633nm低能量红光刺激活性氧增加对人角质形成细胞增殖的影响研究

发布时间：2019-04-18 07:01

【摘要】：目的:观察红光照射人角质形成细胞株(Ha Ca T)后,细胞内活性氧(ROS)浓度的变化对细胞增殖活性的影响。方法:建立人角质形成细胞体外培养模型,分别用0.082J/cm2(10S)、0.164J/cm2(20S)、0.245J/cm2(30S)、0.491J/cm2(60S)、1.472J/cm2(180S)、2.453J/cm2(300S)、4.91 J/cm2(600S)、9.81 J/cm2(1200S)8个不同能量密度波长为633nm的红光照射人角质形成细胞株(Ha Ca T),对照组不照光,DCFH-DA(活性氧检测试剂盒)孵育1小时后用酶标仪分别于孵育后0h、0.5h、1h、2h测定荧光值,反映细胞内ROS的浓度变化;用相同的照射条件照射人角质形成细胞株,每8小时照射1次,照射6次48小时后CCK-8(细胞增殖试剂)孵育4h,酶标仪进行荧光值测定,反映细胞增殖情况,根据细胞内由DCFH-DA探针孵育后检测的荧光结果,设计对照组、0.082J/cm2(10S)组、0.491 J/cm2(60S)组、2.453J/cm2(300S)组、9.81 J/cm2(1200S)组、阳性探针检测组6个组用红色线粒体示踪剂Mito-Tracker染色线粒体,同时每个组细胞内孵育活性氧探针,在共聚焦显微镜下观察ROS的来源和线粒体的关系。结果:DCFH-DA孵育1小时后0h,0.5h,1h,2h四个时相点测定荧光值,能量密度在4.91J/cm2及以下实验组,荧光值均有不同程度的增高,其中2.453 J/cm2能量密度组,1h时相点荧光值升高最明显,和对照组相比,具有明显的统计学意义,能量密度在4.91J/cm2以上的实验组,荧光值升高呈下降趋势,和对照组相比,没有明显的统计学意义。CCK-8孵育4小时后测定荧光值,4.91J/cm2以下实验组荧光值增加明显,其中2.453 J/cm2能量密度组最明显,和对照组相比具有明显的统计学意义,能量密度在4.91J/cm2以上的实验组,荧光值降低,和对照组相比,没有明显的统计学意义。共聚焦显微镜下显示ROS产生情况与荧光值表现一致,且各组与线粒体重合。结论:633nm低能量红光照射对人角质形成细胞增殖具有促进作用,该促进作用与红光照射刺激人角质形成细胞线粒体产生ROS的增加密切相关。
[Abstract]:Aim: to observe the effect of the concentration of reactive oxygen species (ROS) on the proliferation of human keratinocytes (Ha Ca T) after irradiation with red light. Methods: human keratinocytes were cultured in vitro and cultured with 0.082J/cm2 (10s), 0.164J/cm2 (20s), 0.245J/cm2 (30s), 0.491J/cm2 (60s), 1.472J/cm2 (180s), 2.453J/cm2 (300s), respectively. 4.91 J/cm2 (600s), 9.81 J/cm2 (1200s), 8 different energy density wavelengths of red light were used to irradiate human keratinocyte line (Ha Ca T), and no light was observed in the control group. DCFH-DA (reactive oxygen species detection kit) was incubated for 1 hour and the fluorescence values were measured at 0 h, 0.5 h, 1 h, 2 h after incubation with enzyme labeling apparatus, respectively, to reflect the change of intracellular ROS concentration. Human keratinocytes were irradiated with the same irradiation condition, once every 8 hours and 48 hours after 6 times of irradiation, CCK-8 (cell proliferation reagent) was incubated for 4 hours. The fluorescence value of enzyme labeling instrument was measured to reflect the proliferation of human keratinocytes. According to the fluorescent results after incubation with DCFH-DA probe, we designed the control group, 0.082J/cm2 (10s) group, 0.491 J/cm2 (60s) group, 2.453J/cm2 (300s) group, 9.81 J/cm2 (1200s) group, and designed the control group, 0.082J/cm2 (10s) group, 2.453J/cm2 (300s) group, 9.81 J/cm2 (1200s) group. The mitochondria in 6 groups were stained with red mitochondrial tracer Mito-Tracker, and the reactive oxygen species probes were incubated in each group. The relationship between the origin of ROS and mitochondria was observed under confocal microscope. Results: after incubation with DCFH-DA for 1 hour, 0 h, 0.5 h, 1 h, 2 h, the fluorescence values were measured. The energy density of the 4.91J/cm2 group was higher than that of the control group. The fluorescence values of the 2.453 J/cm2 group were higher than those of the control group (P < 0.05), and the fluorescence values of the two groups were higher than those of the control group. Compared with the control group, the fluorescence value of the experimental group with energy density above 4.91J/cm2 showed a decreasing trend, compared with the control group, the fluorescence value of the experimental group was significantly higher than that of the control group, and the fluorescence value of the control group was significantly higher than that of the control group. There was no statistical significance. After 4 hours of incubation with CCK-8, the fluorescence value of the experimental group below 4.91J/cm2 increased significantly, among which the 2.453 J/cm2 energy density group was the most obvious, and the fluorescence value of the experimental group below CCK-8 increased significantly. Compared with the control group, the fluorescent value of the experimental group with energy density above 4.91J/cm2 was lower than that of the control group, and there was no significant difference between the experimental group and the control group. Confocal microscopy showed that the production of ROS was consistent with the fluorescent value, and all the groups were coincident with mitochondria. Conclusion: low energy red light irradiation of 633nm can promote the proliferation of human keratinocytes, which is closely related to the increase of ROS produced by mitochondria of human keratinocytes stimulated by red light irradiation.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R622

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