氧化应激诱导自噬在白癜风发病中的作用及机制研究

发布时间：2018-06-12 02:51

  本文选题：白癜风 + 黑素细胞　； 参考：《第四军医大学》2017年博士论文

【摘要】：背景:白癜风是临床常见的皮肤黏膜色素脱失性疾病,本病皮损多发生在曝光部位,重者可造成损容性表现,严重影响患者的身心健康。白癜风发病的具体机制目前尚不清楚,但造成本病发生的最终环节已经证实为患者皮损局部黑素细胞被破坏。新近研究证实,黑素细胞氧化应激状态是导致白癜风患者表皮黑素细胞破坏的关键因素。黑素细胞在受到内外环境刺激导致的氧化应激状态时,细胞内过量的氧自由基可以干扰细胞的正常生理代谢、增殖和分化,并会进一步诱发机体针对自身黑素细胞的免疫反应,导致黑素细胞出现不可逆性损伤。既往研究发现白癜风患者黑素细胞较正常人更易受到氧化氧化应激,但其中的具体机制尚未阐明。因此,明确白癜风患者黑素细胞易受氧化应激的具体机制对于阐明白癜风发病的具体分子机制、提供针对性的临床精准治疗靶点具有极其重要的意义。细胞自噬是真核生物细胞内降解聚集蛋白、受损细胞器及外源微生物等的一种保守的生物学现象。既往研究指出细胞处于氧化应激时胞内增高的ROS可以通过多种信号途径激活细胞自噬,保护细胞免受氧化损伤。Nrf2-ARE通路已被证实是黑素细胞在氧化应激时被激活的关键抗氧化通路,氧化应激时胞内增高的ROS可促进蛋白Nrf2转移入核,与细胞内多种II相解毒酶和抗氧化分子基因启动子区域的ARE结合,从而转录促进其表达以发挥对抗氧化损伤的作用。新近研究指出:自噬过程中关键蛋白p62启动子区的同样有ARE结合位点,Nrf2可入核与p62启动子区域ARE结合,上调其表达,从而参与细胞自噬完成。我们课题组既往已经证实白癜风黑素细胞存在Nrf2-ARE通路异常,由此,我们提出如下的假说:氧化应激状态下,正常黑素细胞内ROS可通过激活Nrf2-P62通路促进细胞自噬,保护黑素细胞抵抗氧化损伤。而白癜风黑素细胞存在ROS激活Nrf2-P62通路异常,导致细胞自噬水平激活障碍,使白癜风黑素细胞易受氧化损伤。目的:1.明确氧化应激时细胞自噬在正常人黑素细胞抗氧化损伤中的作用;2.分析氧化应激时白癜风黑素细胞自噬水平与正常人黑素细胞间的差异及其作用;3.揭示氧化应激时Nrf2-p62通路对正常黑素细胞及白癜风黑素细胞自噬的调控差异及其机制。方法:1.以不同浓度H_2O_2处理正常人黑素细胞系PIG1,CCK8法检测细胞活性,确定最佳的体外黑素细胞氧化应激模型;2.H_2O_2处理细胞不同时间,以免疫印迹、透射电镜及激光共聚焦显微镜检测正常人原代黑素细胞、正常人黑素细胞系PIG1、白癜风患者黑素细胞系PIG3V细胞自噬的水平;3.H_2O_2、自噬抑制剂及自噬促进剂处理细胞后,以流式细胞技术检测正常人原代黑素细胞、正常人黑素细胞系PIG1、白癜风患者黑素细胞系PIG3V细胞凋亡、线粒体膜电位及胞内ROS的变化情况。4.分别以Nrf2、p62干涉片段或过表达质粒转染正常人黑素细胞系PIG1及白癜风患者黑素细胞系PIG3V,再予以H_2O_2处理细胞后,以免疫印迹、Real-time PCR和激光共聚焦显微镜检测细胞自噬水平变化。5.分别以Nrf2干涉片段及p62过表达质粒转染白癜风患者黑素细胞系PIG3V,再予以H_2O_2处理细胞后,以流式细胞技术、免疫印迹和激光共聚焦显微镜检测细胞凋亡、线粒体膜电位、胞内ROS及细胞自噬水平变化。结果:1.体外培养的正常人原代黑素细胞及正常人黑素细胞系PIG1在H_2O_2作用下细胞活性呈浓度依赖性降低,0.5 mM H_2O_2处理细胞24 h可建立最佳的体外黑素细胞氧化应激模型;2.H_2O_2作用细胞一段时间内可呈时间依赖性显著刺激正常人原代黑素细胞及正常人黑素细胞系PIG1中自噬相关蛋白LC3II/I表达比值升高,细胞自噬溶酶体数量增多;3.自噬促进剂雷帕霉素预处理正常人原代黑素细胞及正常人黑素细胞系PIG1细胞后,可显著降低两种细胞在H_2O_2作用后的细胞凋亡比例及胞内ROS水平,显著提高两种细胞在H_2O_2作用后的细胞线粒体膜电位水平。自噬抑制剂氯喹预处理正常人原代黑素细胞及正常人黑素细胞系PIG1细胞后,可显著增高两种细胞在H_2O_2作用后的细胞凋亡比例及胞内ROS水平,显著降低两种细胞在H_2O_2作用后的细胞线粒体膜电位水平;4.H_2O_2作用正常人黑素细胞系PIG1一定时间后,细胞内自噬相关蛋白LC3II/I表达比值及自噬溶酶体数量显著高于H_2O_2作用白癜风患者黑素细胞系PIG3V后。同时H_2O_2作用正常人黑素细胞系PIG1一定时间后,细胞凋亡比例及胞内ROS水平显著低于H_2O_2作用白癜风患者黑素细胞系PIG3V后,细胞线粒体膜电位水平显著高于H_2O_2作用白癜风患者黑素细胞系PIG3V后;5.敲低正常人黑素细胞系PIG1中蛋白Nrf2表达后,H_2O_2诱导的细胞内自噬相关蛋白LC3II/I表达比值、蛋白p62水平及自噬溶酶体数量显著低于转染空白对照组。敲低正常人黑素细胞系PIG1中蛋白p62表达后,H_2O_2诱导的细胞内自噬相关蛋白LC3II/I表达比值及自噬溶酶体数量显著低于转染空白对照组。在敲低蛋白Nrf2的正常人黑素细胞系PIG1中过表达蛋白p62,H_2O_2诱导的细胞内自噬相关蛋白LC3II/I表达比值及自噬溶酶体数量显著高于单纯敲低蛋白Nrf2的正常人黑素细胞系PIG1组;6.以氯喹预处理细胞以抑制胞内蛋白经自噬降解后,H_2O_2作用细胞不同时间段,正常人黑素细胞系PIG1细胞内蛋白Nrf2及P62的表达显著高于白癜风患者黑素细胞系PIG3V,正常人黑素细胞系PIG1细胞核内蛋白Nrf2表达显著高于白癜风患者黑素细胞系PIG3V;7.在白癜风患者黑素细胞系PIG3V中过表达蛋白p62,H_2O_2诱导的细胞内自噬相关蛋白LC3II/I比值及自噬溶酶体数量显著高于白癜风患者黑素细胞系PIG3V的转染空白对照组。在白癜风患者黑素细胞系PIG3V中过表达蛋白p62,H_2O_2作用后的细胞凋亡比例及胞内ROS水平显著低于白癜风患者黑素细胞系PIG3V的转染空白对照组,细胞线粒体膜电位水平显著高于白癜风患者黑素细胞系PIG3V的转染空白对照组。结论:通过本研究,我们首次明确了氧化应激可促进正常人黑素细胞中细胞自噬水平升高,氧化应激诱导的细胞自噬水平升高可以保护黑素细胞抵抗氧化损伤。在此基础上,我们还进一步解析了Nrf2-p62信号通路调控氧化应激诱导的正常人黑素细胞自噬水平变化的机制,揭示了Nrf2-p62信号通路的激活障碍是白癜风患者黑素细胞更易出现氧化损伤的原因。本研究进一步完善了白癜风氧化应激发病机制的具体环节,并且将可能为白癜风的临床治疗提供新的思路。
[Abstract]:Background: vitiligo is a common skin mucosal pigment loss disease in the clinic. The skin lesion of this disease occurs mostly at the exposure site, the heavy person can cause the loss of capacitive performance, which seriously affects the physical and mental health of the patients. The specific mechanism of the onset of vitiligo is still unclear, but the final link of the occurrence of costing disease has been proved to be the local melanin thin of the patient's skin lesions. Recent studies have confirmed that oxidative stress in melanocytes is a key factor in the damage of melanocytes in vitiligo patients. When melanocytes are subjected to oxidative stress caused by internal and external environmental stimuli, excessive oxygen free radicals in cells can interfere with normal physiological metabolism, proliferation and differentiation of cells and will be further developed. It has been found that melanocytes in vitiligo patients are more susceptible to oxidative stress than normal people, but the specific mechanisms have not been clarified. Therefore, the specific mechanism of melanocytes in vitiligo patients is clear. Clarifying the specific molecular mechanism of the pathogenesis of vitiligo is of great significance to provide targeted targeted therapeutic targets. Autophagy is a conservative biological phenomenon in the degradation of aggregation proteins in eukaryotic cells, damaged organelles and exogenous microbes. Previous studies have pointed out that cells are in the cell of R in oxidative stress. OS can activate cell autophagy through a variety of signaling pathways to protect cells from oxidative damage.Nrf2-ARE pathway has been proved to be a key antioxidant pathway activated by melanocytes during oxidative stress. The elevated ROS in oxidative stress can promote the transfer of protein Nrf2 into the nucleus, and the initiation of a variety of II phase detoxification and antioxidant genes in cells. ARE binding in the subregion of the subregion promotes its expression to play a role in anti oxidative damage. Recently, the new study indicates that the p62 promoter region of the key protein in the process of autophagy also has the ARE binding site, and Nrf2 can join the nucleus with the p62 promoter region ARE to increase its expression and participate in the completion of autophagy. Our group has previously proved that In the substantia melanocytes of vitiligo, the Nrf2-ARE pathway is abnormal. Therefore, we propose the following hypothesis: in oxidative stress, ROS can promote autophagy by activating the Nrf2-P62 pathway in normal melanocytes and protect melanocytes against oxidative damage. Melanocytes in vitiligo are deposited in ROS to activate the Nrf2-P62 pathway to induce autophagic water. Objective: 1. to determine the role of autophagy in the oxidative stress of normal human melanocytes during oxidative stress. 2. analysis of the difference between the autophagy level of melanocytes in vitiligo and the normal human melanocytes during oxidative stress and its role; 3. to reveal the Nrf2-p62 pathway in oxidative stress. Regulation difference and mechanism of autophagy between normal melanocytes and vitiligo melanocytes. Methods: 1. the activity of melanocytes in normal human melanocyte line was treated with different concentrations of H_2O_2, PIG1, CCK8 method was used to detect the activity of cells, and the optimal oxidative stress model of melanocytes in vitro was determined. 2.H_2O_2 treated cells at different time with immunoblotting, transmission electron microscopy and laser confocal Microscopic examination of normal human melanocytes, normal human melanocyte line PIG1, the level of autophagy in the melanocyte line PIG3V cells of vitiligo patients; 3.H_2O_2, autophagy inhibitors and autophagy enhancers to treat cells, the normal human melanocytes were detected by flow cytometry, PIG1 in normal human melanocytes and melanocytes in vitiligo patients. The changes of PIG3V cell apoptosis, mitochondrial membrane potential and intracellular ROS.4. were transfected with Nrf2, p62 interference fragments or overexpressed plasmids to normal human melanocyte line PIG1 and the melanocyte line PIG3V in vitiligo patients. After H_2O_2 processing cells, the autophagic water was detected by immunoblotting, Real-time PCR and laser confocal microscopy. .5. was transfected with Nrf2 interference fragment and p62 overexpression plasmid to transfect melanocyte line PIG3V in vitiligo patients and then H_2O_2 treated cells. Cell apoptosis, mitochondrial membrane potential, intracellular ROS and cellular autophagy were detected by flow cytometry, immunoblotting and laser confocal microscopy. Results: 1. normal culture was normal in vitro. The cell activity of human primary melanocytes and normal human melanocyte line PIG1 decreased in a concentration dependent manner under the action of H_2O_2. 0.5 mM H_2O_2 treated cells 24 h could establish the optimal oxidative stress model of melanocytes in vitro, and 2.H_2O_2 acting cells could stimulate normal human primary melanocytes and normal people in a time dependent manner for a period of time. The expression of autophagy related protein LC3II/I in melanocyte line PIG1 increased and the number of autophagic lysosomes increased. 3. autophagic accelerant, rapamycin pretreated normal human melanocytes and normal human melanocyte line PIG1 cells, significantly reduced the proportion of apoptosis and the intracellular ROS level of two cells after the action of H_2O_2. The mitochondrial membrane potential level of two cells was enhanced after the action of H_2O_2. After chloroquine pretreated normal human melanocytes and normal human melanocyte line PIG1 cells, the percentage of apoptosis and the intracellular ROS level after the action of H_2O_2 were significantly increased by the autophagic inhibitor chloroquine, which significantly reduced the effect of two cells after the action of H_2O_2. The level of cell mitochondrial membrane potential; the expression ratio of autophagy related protein LC3II/I and the number of autophagic lysosomes in the normal human melanocyte line PIG1 after 4.H_2O_2 action in normal people were significantly higher than that of the melanocyte line PIG3V in the patients with vitiligo by H_2O_2. The apoptosis ratio of the melanocyte line in normal human melanocyte line was compared with that of the normal H_2O_2 human melanocyte line. After the melanocyte line PIG3V of patients with vitiligo, the level of intracellular ROS was significantly lower than that of H_2O_2 in patients with vitiligo. The level of mitochondrial membrane potential was significantly higher than that of melanocyte line PIG3V in patients with vitiligo. 5. after the expression of protein Nrf2 in PIG1, the LC3II/I expression ratio of autophagic related proteins induced by H_2O_2 was induced by H_2O_2. The level of protein p62 and the number of autophagy lysosomes were significantly lower than that in the blank control group. After the expression of protein p62 in PIG1, the LC3II/I expression ratio of autophagy related protein and the number of autophagic lysosomes induced by H_2O_2 were significantly lower than that in the blank control group. The PIG1 in the normal human melanocyte line knocking low protein Nrf2 was PIG1 The expression ratio of autophagy related protein LC3II/I expression and the number of autophagic lysosomes induced by H_2O_2 were significantly higher than those of normal human melanocyte PIG1 group with simple knockout Nrf2. 6. chloroquine pretreated cells to inhibit intracellular protein degradation and H_2O_2 cells in different time periods and normal human melanocytes. The expression of protein Nrf2 and P62 in PIG1 cells was significantly higher than that of melanocyte line PIG3V in vitiligo patients. The expression of protein Nrf2 in melanocyte PIG1 in normal human melanocyte line was significantly higher than that of melanocyte line PIG3V in vitiligo patients; 7. in the melanocyte PIG3V of vitiligo patients, the overexpressed protein p62 and H_2O_2 induced intracellular autophagy associated protein LC. The ratio of 3II/I and the number of autophagy lysosomes were significantly higher than that of the blank control group transfected with the melanocyte line PIG3V of vitiligo patients. In the melanocyte line PIG3V of vitiligo patients, the overexpression of protein p62, the proportion of apoptosis and the intracellular ROS level after the action of H_2O_2 were significantly lower than that of the blank control group of the melanocyte line PIG3V in the patients with vitiligo. The mitochondrial membrane potential level is significantly higher than that of the blank control group transfected with melanocyte line PIG3V in vitiligo patients. Conclusion: through this study, we have first made clear that oxidative stress can promote the increase of cell autophagy in normal human melanocytes. The increase of autophagy induced by oxidative stress can protect melanocytes against oxidative damage. On this basis, we further analyzed the mechanism of Nrf2-p62 signaling pathway regulating the changes of autophagy induced by oxidative stress in normal human melanocytes, and revealed that the activation obstacle of Nrf2-p62 signaling pathway is the cause of more oxidative damage to melanocytes in patients with vitiligo. This study further perfected the oxidation of vitiligo to be stimulated. The specific mechanism of the disease mechanism may provide new ideas for the clinical treatment of vitiligo.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R758.41
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本文编号：2007966