中波紫外線對(duì)角質(zhì)形成細(xì)胞自噬和氧化應(yīng)激影響的初步研究
發(fā)布時(shí)間：2018-11-25 08:42
【摘要】：自噬是真核細(xì)胞的一種具有自我保護(hù)功能的生理現(xiàn)象,主要幫助細(xì)胞適應(yīng)各種不良刺激,是細(xì)胞維持內(nèi)環(huán)境的自穩(wěn)并且實(shí)現(xiàn)自我更新的基本途徑。紫外線輻射可以使皮膚角質(zhì)形成細(xì)胞產(chǎn)生活性氧片段(Reactive oxygen species, ROS),進(jìn)一步使組織和細(xì)胞發(fā)生脂質(zhì)過(guò)氧化以及DNA的損害。本課題研究了中波紫外線(Ultraviolet-B, UVB)對(duì)人皮膚角質(zhì)形成細(xì)胞(Keratinocyte, KC)產(chǎn)生的自噬和氧化應(yīng)激現(xiàn)象,并引入抗氧化劑,初步探討自噬和氧化應(yīng)激相關(guān)的可能的信號(hào)通路,為將來(lái)深入發(fā)掘白噬和氧化應(yīng)激之間相互調(diào)控的分子機(jī)制,以及在疾病發(fā)生中的作用奠定實(shí)驗(yàn)基礎(chǔ)。 本研究第一部分首先對(duì)比了不同劑量的UVB和不同濃度的過(guò)氧化氫(H2O2)刺激對(duì)角質(zhì)形成細(xì)胞系HaCaT細(xì)胞和人原代KC增殖活力的影響,確定合適的UVB劑量和H2O2作用濃度；第二部分對(duì)比了相同UVB輻照后以上兩種細(xì)胞自噬囊泡表達(dá)水平上的差異；第三部分檢測(cè)了UVB照射后原代KC氧化應(yīng)激水平的變化,以外源性H2O2孵育為陽(yáng)性對(duì)照,并引入抗氧化劑α-硫辛酸(alfa-lipoic acid, LA),了解其是否能夠改變UVB輻照相關(guān)的氧化壓力；第四部分檢測(cè)了UVB照射以及UVB照射+LA處理后原代KC自噬表達(dá)水平的變化。 本課題研究目的： 1.了解兩種角質(zhì)形成細(xì)胞對(duì)紫外線輻射和外源性H2O2刺激耐受程度。 2.對(duì)比UVB輻射后兩種角質(zhì)形成細(xì)胞自噬囊泡表達(dá)水平的差異。 3.抗氧化劑干預(yù)是否改變UVB造成的原代KC氧化應(yīng)激水平和自噬水平,分析自噬與氧化壓力水平的相關(guān)性及可能的分子機(jī)制。 研究方法： 1.建立UVB輻射和外源性H2O2相關(guān)的氧化應(yīng)激模型,倒置顯微鏡下觀察UVB和H2O2刺激后細(xì)胞形態(tài)和顯微結(jié)構(gòu)的變化,并用MTT法檢測(cè)UVB、H2O2對(duì)細(xì)胞增殖活力的影響。 2.采用MDC染色法對(duì)自噬體和晚期自噬囊泡進(jìn)行染色,運(yùn)用倒置熒光顯微鏡在UVB輻照和(或)抗氧化劑孵育后相應(yīng)的時(shí)間點(diǎn)對(duì)染色后的細(xì)胞進(jìn)行攝片,統(tǒng)計(jì)每個(gè)視野自噬囊泡表達(dá)陽(yáng)性的百分比。 3.運(yùn)用化學(xué)法檢測(cè)細(xì)胞內(nèi)脂質(zhì)過(guò)氧化物MDA和總抗氧化能力TAOC。 4.2'7'-二氯熒光乙酰乙酸鈉(DCFH-DA)熒光探針對(duì)細(xì)胞內(nèi)ROS進(jìn)行染色,倒置熒光顯微鏡下觀察熒光強(qiáng)度的變化,并用流式細(xì)胞術(shù)對(duì)綠色熒光進(jìn)行定量。采用免疫印跡法檢測(cè)自噬自噬相關(guān)蛋白磷酸化mTOR、LC3、p62,對(duì)自噬表達(dá)水平進(jìn)行半定量分析。 研究結(jié)果： 1.UVB和H202對(duì)原代KC、HaCaT細(xì)胞形態(tài)和增殖活力的損傷成劑量相關(guān)性,原代角質(zhì)形成細(xì)胞更耐受UVB和H2O2的損傷。 2.MDC染色法表明相同劑量的UVB照射對(duì)原代KC和HaCaT細(xì)胞自噬水平產(chǎn)生不同的結(jié)果,5、10、20mJ/cm2UVB照射能促進(jìn)HaCaT細(xì)胞自噬囊泡的表達(dá)水平增加,且有劑量依賴性,而對(duì)原代角質(zhì)形成細(xì)胞自噬囊泡表達(dá)水平未產(chǎn)生顯著影響；40mJ/cm2UVB照射后HaCaT細(xì)胞自噬囊泡表達(dá)水平高于未照射組但較20mJ/cm2組低,而40mJ/cm2UVB照射顯著抑制原代KC自噬水平的表達(dá)。 3.10mM的H2O2和40mJ/cm2的UVB均能顯著誘導(dǎo)角質(zhì)形成細(xì)胞ROS和MDA的生成,且照射后4h細(xì)胞內(nèi)ROS、MDA水平較照射后12h為高,照射后12hROS、MDA水平基本恢復(fù)正常；加抗氧化劑LA孵育后能減輕照射后4h的ROS、MDA水平。 4.MDC染色顯示UVB照射+LA孵育4h或12h原代KC自噬囊泡表達(dá)陽(yáng)性的細(xì)胞比例較單純UVB照射組增加,以UVB照射+LA孵育12h者增加更為顯著；單純UVB照射后12h細(xì)胞自噬囊泡表達(dá)陽(yáng)性的細(xì)胞比例及LC3水平較照射后4h上調(diào)。 5.LA和H202均有誘導(dǎo)原代KC自噬囊泡形成及LC3水平上調(diào)的作用,免疫印跡法檢測(cè)磷酸化mTOR和p62均未顯示明顯差異。 結(jié)論： 1. HaCaT細(xì)胞和原代KC對(duì)相同UVB輻射體現(xiàn)了不一致的增殖抑制效應(yīng)和自噬表達(dá)結(jié)果,原代KC更耐受UVB的輻射損傷,UVB照射抑制了原代角質(zhì)形成細(xì)胞自噬囊泡的形成。 2.UVB輻射和外源性H202孵育均能夠使原代KC氧化應(yīng)激壓力增加,照射后隨著時(shí)間推移細(xì)胞有自行修復(fù)的能力,表現(xiàn)為ROS和MDA水平恢復(fù)正常,及UVB對(duì)自噬的抑制效應(yīng)減輕。 3.LA能夠減輕UVB照射相關(guān)的氧化應(yīng)激損傷,并且能夠減輕UVB照射導(dǎo)致的自噬抑制現(xiàn)象,LA的保護(hù)作用可能通過(guò)誘導(dǎo)自噬上調(diào)來(lái)實(shí)現(xiàn)。 4.抗氧化劑LA和促氧化劑H202上調(diào)LC3的表達(dá)均通過(guò)非mTOR途徑實(shí)現(xiàn),p62可能沒(méi)有參與兩者相關(guān)的自噬調(diào)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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R751
本文編號(hào)：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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R751
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 閔瑋,駱丹,林向飛,繆旭,吉璽,朱潔;中波紫外線輻射對(duì)原代及永生化角質(zhì)形成細(xì)胞損傷能力的比較研究[J];中國(guó)麻風(fēng)皮膚病雜志;2004年06期
2 張青松;顧恒;;UVB對(duì)人皮膚成纖維細(xì)胞自噬影響的初步研究[J];中國(guó)麻風(fēng)皮膚病雜志;2008年07期
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