【作者】 坚哲；

【导师】 高天文； 李春英；

【作者基本信息】 第四军医大学 ， 皮肤病与性病学， 2013， 博士

【摘要】 背景：白癜风是一种常见的色素脱失性皮肤病，发病率呈逐年上升趋势，皮损局部黑素细胞减少或消失是其色素脱失的主要原因。由于白癜风发病的确切机制尚不清楚，目前仍缺乏有效的治疗方法。近年来的研究发现，氧化应激在诱发表皮黑素细胞破坏或功能障碍方面发挥关键作用。氧化应激可通过产生大量的氧自由基来攻击细胞，干扰其正常代谢、增殖和分化，并可能进一步诱发机体自身免疫反应，形成瀑布式效应，造成表皮黑素细胞不可逆性损伤。与其它表皮细胞相比，白癜风患者黑素细胞更易于受到氧化攻击而发生损伤，然而这其中的原因和机制并不清楚。因此，阐明白癜风患者黑素细胞易受到氧化损伤的原因和具体分子机制对于完善白癜风氧化应激发病机理、指导临床治疗具有重要的意义。Nrf2-ARE通路是细胞对抗外源性刺激和氧化损伤的主要机制。Nrf2属于“CNC”家族的亮氨酸拉链蛋白，它可与胞核内的抗氧化反应元件（ARE）的DNA序列结合，调控下游一系列II相解毒酶和抗氧化基因的表达。在无任何刺激的状况下，Nrf2与胞浆伴侣蛋白Keap1相结合，处于相对抑制的状态，并被铆钉在胞浆中。当处于氧化应激条件时，Nrf2将与Keap1解偶联并转移入核，与核内的抗氧化反应元件结合，启动一系列II相解毒酶和抗氧化基因的表达。这些基因主要包括：血红素氧合酶-1（HO-1）、NADP（H）醌氧化还原酶1（NQO1）、谷氨酸-半胱氨酸连接酶催化亚基（GCLC）和谷氨酸-半胱氨酸连接酶调节亚基（GCLM）。Nrf2-ARE信号通路及其下游基因已被证实在多种细胞、组织和器官抗氧化损伤中发挥重要作用。由此，我们提出如下的假说：第一，Nrf2-ARE通路在保护黑素细胞抵抗氧化损伤中发挥重要作用；第二，白癜风患者黑素细胞中Nrf2-ARE通路存在异常，导致其对氧化应激更为敏感。目的：1.研究Nrf2-ARE通路及其下游分子在人黑素细胞中的表达情况，明确该通路在黑素细胞抗氧化应激中的作用；2.分析Nrf2-ARE通路在正常人和白癜风患者黑素细胞中的表达及功能差异；3.探讨Nrf2-ARE通路及其下游分子在白癜风患者黑素细胞中表达及功能差异的分子机制。方法：1.首先利用不同浓度的H₂O₂处理人黑素细胞PIG1，MTS实验检测细胞活性，摸索建立体外黑素细胞氧化应激模型的最佳条件；2.在PIG1中通过转染Nrf2干涉片段及过表达质粒下调或者上调Nrf2的表达，氧化应激下MTS和流式细胞术检测细胞活性和凋亡情况。3. H₂O₂预处理后，Real-time PCR检测Nrf2下游主要的4个抗氧化基因（HO-1,NQO-1, GCLC和GCLM）表达水平，免疫细胞化学及Real-time PCR检测原代黑素细胞中Nrf2及HO-1的表达情况。4.分别利用MTS实验、Real-time PCR、免疫印迹、激光共聚焦显微镜、双荧光素酶报告基因实验、流式细胞术以及酶联免疫实验检测氧化应激下正常人及白癜风患者黑素细胞活性、HO-1表达水平、Nrf2的核转位情况、转录活性、细胞内ROS和MDA水平以及SOD、GPx、CAT和GSH的含量。5.用免疫组化法检测白癜风患者皮损处Nrf2、p-Nrf2和HO-1的表达分布模式，ELISA检测患者血清中IL-2和HO-1表达水平。6.应用ATM干涉片段或抑制剂KU55933抑制ATM的表达后，Real Time-PCR和免疫印迹实验检测Nrf2、HO-1和p-Nrf2的表达情况。H₂O₂预处理后，MTS实验、流式细胞术以及酶联免疫实验检测氧化应激下黑素细胞活性、细胞内ROS和MDA水平，免疫组化检测白癜风患者皮损处ATM和p-ATM表达分布模式。结果：1. H₂O₂可以浓度依赖性的方式诱导黑素细胞死亡，1.0mM H₂O₂处理24h是体外建立黑素细胞氧化应激模型的最佳条件；2. MTS实验及流式结果证实，下调Nrf2的表达可导致氧化应激下细胞活性下降，增加细胞凋亡和坏死的比率；而上调Nrf2的表达则可提高细胞活性，显著降低细胞凋亡的比率，说明调节Nrf2-ARE通路可影响黑素细胞对氧化应激的抵抗性；3. Real-time PCR结果表明，H₂O₂可诱导Nrf2通路下游4个主要抗氧化基因表达升高，其中HO-1变化最显著（升高了近5倍）。用HO-1抑制剂ZnPP预处理可增加过氧化氢对细胞的损伤，而用其激动剂hemin预处理可提高细胞抗氧化能力，进一步研究显示，HO-1的表达水平与Nrf2的表达呈正相关，说明HO-1可通过Nrf2通路保护黑素细胞避免氧化损伤；4. MTS实验结果显示，在未使用H₂O₂处理时，正常人和白癜风患者黑素细胞活性无显著差异，但在H₂O₂作用下，白癜风患者黑素细胞活性较正常人相比显著下降，说明白癜风患者黑素细胞对氧化应激的敏感性更高；5.激光共聚焦显示，Nrf2在正常人黑素细胞（PIG1）中主要位于胞核，而在白癜风患者黑素细胞（PIG3V）中则主要位于胞浆。在正常状态下，PIG1细胞胞核中Nrf2的含量要高于PIG3V中，而胞浆中的含量则明显低于PIG3V中；免疫印迹实验结果显示，H₂O₂刺激后两组黑素细胞胞核中Nrf2的含量均有所增加，但PIG3V细胞胞核中Nrf2的增加量显著低于PIG1中；双荧光素酶报告基因实验显示，H₂O₂可在两组黑素细胞中以时间依赖性的方式诱导Nrf2转录活性升高，而在PIG3V中Nrf2转录活性的升高幅度显著低于PIG1中；进一步研究显示，在氧化应激下，白癜风患者黑素细胞中HO-1表达增高幅度显著低于正常人黑素细胞中，说明PIG3V中Nrf2存在激活障碍导致HO-1表达水平降低；6.在PIG3V中上调Nrf2的表达可显著减少氧化应激下细胞死亡率，说明激活Nrf2可降低PIG3V对氧化应激的敏感性；7.与PIG1相比，PIG3V细胞中ROS和MDA水平升高、SOD和GPx活性及总GSH和GSH含量降低，但是CAT活性在两组细胞中无显著差异，说明PIG3V细胞中氧化-抗氧化失衡；8.白癜风患者皮损处表皮细胞中Nrf2、p-Nrf2和HO-1表达分布异常，细胞核内Nrf2、p-Nrf2和HO-1含量减少，氧化应激并未引起细胞核内Nrf2、p-Nrf2和HO-1核转位增加；9.与健康对照相比，白癜风患者血清中HO-1水平降低，而IL-2水平升高，同时发现血清HO-1水平与IL-2水平呈显著负相关；10.免疫组化结果表明，白癜风患者皮损处表皮细胞中ATM表达降低，而p-ATM表达升高；在PIG1中下调ATM的表达可显著抑制Nrf2、HO-1和p-Nrf2的表达，降低氧化应激下黑素细胞活性，提高细胞内ROS和MDA的水平，而对黑素合成及酪氨酸酶活性则无影响，提示ATM可通过作用于Nrf2通路影响细胞氧化应激水平，ATM表达量降低可能是白癜风患者黑素细胞中Nrf2通路存在激活障碍的原因。结论：通过本课题的研究，我们首次明确了Nrf2-ARE信号通路在黑素细胞抗氧化应激中的重要作用，发现Nrf2激活障碍是白癜风患者黑素细胞易于发生氧化损伤的原因，初步阐明了Nrf2存在激活障碍的分子机制，为进一步认识白癜风中黑素细胞的损伤机制奠定了基础。同时，本研究不仅丰富和完善了白癜风氧化应激发病机制，而且为临床治疗提供了新靶点。更多还原

【Abstract】 Background:Vitiligo is a well-known skin depigmenting disorder characterized by the death ofmelanocytes from the lesional epidermis, and with an increasing incidence rate. Due to thepoorly understood in the pathogenesis, vitiligo is still lack of effective treatment. Recently,experimental and clinical evidences suggest that oxidative stress in the epidermis ofaffected skin lead to melanocyte degeneration. Oxidative stress can generate largeamounts of oxygen free radicals that affect the metabolism, proliferation anddifferentiation of normal cells. It may also further induce the autoimmune response toform a waterfall effect, causing irreversible damage of the epidermal melanocytes. Recentexperimental data underline that melanocytes involved in vitiligo may have inherentaberrations that make them more vulnerable to extracellular insults. However, the processand the executive disappearance mechanism lack a clear explanation. Therefore, it hasgreat significance in improving the pathogenesis and treatment of vitiligo to clarify the causes that vitiligo melanocytes is susceptible to oxidative damage and its molecularmechanism.Nrf2-ARE （Nuclear factor E2-related factor2-Antioxidant response element）pathway plays an important function in the defense against oxidative stress. Nrf2belongsto the cap’n’collar （CNC） family of basic leucine zipper proteins, and Nrf2regulates theexpression of phase II detoxifying and antioxidant genes by binding to the ARE sequence.Under unstimulated conditions, Nrf2is sequestered in the cytosol, where it is associatedwith Kelch-like ECH-associated protein1（Keap1）. When under oxidative stress, Nrf2escapes from Keap1and then translocates to the nucleus where it binds to ARE andinduces the phase II antioxidant genes. These genes encode heme oxygenase-1（HO-1）,catalase （CAT）, superoxide dismutase （SOD）, glutathione-S-transferase （GST）, glutathioneperoxidase （GPx）, NADH quinone oxidoreductase1（NQO1）, glutamate-cysteine ligasecatalytic subunit （GCLC）, and glutamyl-cystine ligase modulatory subunit （GCLM）.Recently, the functions of Nrf2and its downstream genes have been shown to beimportant for protection against oxidative stress and chemical-induced cellular damage inmany cells, tissues and organs. Therefore, we hypothesized that the Nrf2-ARE pathwaymay serve as a key mechanism of cytoprotection during H₂O₂-mediated oxidative stress inhuman melanocytes, and hypothesize that the increased sensitivity to H₂O₂-inducedoxidative insults of vitiligo melanocytes is due to the dysfunction in Nrf2-ARE redoxsignaling pathway and the activation of Nrf2can decrease the sensitivity of vitiligomelanocytes to H₂O₂-induced oxidative damage.Aims:1. To study the expression of Nrf2-ARE signaling pathway and its downstream genesin human melanocytes and to determine the role of Nrf2-ARE signaling pathway in copingwith oxidative stress in melanocytes;2. To analyze the expression and functional changes of Nrf2-ARE signaling pathwaybetween normal human melanocytes and vitiligo melanocytes;3. To explore the underlying mechanism for the aberrant expression of Nrf2-AREsignaling pathway and its downstream genes in vitiligo melanocytes. Methods:1. Firstly, we utilized H₂O₂in our studies and performed a series of dose-responseassays to determine the optimal concentration that would lead to a consistent and highdegree of cytotoxicity.2. Then, we used Nrf2siRNA and pCMV6-XL5-Nrf2to downregulate or upregulateNrf2expression in immortalized human melanocyte cell line PIG1. The melanocytes werethen analyzed under oxidative stress conditions for cell viability and apoptosis.3. After treatment with H₂O₂in PIG1cells, the expression of four main downstreamgenes （HO-1, NQO-1, GCLC and GCLM） of Nrf2pathway was determined by Real-timePCR. Then, immunocytochemistry and Real-time PCR analysis were carried out toexamine the expression of Nrf2and HO-1in the normal primary human melanocytes afterthe treatment with H₂O₂.4. Next, the cell viability, HO-1expression, the location of Nrf2, Nrf2-AREtranscriptional activity, the levels of ROS and MDA, the activities or contents of SOD,GPx, CAT and GSH were determined respectively by MTS aasay, Real-time PCR, westernblot, laser confocal scanning microscopy, dual luciferase reporter assay, flow cytometerand enzyme-linked immunosorbent assay between control and vitiligo melanocytes.5. Moreover, the expression patterns of Nrf2, p-Nrf2and HO-1in lesional skin weredetected by immunohistochemistry, and the serum levels of HO-1and interleukin （IL）-2inpatients with vitiligo were determined by ELISA.6. Finally, we used ATM siRNA or KU55933to downregulate ATM expression inPIG1cells and used Real-time PCR and western blot to determine the expression of Nrf2,HO-1and p-Nrf2. In order to observe the effect about ATM downregulation, the cellviability, the levels of ROS and MDA, melanin synthesis and tyrosinase activity weredetermined respectively by MTS aasay, flow cytometer and enzyme-linkedimmunosorbent assay. The expression patterns of ATM and p-ATM in lesional skin werealso detected by immunohistochemistry.Results:1. As determined by MTS assay, the H₂O₂-induced cell death of normal human melanocytes was concentration-dependent, and1.0mM H₂O₂caused consistentcytotoxicity but maintained high cell viability.2. MTS assay results showed that suppressed Nrf2expression led to significant celldeath under oxidative stress, and the percentage of dead cells was low in cells withupregulated Nrf2expression. Compared with the control group, the proportion ofapoptotic and necrotic cells increased after Nrf2was downregulated by Nrf2siRNA, butthere was a statistically significant induction in apoptosis48h after transfection withpCMV6-XL5-Nrf2. It suggests that modulation of Nrf2-ARE pathway can affect thecapability of melanocytes to cope with oxidative stress.3. The RT-PCR results shown that the expression levels of four main phase IIdetoxifying genes （HO-1, NQO-1, GCLC and GCLM） were upregulated after H₂O₂treatment. The most significant change was found in the HO-1gene with an approximate5-fold increase when compared to the control. ZnPP treatment exacerbated the damageinduced by H₂O₂in melanocytes, and hemin treatment enhanced cells viability comparedwith the control. The reduction of HO-1expression was associated with Nrf2knockdown,while the increase in both mRNA and protein levels of HO-1coincided with increasedNrf2levels. It suggests that HO-1can protect human melanocytes against H₂O₂-inducedoxidative damage through an Nrf2-dependent pathway.4. MTS assay results showed that no remarkable difference in cell viability was foundbetween normal human melanocytes from vitiligo melanocytes when H₂O₂was absent,while the present of1.0mM H₂O₂caused statistically significant reduction in cell viabilityof vitiligo melanocytes compared with control melanocytes. It suggests that vitiligomelanocytes showing more hypersensitive to H₂O₂-induced oxidative injury.5. Laser confocal scanning microscopy showed that nuclear Nrf2localization wasobserved mainly in control melanocytes, while vitiligo melanocytes produced primarilycytoplasmic staining. Under normal condition, the expression of nuclear Nrf2in controlmelanocytes are higher than that in vitiligo melanocytes, while the expression ofcytoplasmic Nrf2in vitiligo melanocytes are higher than that in control melanocytes.Moreover, western blot showed that in the nuclei-enriched fractions of control and vitiligo melanocytes, Nrf2content increased in response to H₂O₂. Far smaller Nrf2increases wereseen in the nuclei-enriched fractions of vitiligo melanocytes. Dual luciferase reporter assayshowed that treatment with H₂O₂increased transcriptional activity of Nrf2in terms ofARE luciferase activity in a time-dependent manner in both groups, whereas AREluciferase activity of PIG3V was much lower than that of PIG1in the presence of H₂O₂. Incontrol melanocytes, H₂O₂led to greater increases in HO-1. While in vitiligo melanocytes,the increases were far smaller compared with H₂O₂-untreated group. These data clearlyindicate that Nrf2-driven transcriptional activation of HO-1is aberrant in vitiligomelanocytes.6. Compared with normal control group, up-regulated Nrf2expression by transfectedwith pCMV6-XL5-Nrf2led to a less cell death under oxidative stress, whereas suchchanges were not observed in mock group. These data indicate that up-regulation of Nrf2restores the antioxidative ability of vitiligo melanocytes.7. Vitiligo melanocytes present a significantly higher intracellular ROS and MDAlevels. The SOD and GPx activities and low contents of total GSH and GSH in vitiligomelanocytes were comparatively lower than that in control melanocytes. Interestingly,there was no signifcant difference in activity of CAT between two groups. These dataindicate that antioxidant enzymes system is impaired in vitiligo melanocytes.8. In vitiligo lesional skin, we observed a dramatic reduction in nuclear Nrf2, p-Nrf2and HO-1in epidermal cells, which was different from the expected localizationresponding to oxidative stress.9. Serum HO-1levels were signifcantly lower in patients with vitiligo than in healthycontrols and the overall levels of serum IL-2of the patients with vitiligo were obviouslyhigher than that of healthy controls. The level of serum HO-1was inversely correlatedwith serum IL-2in vitiligo patients.10. Immunohistochemistry showed that decreased expression of ATM and increasedexpression of p-ATM were observed in epidermal cells of vitiligo lesional skin.Transfection with ATM-siRNA2lead to decreased expression of Nrf2, HO-1and p-Nrf2.Downregulation of ATM present a significantly lower cell viability and higher intracellular ROS and MDA levels, but have no effect on melanin synthesis and tyrosinase activity. Itsuggests that ATM regulates the oxidative stress level via Nrf2-ARE signaling pathway inmelanocytes, and the lack of ATM in epidermal cells of lesional skin may be the reasonthat the Nrf2activation is impaired under oxidative stress.Conclusion:In view of the above data, we demonstrate for the first time that Nrf2-ARE pathwayplays an important function in the defense against oxidative stress in human melanocytesand impaired Nrf2-ARE signaling pathway in vitiligo melanocytes shown for the first timehere contributes to the severity of oxidative stress and the failure of antioxidant inductionin response to oxidative stress. Furthermore, the underlying mechanism for the aberrantactivation of Nrf2in vitiligo melanocytes is clarified. Our work laid the foundation for thefurther understanding of the injury mechanism of vitiligo melanocytes. Meanwhile, ourstudy not only enriches and improves the oxidative stress pathogenesis in vitiligo, but alsoprovides a new target for clinical treatment.更多还原