中波紫外线对角质形成细胞自噬和氧化应激影响的初步研究
发布时间：2018-11-25 08:42
【摘要】：自噬是真核细胞的一种具有自我保护功能的生理现象,主要帮助细胞适应各种不良刺激,是细胞维持内环境的自稳并且实现自我更新的基本途径。紫外线辐射可以使皮肤角质形成细胞产生活性氧片段(Reactive oxygen species, ROS),进一步使组织和细胞发生脂质过氧化以及DNA的损害。本课题研究了中波紫外线(Ultraviolet-B, UVB)对人皮肤角质形成细胞(Keratinocyte, KC)产生的自噬和氧化应激现象,并引入抗氧化剂,初步探讨自噬和氧化应激相关的可能的信号通路,为将来深入发掘白噬和氧化应激之间相互调控的分子机制,以及在疾病发生中的作用奠定实验基础。 本研究第一部分首先对比了不同剂量的UVB和不同浓度的过氧化氢(H2O2)刺激对角质形成细胞系HaCaT细胞和人原代KC增殖活力的影响,确定合适的UVB剂量和H2O2作用浓度；第二部分对比了相同UVB辐照后以上两种细胞自噬囊泡表达水平上的差异；第三部分检测了UVB照射后原代KC氧化应激水平的变化,以外源性H2O2孵育为阳性对照,并引入抗氧化剂α-硫辛酸(alfa-lipoic acid, LA),了解其是否能够改变UVB辐照相关的氧化压力；第四部分检测了UVB照射以及UVB照射+LA处理后原代KC自噬表达水平的变化。 本课题研究目的： 1.了解两种角质形成细胞对紫外线辐射和外源性H2O2刺激耐受程度。 2.对比UVB辐射后两种角质形成细胞自噬囊泡表达水平的差异。 3.抗氧化剂干预是否改变UVB造成的原代KC氧化应激水平和自噬水平,分析自噬与氧化压力水平的相关性及可能的分子机制。 研究方法： 1.建立UVB辐射和外源性H2O2相关的氧化应激模型,倒置显微镜下观察UVB和H2O2刺激后细胞形态和显微结构的变化,并用MTT法检测UVB、H2O2对细胞增殖活力的影响。 2.采用MDC染色法对自噬体和晚期自噬囊泡进行染色,运用倒置荧光显微镜在UVB辐照和(或)抗氧化剂孵育后相应的时间点对染色后的细胞进行摄片,统计每个视野自噬囊泡表达阳性的百分比。 3.运用化学法检测细胞内脂质过氧化物MDA和总抗氧化能力TAOC。 4.2'7'-二氯荧光乙酰乙酸钠(DCFH-DA)荧光探针对细胞内ROS进行染色,倒置荧光显微镜下观察荧光强度的变化,并用流式细胞术对绿色荧光进行定量。采用免疫印迹法检测自噬自噬相关蛋白磷酸化mTOR、LC3、p62,对自噬表达水平进行半定量分析。 研究结果： 1.UVB和H202对原代KC、HaCaT细胞形态和增殖活力的损伤成剂量相关性,原代角质形成细胞更耐受UVB和H2O2的损伤。 2.MDC染色法表明相同剂量的UVB照射对原代KC和HaCaT细胞自噬水平产生不同的结果,5、10、20mJ/cm2UVB照射能促进HaCaT细胞自噬囊泡的表达水平增加,且有剂量依赖性,而对原代角质形成细胞自噬囊泡表达水平未产生显著影响；40mJ/cm2UVB照射后HaCaT细胞自噬囊泡表达水平高于未照射组但较20mJ/cm2组低,而40mJ/cm2UVB照射显著抑制原代KC自噬水平的表达。 3.10mM的H2O2和40mJ/cm2的UVB均能显著诱导角质形成细胞ROS和MDA的生成,且照射后4h细胞内ROS、MDA水平较照射后12h为高,照射后12hROS、MDA水平基本恢复正常；加抗氧化剂LA孵育后能减轻照射后4h的ROS、MDA水平。 4.MDC染色显示UVB照射+LA孵育4h或12h原代KC自噬囊泡表达阳性的细胞比例较单纯UVB照射组增加,以UVB照射+LA孵育12h者增加更为显著；单纯UVB照射后12h细胞自噬囊泡表达阳性的细胞比例及LC3水平较照射后4h上调。 5.LA和H202均有诱导原代KC自噬囊泡形成及LC3水平上调的作用,免疫印迹法检测磷酸化mTOR和p62均未显示明显差异。 结论： 1. HaCaT细胞和原代KC对相同UVB辐射体现了不一致的增殖抑制效应和自噬表达结果,原代KC更耐受UVB的辐射损伤,UVB照射抑制了原代角质形成细胞自噬囊泡的形成。 2.UVB辐射和外源性H202孵育均能够使原代KC氧化应激压力增加,照射后随着时间推移细胞有自行修复的能力,表现为ROS和MDA水平恢复正常,及UVB对自噬的抑制效应减轻。 3.LA能够减轻UVB照射相关的氧化应激损伤,并且能够减轻UVB照射导致的自噬抑制现象,LA的保护作用可能通过诱导自噬上调来实现。 4.抗氧化剂LA和促氧化剂H202上调LC3的表达均通过非mTOR途径实现,p62可能没有参与两者相关的自噬调控。
[Abstract]:Autophagy is a kind of physiological phenomenon with self-protection function, which mainly helps the cells to adapt to various adverse stimuli, and is the basic way to maintain the homeostasis of the environment and to realize self-renewal. Ultraviolet radiation can cause skin keratinocytes to form reactive oxygen species (ROS), and further cause lipid peroxidation and DNA damage to tissues and cells. The autophagy and oxidative stress of human skin keratinocytes (Kratinocyte, KC) were studied in this paper, and antioxidants were introduced to study the possible signal pathways associated with autophagy and oxidative stress. In order to explore the molecular mechanism of mutual regulation between the white and the oxidative stress in the future, and to lay the foundation for the role of the disease in the future. The first part of this study first compared the effects of different doses of UVB and different concentrations of hydrogen peroxide (H2O2) on the proliferation of HaCaT cells and primary KC of human keratinocytes, and determined the appropriate UVB dose and H2O2 effect. The second part was compared with the difference in the level of the expression of autophagy in the two cells after the same UVB irradiation, and the third part detected the change of the oxidative stress level of the primary KC after the UVB irradiation, and the exogenous H2O2 was incubated as a positive control and the antioxidant was introduced into alfa-lipoic acid, L. A) It is known whether it can change the oxidation pressure related to UVB irradiation, and the fourth part detects the level of primary KC autophagy expression after UVB irradiation and UVB irradiation + LA treatment. Change. This lesson Objective: To study the effects of two kinds of keratinocytes on ultraviolet radiation and exogenous H2 O2 stimulation tolerance. 2. Two keratinocytes after UVB radiation. The difference in the level of autophagy expression. 3. Does the antioxidant intervention change the level of primary KC oxidative stress and autophagy caused by UVB, and analyze autophagy and oxidative stress level correlation Sex and possible molecular mechanism. Methods: 1. The oxidative stress model related to UVB radiation and exogenous H2O2 was established. The changes of cell morphology and microstructure of UVB and H2O2 stimulated by UVB and H2O2 were observed under inverted microscope. The effect of UVB and H2O2 on the cell proliferation was detected. 2. The autophagy and the late autophagy were stained with the MDC staining method, and the cells were irradiated with an inverted fluorescence microscope after incubation with UVB and/ or the antioxidant. taking a shot, counting the percentage of positive percentage of autophagy expression for each field of view. 3. Application of chemistry Method for detecting cells Internal lipid peroxide MDA and total antioxidant capacity TAOC. 4. 2 '7'-Dichlorofluorescein sodium acetate (DCFH-DA) fluorescent probe were used to dye the ROS in the cells and the fluorescence was inverted. The changes of the fluorescence intensity were observed under the micro-mirror, and the green fluorescence was quantified by flow cytometry. The autophagy-related protein P was detected by immunoblotting. Acidified mTO R, LC3, p62, semi-quantitative analysis of autophagy expression level. The results were as follows: 1. UVB and H202 in primary KC, HaCaT cell morphology and increase The effects of UVB irradiation on the autophagy of primary KC and HaCaT cells were more resistant to UVB and H2O2 damage. The results showed that the irradiation of 5, 10, 20mJ/ cm2UVB could promote the expression of the autophagy of HaCaT cells. There was a dose-dependent increase in the level of autophagy in primary cutin-forming cells and no significant effect on the level of autophagy expression of primary cutin-forming cells; the level of autophagy in HaCaT cells after irradiation with 40mJ/ cm2UVB was higher than that of the non-irradiated group, but 20 The levels of ROS and MDA of the primary KC were significantly inhibited by the irradiation of 40mJ/ cm2UVB, and the ROS and MDA levels in the cells after irradiation were higher than that of 12h after the irradiation and 12hROS after irradiation. The level of MDA was normal; after incubation with antioxidant LA, the ROS and MDA level of 4h after irradiation could be reduced. The percentage of cell positive cells increased with the increase of UVB irradiation group, and increased by UVB irradiation + LA incubation for 12h. The cell ratio and LC3 level of the cell autophagy were up-regulated after the irradiation of UVB and the level of LC3 was up-regulated after exposure to LC3. generation KC The results showed that 1. HaCaT cells and primary KC were not consistent with the same UVB radiation. The results showed that the primary KC was more resistant to the radiation injury of UVB, and UVB irradiation inhibited the formation of primary keratinocytes from the autophagy. The increase of pressure and the ability of the cells to self-repair over time after irradiation showed that the level of ROS and MDA returned to normal, and the inhibitory effect of UVB on autophagy was reduced. It is sufficient to reduce the oxidative stress damage associated with UVB irradiation, and to reduce the autophagy inhibition caused by UVB irradiation, and the protective effect of LA may be induced by induction of autophagy.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R751
本文编号：2355487
[Abstract]:Autophagy is a kind of physiological phenomenon with self-protection function, which mainly helps the cells to adapt to various adverse stimuli, and is the basic way to maintain the homeostasis of the environment and to realize self-renewal. Ultraviolet radiation can cause skin keratinocytes to form reactive oxygen species (ROS), and further cause lipid peroxidation and DNA damage to tissues and cells. The autophagy and oxidative stress of human skin keratinocytes (Kratinocyte, KC) were studied in this paper, and antioxidants were introduced to study the possible signal pathways associated with autophagy and oxidative stress. In order to explore the molecular mechanism of mutual regulation between the white and the oxidative stress in the future, and to lay the foundation for the role of the disease in the future. The first part of this study first compared the effects of different doses of UVB and different concentrations of hydrogen peroxide (H2O2) on the proliferation of HaCaT cells and primary KC of human keratinocytes, and determined the appropriate UVB dose and H2O2 effect. The second part was compared with the difference in the level of the expression of autophagy in the two cells after the same UVB irradiation, and the third part detected the change of the oxidative stress level of the primary KC after the UVB irradiation, and the exogenous H2O2 was incubated as a positive control and the antioxidant was introduced into alfa-lipoic acid, L. A) It is known whether it can change the oxidation pressure related to UVB irradiation, and the fourth part detects the level of primary KC autophagy expression after UVB irradiation and UVB irradiation + LA treatment. Change. This lesson Objective: To study the effects of two kinds of keratinocytes on ultraviolet radiation and exogenous H2 O2 stimulation tolerance. 2. Two keratinocytes after UVB radiation. The difference in the level of autophagy expression. 3. Does the antioxidant intervention change the level of primary KC oxidative stress and autophagy caused by UVB, and analyze autophagy and oxidative stress level correlation Sex and possible molecular mechanism. Methods: 1. The oxidative stress model related to UVB radiation and exogenous H2O2 was established. The changes of cell morphology and microstructure of UVB and H2O2 stimulated by UVB and H2O2 were observed under inverted microscope. The effect of UVB and H2O2 on the cell proliferation was detected. 2. The autophagy and the late autophagy were stained with the MDC staining method, and the cells were irradiated with an inverted fluorescence microscope after incubation with UVB and/ or the antioxidant. taking a shot, counting the percentage of positive percentage of autophagy expression for each field of view. 3. Application of chemistry Method for detecting cells Internal lipid peroxide MDA and total antioxidant capacity TAOC. 4. 2 '7'-Dichlorofluorescein sodium acetate (DCFH-DA) fluorescent probe were used to dye the ROS in the cells and the fluorescence was inverted. The changes of the fluorescence intensity were observed under the micro-mirror, and the green fluorescence was quantified by flow cytometry. The autophagy-related protein P was detected by immunoblotting. Acidified mTO R, LC3, p62, semi-quantitative analysis of autophagy expression level. The results were as follows: 1. UVB and H202 in primary KC, HaCaT cell morphology and increase The effects of UVB irradiation on the autophagy of primary KC and HaCaT cells were more resistant to UVB and H2O2 damage. The results showed that the irradiation of 5, 10, 20mJ/ cm2UVB could promote the expression of the autophagy of HaCaT cells. There was a dose-dependent increase in the level of autophagy in primary cutin-forming cells and no significant effect on the level of autophagy expression of primary cutin-forming cells; the level of autophagy in HaCaT cells after irradiation with 40mJ/ cm2UVB was higher than that of the non-irradiated group, but 20 The levels of ROS and MDA of the primary KC were significantly inhibited by the irradiation of 40mJ/ cm2UVB, and the ROS and MDA levels in the cells after irradiation were higher than that of 12h after the irradiation and 12hROS after irradiation. The level of MDA was normal; after incubation with antioxidant LA, the ROS and MDA level of 4h after irradiation could be reduced. The percentage of cell positive cells increased with the increase of UVB irradiation group, and increased by UVB irradiation + LA incubation for 12h. The cell ratio and LC3 level of the cell autophagy were up-regulated after the irradiation of UVB and the level of LC3 was up-regulated after exposure to LC3. generation KC The results showed that 1. HaCaT cells and primary KC were not consistent with the same UVB radiation. The results showed that the primary KC was more resistant to the radiation injury of UVB, and UVB irradiation inhibited the formation of primary keratinocytes from the autophagy. The increase of pressure and the ability of the cells to self-repair over time after irradiation showed that the level of ROS and MDA returned to normal, and the inhibitory effect of UVB on autophagy was reduced. It is sufficient to reduce the oxidative stress damage associated with UVB irradiation, and to reduce the autophagy inhibition caused by UVB irradiation, and the protective effect of LA may be induced by induction of autophagy.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R751
【参考文献】
相关期刊论文 前2条
1 闵玮,骆丹,林向飞,缪旭,吉玺,朱洁;中波紫外线辐射对原代及永生化角质形成细胞损伤能力的比较研究[J];中国麻风皮肤病杂志;2004年06期
2 张青松;顾恒;;UVB对人皮肤成纤维细胞自噬影响的初步研究[J];中国麻风皮肤病杂志;2008年07期
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