白对照组相近（t值分别为－2.854、0.711，P值均大于0.05）。（2）培养后0、30 min，与空白对照组比较，0.164～2.453 J/cm^2照射组细胞内活性氧水平明显增高（t值为－12.453～－4.684，P〈0.05或P〈0.01），0.082、4.910、9.810 J/cm^2照射组细胞内活性氧水平无明显变化（t值为－3.925～－0.672，P值均大于0.05）。培养后60 min，与空白对照组比较，0.082～2.453 J/cm^2照射组细胞内活性氧水平明显增高（t值为－11.387～－4.717，P〈0.05或P〈0.01）。培养后120 min，与空白对照组比较，0.491～2.453 J/cm^2照射组细胞内活性氧水平明显增高（t值为－10.657～－6.644，P〈0.05或P〈0.01）。（3）与空白对照组比较，0.082、0.491、2.453 J/cm^2照射组和阳性对照组细胞内活性氧的表达均增强。9.810 J/cm^2照射组细胞内活性氧的表达较其他照射组明显减弱。各组细胞内活性氧均表达于线粒体中。结论 低能量633 nm红光照射对人表皮细胞株HaCaT增殖具有促进作用，该促进作用与红光照射刺激细胞线粒体产生活性氧关系密切。

Objective To investigate the changes of proliferative activity and reactive oxygen species level of human epidermal cell line HaCaT after being irradiated with low-energy 633 nm red light.Methods Irradiation distance was determined through preliminary experiment. HaCaT cells were conventionally sub-cultured with RPMI 1640 culture medium containing 10% fetal calf serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells of the third passage were used in the following experiments. （1） Cells were divided into blank control group and 0.082, 0.164, 0.245, 0.491, 1.472, 2.453, 4.910, and 9.810 J/cm^2 irradiation groups according to the random number table, with 3 wells in each group. Cells in blank control group were not irradiated, while cells in the latter 8 irradiation groups were irradiated with 633 nm red light for 10, 20, 30, 60, 180, 300, 600, and 1 200 s in turn. Cells were reirradiated once every 8 hours. After being irradiated for 48 hours （6 times） in irradiation groups, the proliferative activity of cells in 9 groups was determined with cell counting kit 8 and microplate reader （denoted as absorbance value）. （2） Another batch of cells were grouped and irradiated as in experiment （1）. After being irradiated for once in irradiation groups, cells in 9 groups were conventionally cultured for 60 min with detection reagent of reactive oxygen species. At post culture minute （PCM） 0 （immediately）, 30, 60, and 120, reactive oxygen species level of cells was determined with microplate reader （denoted as absorbance value）. （3） Another batch of cells were divided into blank control group, 0.082, 0.491, 2.453, and 9.810 J/cm^2 irradiation groups, and positive control group. Cells in blank control group and positive control group were not irradiated （positive control reagent of reactive oxygen species was added to cells in positive control group）, and cells in irradiation groups were irradiated as in experiment （1） for once. The expression of reactive oxygen species in cells of each group was observed by confocal laser scanning microscope. Data were processed with one-way analysis of variance, analysis of variance for repeated measurement, and t test.Results （1） Irradiation distance was 10 cm. Proliferative activity of cells in blank control group and 0.082, 0.164, 0.245, 0.491, 1.472, 2.453, 4.910, and 9.810 J/cm^2 irradiation groups was 1.000, 1.116±0.031, 1.146±0.016, 1.162±0.041, 1.179±0.016, 1.207±0.016, 1.247±0.040, 1.097±0.059, and 0.951±0.118, respectively. Compared with that in blank control group, proliferative activity of cells in 0.082-2.453 J/cm^2 irradiation groups was significantly higher （with t values from -22.803 to -6.779, P values below 0.05）. Proliferative activity of cells in 4.910 and 9.810 J/cm^2 irradiation groups was similar to that in blank control group （with t values respectively -2.854 and 0.711, P values above 0.05）. （2） Compared with that in blank control group, reactive oxygen species level of cells was significantly enhanced at PCM 0 and 30 in 0.164-2.453 J/cm^2 irradiation groups （with t values from -12.453 to -4.684, P〈0.05 or P〈0.01）, while that showed no significant change in 0.082, 4.910, and 9.810 J/cm^2 irradiation groups （with t values from -3.925 to -0.672, P values above 0.05）. Compared with that in blank control group, reactive oxygen species level of cells was significantly enhanced at PCM 60 in 0.082-2.453 J/cm^2 irradiation groups （with t values from -11.387 to -4.717, P〈0.05 or P〈0.01）. Compared with that in blank control group, reactive oxygen species level of cells was significantly enhanced at PCM 120 in 0.491-2.453 J/cm^2 irradiation groups （with t values from -10.657 to -6.644, P〈0.05 or P〈0.01）. （3） Compared with that in blank control group, the expression of reactive oxygen species of cells was increased in 0.082, 0.491, and 2.453 J/cm^2 irradiation groups and positive control group. The expression of reactive oxygen species of cells in 9.810 J/cm^2 irradiation group was attenuated when compared with the expressions in the other irradiation groups. Reactive oxygen species expressed in mitochondria of cells in each group.Conclusions Low-energy 633 nm red light can enhance the proliferation of human epidermal cell line HaCaT, and the effect is closely related to the increase of reactive oxygen species produced by mitochondria after being stimulated by red light irradiation.