【作者】 王旭东；

【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， 2010， 硕士

【摘要】 白癜风是一种以皮损处色素脱失为特征的慢性皮肤疾病,其发病机制目前尚不完全清楚。以往的研究发现白癜风发病机制与遗传、自身免疫、氧化应激等因素有关,近年来氧化应激成为白癜风研究领域的一大热点。多巴胺氧化是黑素细胞和多巴胺能神经细胞特有的氧化应激形式,多项研究证实多巴胺氧化可以影响多巴胺能神经元线粒体的正常形态和功能。线粒体是真核细胞中最重要的细胞器之一,是细胞有氧呼吸的主要场所,在细胞代谢和能量产生过程中扮演着重要角色。同时线粒体也是细胞内氧自由基的最重要来源,更是活性氧的主要攻击对象。线粒体的形态异常会加速氧自由基的产生,当线粒体受到氧化损伤不能代偿时,会激活线粒体凋亡途径诱导细胞凋亡。多位学者从与多巴胺氧化相关的基因多态性、多巴胺氧化产物在皮损局部和全身的蓄积、多巴胺氧化对黑素细胞的影响等不同方面进行研究,提示多巴胺氧化与白癜风发病关系密切。本课题组的前期工作发现:白癜风患者血液循环中存在高滴度针对Mitofilin的自身抗体,Mitofilin是定位于线粒体内膜的锚定蛋白,虽然Mitofilin的功能目前尚不完全清楚,但其在维持线粒体内膜和嵴的正常形态方面的关键性作用已为世人所公认。在HeLa细胞中特异性下调Mitofilin时,线粒体内膜重构,嵴形态消失,线粒体内活性氧增高,细胞凋亡增加。同时Mitofilin在修复mtDNA、维护线粒体内基因组稳定方面扮演着重要的角色。新近的研究发现白癜风皮损区周围的黑素细胞线粒体变得非常巨大,同时线粒体嵴的形态也发生了明显的变化,进一步提示Mitofilin可能在白癜风发病中发挥了重要作用。黑素细胞与多巴胺能神经元共同起源于外胚层神经嵴,并且都具有多巴胺代谢功能。我们推测多巴胺氧化条件下黑素细胞Mitofilin表达水平将下降,进而导致其线粒体结构和功能紊乱,产生更多的活性氧,导致细胞陷入氧化应激的恶性循环之中,最终激活包括线粒体途径在内的多条凋亡通路,导致黑素细胞凋亡,这可能是白癜风中黑素细胞丧失的机制之一。第一部分实验: Mitofilin作为白癜风相关抗原的确认1.主要方法提取黑素瘤细胞膜蛋白,利用白癜风自身抗体阳性血清和Mitofilin抗体分别与膜蛋白行免疫沉淀实验,所得样品再行Western blot检测线粒体内膜蛋白Mitofilin。2.主要结果Western blot结果显示在90kDa附近,白癜风血清组和Mitofilin抗体组均出现了阳性条带。3.主要结论白癜风阳性血清中包含有针对Mitofilin的自身抗体,提示Mitofilin在白癜风发病中起重要作用。第二部分实验:多巴胺对黑素细胞、Mitofilin及线粒体的影响1.主要方法300μM、500μM、1000μM多巴胺处理永生化的人表皮黑素细胞系PIG1,于处理后24h、24h及48h分别进行结晶紫染色、流式细胞术及Westernblot检测黑素细胞增殖活性、凋亡及Mitofilin被抑制的情况。500μM多巴胺处理黑素细胞PIG1,24h后利用JC-1染料检测线粒体内膜电位变化。300μM多巴胺处理黑素细胞PIG1,72h后透射电镜观察细胞器变化。每次处理均设空白对照组和实验组。2.主要结果多巴胺抑制黑素细胞增殖活性并诱导黑素细胞凋亡,与空白对照组相比具有统计学意义。多巴胺处理明显降低黑素细胞Mitofilin蛋白及线粒体内膜电位水平。多巴胺处理使黑素细胞线粒体内膜、嵴明显肿胀,内质网明显肿胀,染色质边集,凋亡小体形成。3.主要结论多巴胺处理后,黑素细胞Mitofilin蛋白明显下降,并因此导致线粒体形态和线粒体电位水平的异常,可能激活线粒体途径诱导细胞凋亡。第三部分实验:多巴胺通过内质网应激及线粒体途径诱导黑素细胞凋亡1.主要方法500μM多巴胺分别处理黑素细胞PIG1 1h、3h、6h、12h,Western blot检测GRP78、PKR、P-PKR、P-eIF2α、Caspase-3、Caspase-9、Bax、Bcl-2、β-actin等蛋白表达水平。2.主要结果在内参照β-actin不变的情况下,GRP78、P-PKR、P-eIF2α、Caspase-3、Caspase-9、Bax较对照组明显升高, Bcl-2水平明显降低, PKR水平未发生明显变化。3.主要结论多巴胺激活内质网途径及线粒体途径诱导黑素细胞凋亡。本研究首次证实白癜风患者体内存在高滴度针对Mitofilin的自身抗体;明确Mitofilin为黑素细胞多巴胺氧化的靶蛋白之一;证实多巴胺通过下调Mitofilin影响黑素细胞线粒体的正常形态和功能,并激活内质网途径和线粒体途径诱导黑素细胞凋亡。我们推测在白癜风中存在着一个恶性循环:黑素细胞中多巴胺或多巴醌代谢紊乱,产生过量的氧自由基,超出了机体自身抗氧化系统处理能力,抑制抗氧化系统功能,从而攻击包括线粒体在内的生物膜系统,发生脂质过氧化作用,产生大量丙二醛类小分子。丙二醛等小分子与细胞蛋白质、DNA等非特异性结合,影响细胞正常功能。同时,多巴醌也能直接与蛋白质结合,影响包括Mitofilin、SOD在内的蛋白质数量和功能,从而影响到线粒体的形态、功能和复制。异常的线粒体功能导致更大量氧自由基的产生,使细胞处于氧化应激的恶性循环之中,最终激活包括线粒体途径在内的多条通路,导致细胞的坏死和凋亡。而细胞中蓄积的大量被变异调节的蛋白质如Mitofilin,释放到细胞外,在局部炎症因子的参与下,被抗原提呈细胞所识别,激活B细胞,最终产生了针对黑素细胞的自身抗体。本研究为揭示白癜风发病机制进行了有益的探索,并可能为白癜风的治疗提供新的思路。更多还原

【Abstract】 Vitiligo is a chronic acquired depigmentation disorder characterized by generalized or circumscribed depigmented macules resulting from the loss of functional melanocytes. Theories concerning the cause of vitiligo have focused on several different mechanisms: autoimmune, biochemical, neural , self-destructive and genetic hypotheses, etc. In recent years, the relationship between oxidative stress and vitiligo has been extensively studied and oxidative stress now is recognized as an integral part contributing to vitiligo. Dopamine oxidation is one of special oxidative form in melanocytes and dopaminergic neurons. Several studies document that dopamine oxidation can alter mitochondrial respiration and induce permeability transition in brain mitochondria, which involves the down-regulation/dysfunction of mitofilin.The critical role of mitochondria for cellular survival is well known. Meanwhile, increasing reports indicate that mitochondria are the key in the regulation of apoptosis during oxidative stress. On one hand, ROS were mainly generated during mitochondrial redox process under physiological conditions. On the other hand, mitochondria may be the most sensitive primary cellular targets of oxidative stress. Numerous studies have indicated that mitochondria are the major source of oxidative damage in degenerative diseases. Dysfunction of mitochondria will lead to the abnormal of mitochondrial transmembrane potential and the release of cytochrome c, which will cause caspases activation and apoptosis.Several studies from gene polymorphism to dopamine metabolism have revealed that dopamine oxidation may play an important role in the disease. Our group has observed that there are an increasing level of mitofilin autoantibody in vitiligo patients, which indicates that dysfunction of mitofilin may play a crucial role in the pathogenesis of vitiligo.Though the exact role of inner mitochondrial membrane protein mitofilin has not been evaluated, the presence of mitofilin has been shown to be critical for maintenance of mitochondrial cristae structure. A recent study suggested that mitofilin forms a complex with PARP-1, which plays a role in mtDNA damage signaling and/or repair. It was reported that an ultrastructural study of the melanocytes mitochondria in the vitiligo perilesional skin appeared alterations of the cristae, reflecting dysfunction of mitofilin.As melanocyte is derived from neural crest cells, it shares several characteristic with neural cells. Here, we postulate that inner mitochondrial membrane protein mitofilin is one of the critical protein targets to the toxicity induced by dopamine .The down-regulation /dysfunction of mitofilin will induce disorder of mitochondria, increasing ROS generation and cell apoptosis, which may be a possible cause for melanocytes loss in vitiligo. PartⅠ: Identification of mitofilin as a vitiligo associated antigen1. Main methodsImmunoprecipitation experiment was carried out using vitiligo serum and membrane proteins which were extracted from cultured A375 cells (human melanoma cell lines), and positive control (using mitofilin antibody) were performed meanwhile. The present of mitofilin was checked by western blot analysis, respectively.2. Main resultsCompared with positive control, there were positive bands near 90kDa in western blot analysis.3. Main conclusionsPatients with vitiligo have circulating antibodies to mitofilin, and mitofilin is documented to be a vitiligo-associated antigen.PartⅡ: Dopamine induces melanocytes apoptosis involving dysfunction of mitofilin and mitochondria1. Main methodsImmortalized human epidermal melanocytes cell line PIG1 was treated with different concentration of dopamine for 24h or 48h, proliferating activity, apoptosis and mitofilin were assessed with crystal violet assay, flow cytometry and western blot. After treated with dopamine, mitochondrial membrane potential and ultrastructural analysis of melanocytes were investigated. The data was analyzed by unpaired two-tailed Student t test.2. Main resultsCompared with negative controls, dopamine significantly inhibited the proliferation of PIG1 cells in a dose dependent manner; meanwhile the apoptosis rate of the cells was obviously increased. With no difference in the expression of internal standardβ-actin, mitofilin expression was remarkably decreased in cells treated with dopamine. Mitochondrial transmembrane potential is significantly damaged in dopamine treated melanocytes. Ultrastructural study of the mitochondria of treated cells appeared very large, reflecting swelling of the mitochondrial cristae.3. Main conclusionsInner mitochondrial membrane protein mitofilin is one of the critical protein targets of dopamine oxidation. Dopamine oxidation significantly damages the cristae structure and mitochondrial transmembrane potential of melanocytes though down regulation of mitofilin.Part III: Dopamine induced melanocytes apoptosis involving activation of endoplasmic reticulum and mitochondria pathway.1. Main methodsAfter PIG1 cells were treated with 500μM dopamine for 1h, 3h, 6h or 12h, GRP78, PKR, P-PKR, P-eIF2α, Caspase3, Caspase9, Bax, and Bcl-2 were assessed with western blot analysis.2. Main resultsCompared with negative controls with no difference in the expression of internal standardβ-actin, GRP78, P-PKR, P-eIF2α, Caspase3, Caspase9, and Bax expression was remarkably increased in cells treated with dopamine. Meanwhile, Bcl-2 was significant decreased, and PKR was no difference in dopamine treated cells.3. Main conclusionsDopamine induced melanocytes apoptosis involving activation of endoplasmic reticulum and mitochondria pathway.The study documents that vitiligo patients have circulating auto-antibodies to mitofilin, and reveals that mitofilin is one of the critical protein targets to the toxicity induced by DA. The dysfunction/loss of mitofilin induce disorder of mitochondria, increasing generation of ROS and apoptosis of melanocytes, which involving the activation of endoplasmic reticulum and mitochondria pathway.We postulate that there exists a vicious circle in vitiligo. Excessive ROS, which partly produced by metabolic disorder of dopamine, disturbs normal pathways such as antioxidant system, pigmentation, synthesis/recycling of the essential cofactor by oxidizing proteins, DNA, and membranes. Some of these products, such as MDA and dopaquinone, are unstable and can react with mitochondrial molecules in the cell, which induce the disorder of mitochondria. Dysfunction pathways lead to compromised ability to remove ROS and abnormal endogenous ROS generation. Dopamine mediated oxidative stress not only causes decreased mitofilin expression but also deactivates mitofilin. The consequences of it include swelling of the mitochondrial cristae, losing of mitochondrial membrane potential and an increasing of intracellular ROS level. All those further exacerbate oxidative stress and ultimately result in loss of function, even melanocytes death. And the nonenzymatic oxidative modification of proteins and the subsequent accumulation of the modified proteins, such as mitofilin, will released to serum, which will break down the tolerance to self proteins and lead to autoimmune.更多还原