發(fā)布時(shí)間：2018-08-28 13:00

【摘要】：背景： 紫外線A (ultravioletA，UVA)照射皮膚可產(chǎn)生紅斑等急性炎癥反應(yīng)，導(dǎo)致皮膚光老化、皮膚光敏作用和多種皮膚疾病的發(fā)生，甚至誘發(fā)皮膚癌。血紅素氧合酶(heme oxygenase，HO)是一種廣泛存在于機(jī)體內(nèi)的抗氧化酶，能保護(hù)多種組織和器官抵抗外界刺激引起的氧化損傷，但其在抵抗UVA照射引起的皮膚細(xì)胞損傷中的作用研究較少。 目的： 檢測(cè)不同劑量的UVA照射(100、250、500kJ/m~2)對(duì)人體皮膚角質(zhì)形成細(xì)胞(HaCaT細(xì)胞)的氧化損傷作用；觀察Bach1(BTB and CNC-homology1transcriptionfactor)的抑制對(duì)不同劑量UVA照射(250、500kJ/m~2)引起的HaCaT細(xì)胞損傷的影響，觀察HO缺失對(duì)生理劑量250kJ/m~2UVA照射引起的HaCaT細(xì)胞損傷的影響，以探究Bach1和HO是否能調(diào)節(jié)UVA照射導(dǎo)致的細(xì)胞損傷。 方法： ①細(xì)胞培養(yǎng)：用含有10%體積胎牛血清的RMPI l640培養(yǎng)基培養(yǎng)HaCaT細(xì)胞，接種于六孔板、培養(yǎng)皿或96孔板中。 ②紫外線照射：根據(jù)實(shí)驗(yàn)設(shè)計(jì)定時(shí)定量照射培養(yǎng)細(xì)胞，并分別于UVA照射前進(jìn)行基因干擾預(yù)處理。 ③細(xì)胞形態(tài)檢測(cè)：用鬼筆環(huán)肽和DAPI對(duì)各組HaCaT細(xì)胞進(jìn)行細(xì)胞染色，并在熒光顯微鏡下觀察細(xì)胞骨架和細(xì)胞核。 ④細(xì)胞活性檢測(cè)：采用MTS法檢測(cè)各實(shí)驗(yàn)組HaCaT細(xì)胞的增殖活性。 ⑤細(xì)胞活性氧族(reactive oxygen species，ROS)水平檢測(cè)：熒光探針DHE染色檢測(cè)各組HaCaT細(xì)胞內(nèi)的ROS水平。 ⑥細(xì)胞DNA損傷檢測(cè)：運(yùn)用單細(xì)胞凝膠電泳法檢測(cè)各實(shí)驗(yàn)組HaCaT細(xì)胞的DNA損傷。 結(jié)果： ①不同劑量UVA照射對(duì)HaCaT細(xì)胞損傷的影響： 與未照射組細(xì)胞相比，小劑量100kJ/m~2UVA照射組細(xì)胞形態(tài)、細(xì)胞活性均無(wú)明顯的差異，細(xì)胞ROS水平微弱升高，細(xì)胞DNA損傷較輕；生理劑量250kJ/m~2UVA照射組細(xì)胞出現(xiàn)輕微皺縮和少量細(xì)胞碎片，細(xì)胞存活率明顯下降，細(xì)胞ROS水平和細(xì)胞DNA損傷明顯增加，差異有統(tǒng)計(jì)學(xué)意義(p0.05)；大劑量500kJ/m~2UVA照射組細(xì)胞受損傷情況較為嚴(yán)重，與250kJ/m~2UVA照射組相比，細(xì)胞皺縮情況和細(xì)胞碎片明顯增加，，細(xì)胞存活率進(jìn)一步降低，細(xì)胞ROS水平和DNA損傷明顯增加(p0.05)。 ②基因干擾Bach1對(duì)UVA照射導(dǎo)致的HaCaT細(xì)胞損傷的影響： 與非特異性基因干擾對(duì)照組細(xì)胞相比，2nM、10nM基因干擾Bach1處理對(duì)HaCaT細(xì)胞形態(tài)、增殖活性、ROS水平、DNA損傷無(wú)明顯影響。預(yù)先進(jìn)行2nM基因干擾Bach1處理對(duì)250kJ/m~2和500kJ/m~2UVA照射后HaCaT細(xì)胞的活性、形態(tài)、ROS水平及DNA損傷均無(wú)明顯影響。預(yù)先進(jìn)行10nM基因干擾Bach1處理可以提高250kJ/m~2和500kJ/m~2UVA照射后HaCaT細(xì)胞的活性，細(xì)胞形態(tài)有所恢復(fù)，減少UVA照射引起的ROS生成量和減輕DNA損傷程度，其中與500kJ/m~2UVA照射組細(xì)胞相比，10nM預(yù)處理組差異有統(tǒng)計(jì)學(xué)意義(p0.05)。 ③基因干擾HO(同時(shí)干擾HO-1和HO-2)對(duì)生理劑量UVA照射導(dǎo)致的HaCaT 細(xì)胞損傷的影響： 與非特異性基因干擾對(duì)照組細(xì)胞相比，10nM、20nM基因干擾HO處理組HaCaT細(xì)胞生長(zhǎng)變緩，細(xì)胞活性有所降低，細(xì)胞內(nèi)ROS水平和DNA損傷無(wú)明顯改變。預(yù)先進(jìn)行10nM、20nM基因干擾HO處理可以抑制生理劑量UVA照射后HaCaT細(xì)胞的活性和細(xì)胞形態(tài)，并增加了UVA照射引起的ROS生成量和DNA損傷。 結(jié)論： 血紅素氧合酶HO可能減輕UVA照射對(duì)人皮膚角質(zhì)形成細(xì)胞HaCaT的損傷效應(yīng)，該保護(hù)機(jī)制可能與其提高細(xì)胞增殖活性、降低細(xì)胞內(nèi)ROS水平和DNA損傷有關(guān)。
[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.
【學(xué)位授予單位】：重慶大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R751

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 李春雨;張麗宏;張寧;楊智榮;;紫外線誘導(dǎo)皮膚光老化的形成機(jī)制[J];中國(guó)美容醫(yī)學(xué);2009年03期

2 蒲愛萍,宋琦如,汪嶺;紫外線對(duì)皮膚的損傷及其防護(hù)[J];寧夏醫(yī)學(xué)院學(xué)報(bào);2003年04期

本文編號(hào)：2209468