血红素氧合酶对长波紫外线导致人体皮肤角质形成细胞损伤作用的初步研究
发布时间：2018-08-28 13:00
【摘要】：背景： 紫外线A (ultravioletA，UVA)照射皮肤可产生红斑等急性炎症反应，导致皮肤光老化、皮肤光敏作用和多种皮肤疾病的发生，甚至诱发皮肤癌。血红素氧合酶(heme oxygenase，HO)是一种广泛存在于机体内的抗氧化酶，能保护多种组织和器官抵抗外界刺激引起的氧化损伤，但其在抵抗UVA照射引起的皮肤细胞损伤中的作用研究较少。 目的： 检测不同剂量的UVA照射(100、250、500kJ/m~2)对人体皮肤角质形成细胞(HaCaT细胞)的氧化损伤作用；观察Bach1(BTB and CNC-homology1transcriptionfactor)的抑制对不同剂量UVA照射(250、500kJ/m~2)引起的HaCaT细胞损伤的影响，观察HO缺失对生理剂量250kJ/m~2UVA照射引起的HaCaT细胞损伤的影响，以探究Bach1和HO是否能调节UVA照射导致的细胞损伤。 方法： ①细胞培养：用含有10%体积胎牛血清的RMPI l640培养基培养HaCaT细胞，接种于六孔板、培养皿或96孔板中。 ②紫外线照射：根据实验设计定时定量照射培养细胞，并分别于UVA照射前进行基因干扰预处理。 ③细胞形态检测：用鬼笔环肽和DAPI对各组HaCaT细胞进行细胞染色，并在荧光显微镜下观察细胞骨架和细胞核。 ④细胞活性检测：采用MTS法检测各实验组HaCaT细胞的增殖活性。 ⑤细胞活性氧族(reactive oxygen species，ROS)水平检测：荧光探针DHE染色检测各组HaCaT细胞内的ROS水平。 ⑥细胞DNA损伤检测：运用单细胞凝胶电泳法检测各实验组HaCaT细胞的DNA损伤。 结果： ①不同剂量UVA照射对HaCaT细胞损伤的影响： 与未照射组细胞相比，小剂量100kJ/m~2UVA照射组细胞形态、细胞活性均无明显的差异，细胞ROS水平微弱升高，细胞DNA损伤较轻；生理剂量250kJ/m~2UVA照射组细胞出现轻微皱缩和少量细胞碎片，细胞存活率明显下降，细胞ROS水平和细胞DNA损伤明显增加，差异有统计学意义(p0.05)；大剂量500kJ/m~2UVA照射组细胞受损伤情况较为严重，与250kJ/m~2UVA照射组相比，细胞皱缩情况和细胞碎片明显增加，，细胞存活率进一步降低，细胞ROS水平和DNA损伤明显增加(p0.05)。 ②基因干扰Bach1对UVA照射导致的HaCaT细胞损伤的影响： 与非特异性基因干扰对照组细胞相比，2nM、10nM基因干扰Bach1处理对HaCaT细胞形态、增殖活性、ROS水平、DNA损伤无明显影响。预先进行2nM基因干扰Bach1处理对250kJ/m~2和500kJ/m~2UVA照射后HaCaT细胞的活性、形态、ROS水平及DNA损伤均无明显影响。预先进行10nM基因干扰Bach1处理可以提高250kJ/m~2和500kJ/m~2UVA照射后HaCaT细胞的活性，细胞形态有所恢复，减少UVA照射引起的ROS生成量和减轻DNA损伤程度，其中与500kJ/m~2UVA照射组细胞相比，10nM预处理组差异有统计学意义(p0.05)。 ③基因干扰HO(同时干扰HO-1和HO-2)对生理剂量UVA照射导致的HaCaT 细胞损伤的影响： 与非特异性基因干扰对照组细胞相比，10nM、20nM基因干扰HO处理组HaCaT细胞生长变缓，细胞活性有所降低，细胞内ROS水平和DNA损伤无明显改变。预先进行10nM、20nM基因干扰HO处理可以抑制生理剂量UVA照射后HaCaT细胞的活性和细胞形态，并增加了UVA照射引起的ROS生成量和DNA损伤。 结论： 血红素氧合酶HO可能减轻UVA照射对人皮肤角质形成细胞HaCaT的损伤效应，该保护机制可能与其提高细胞增殖活性、降低细胞内ROS水平和DNA损伤有关。
[Abstract]:Background:
Ultraviolet A (UVA) irradiation can produce acute inflammation such as erythema, which can lead to skin photoaging, skin photosensitization and the occurrence of various skin diseases, and even skin cancer. Heme oxygenase (HO) is an antioxidant enzyme that widely exists in the body and can protect many tissues and organs from the outside world. Stimulation-induced oxidative damage, but its role in resisting UVA-induced skin cell damage has been less studied.
Objective:
The effects of different doses of UVA irradiation (100,250,500 kJ/m~2) on the oxidative damage of human skin keratinocytes (HaCaT cells) were examined, and the effects of inhibition of Bach 1 (BTB and CNC-homology 1 transcription factor) on the damage of HaCaT cells induced by different doses of UVA irradiation (250,500 kJ/m~2) were observed. To explore whether Bach1 and HO can regulate the damage of HaCaT cells induced by UVA irradiation.
Method:
(1) Cell culture: HaCaT cells were cultured in RMPIl640 medium containing 10% fetal bovine serum and inoculated in six-well plate, culture dish or 96-well plate.
(2) Ultraviolet irradiation: According to the experimental design, the cultured cells were irradiated regularly and quantitatively, and gene interference pretreatment was performed before UVA irradiation.
(3) Cell morphology: HaCaT cells were stained with GNP and DAPI, and cytoskeleton and nucleus were observed under fluorescence microscope.
Cell activity assay: MTS assay was used to detect the proliferative activity of HaCaT cells in each experimental group.
_Detection of reactive oxygen species (ROS) level in cells: Fluorescence probe DHE staining was used to detect the ROS level in HaCaT cells of each group.
Cell DNA damage detection: single cell gel electrophoresis was used to detect the DNA damage of HaCaT cells in each experimental group.
Result:
(1) effects of different doses of UVA irradiation on HaCaT cell injury:
Compared with the non-irradiated group, there was no significant difference in cell morphology and cell viability between the low dose 100 kJ/m~2 UVA irradiation group and the irradiation group. The level of ROS was slightly elevated and DNA damage was mild. Compared with 250 kJ/m~2 UVA irradiation group, cell shrinkage and cell fragments were significantly increased, cell survival rate was further decreased, ROS level and DNA damage were significantly increased (p0.05).
(2) effects of Bach1 interference on HaCaT cell injury induced by UVA irradiation:
Compared with non-specific gene interference control group, 2nM and 10nM gene interference with Bach1 had no significant effect on the morphology, proliferation activity, ROS level and DNA damage of HaCaT cells. 10nM gene interference with Bach1 treatment can increase the activity of HaCaT cells after 250kJ/m2 and 500kJ/m2 UVA irradiation, restore the cell morphology, reduce the ROS production and reduce the degree of DNA damage caused by UVA irradiation. Compared with 500kJ/m2 UVA irradiation group, 10nM pretreatment group has significant difference (p0.05).
(3) gene interference with HO (simultaneous interference of HO-1 and HO-2) on physiological dose of UVA induced HaCaT
Effects of cell injury:
Compared with the non-specific gene interference control group, the growth of HaCaT cells slowed down, the cell activity decreased, and the level of ROS and DNA damage did not change significantly in the 10 nM and 20 nM gene interference HO treatment group. The ROS production and DNA damage induced by UVA irradiation were also investigated.
Conclusion:
Heme oxygenase HO may alleviate the damage effect of UVA irradiation on human keratinocyte HaCaT. The protective mechanism may be related to the increase of cell proliferation activity and the decrease of ROS level and DNA damage.
【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R751
本文编号：2209468
[Abstract]:Background:
Ultraviolet A (UVA) irradiation can produce acute inflammation such as erythema, which can lead to skin photoaging, skin photosensitization and the occurrence of various skin diseases, and even skin cancer. Heme oxygenase (HO) is an antioxidant enzyme that widely exists in the body and can protect many tissues and organs from the outside world. Stimulation-induced oxidative damage, but its role in resisting UVA-induced skin cell damage has been less studied.
Objective:
The effects of different doses of UVA irradiation (100,250,500 kJ/m~2) on the oxidative damage of human skin keratinocytes (HaCaT cells) were examined, and the effects of inhibition of Bach 1 (BTB and CNC-homology 1 transcription factor) on the damage of HaCaT cells induced by different doses of UVA irradiation (250,500 kJ/m~2) were observed. To explore whether Bach1 and HO can regulate the damage of HaCaT cells induced by UVA irradiation.
Method:
(1) Cell culture: HaCaT cells were cultured in RMPIl640 medium containing 10% fetal bovine serum and inoculated in six-well plate, culture dish or 96-well plate.
(2) Ultraviolet irradiation: According to the experimental design, the cultured cells were irradiated regularly and quantitatively, and gene interference pretreatment was performed before UVA irradiation.
(3) Cell morphology: HaCaT cells were stained with GNP and DAPI, and cytoskeleton and nucleus were observed under fluorescence microscope.
Cell activity assay: MTS assay was used to detect the proliferative activity of HaCaT cells in each experimental group.
_Detection of reactive oxygen species (ROS) level in cells: Fluorescence probe DHE staining was used to detect the ROS level in HaCaT cells of each group.
Cell DNA damage detection: single cell gel electrophoresis was used to detect the DNA damage of HaCaT cells in each experimental group.
Result:
(1) effects of different doses of UVA irradiation on HaCaT cell injury:
Compared with the non-irradiated group, there was no significant difference in cell morphology and cell viability between the low dose 100 kJ/m~2 UVA irradiation group and the irradiation group. The level of ROS was slightly elevated and DNA damage was mild. Compared with 250 kJ/m~2 UVA irradiation group, cell shrinkage and cell fragments were significantly increased, cell survival rate was further decreased, ROS level and DNA damage were significantly increased (p0.05).
(2) effects of Bach1 interference on HaCaT cell injury induced by UVA irradiation:
Compared with non-specific gene interference control group, 2nM and 10nM gene interference with Bach1 had no significant effect on the morphology, proliferation activity, ROS level and DNA damage of HaCaT cells. 10nM gene interference with Bach1 treatment can increase the activity of HaCaT cells after 250kJ/m2 and 500kJ/m2 UVA irradiation, restore the cell morphology, reduce the ROS production and reduce the degree of DNA damage caused by UVA irradiation. Compared with 500kJ/m2 UVA irradiation group, 10nM pretreatment group has significant difference (p0.05).
(3) gene interference with HO (simultaneous interference of HO-1 and HO-2) on physiological dose of UVA induced HaCaT
Effects of cell injury:
Compared with the non-specific gene interference control group, the growth of HaCaT cells slowed down, the cell activity decreased, and the level of ROS and DNA damage did not change significantly in the 10 nM and 20 nM gene interference HO treatment group. The ROS production and DNA damage induced by UVA irradiation were also investigated.
Conclusion:
Heme oxygenase HO may alleviate the damage effect of UVA irradiation on human keratinocyte HaCaT. The protective mechanism may be related to the increase of cell proliferation activity and the decrease of ROS level and DNA damage.
【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R751
【参考文献】
相关期刊论文 前2条
1 李春雨;张丽宏;张宁;杨智荣;;紫外线诱导皮肤光老化的形成机制[J];中国美容医学;2009年03期
2 蒲爱萍,宋琦如,汪岭;紫外线对皮肤的损伤及其防护[J];宁夏医学院学报;2003年04期
本文编号：2209468
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