本文選題：白癜風(fēng) + 黑素細胞�。� 參考：《第四軍醫(yī)大學(xué)》2017年博士論文

【摘要】：背景:白癜風(fēng)是臨床常見的皮膚黏膜色素脫失性疾病,本病皮損多發(fā)生在曝光部位,重者可造成損容性表現(xiàn),嚴重影響患者的身心健康。白癜風(fēng)發(fā)病的具體機制目前尚不清楚,但造成本病發(fā)生的最終環(huán)節(jié)已經(jīng)證實為患者皮損局部黑素細胞被破壞。新近研究證實,黑素細胞氧化應(yīng)激狀態(tài)是導(dǎo)致白癜風(fēng)患者表皮黑素細胞破壞的關(guān)鍵因素。黑素細胞在受到內(nèi)外環(huán)境刺激導(dǎo)致的氧化應(yīng)激狀態(tài)時,細胞內(nèi)過量的氧自由基可以干擾細胞的正常生理代謝、增殖和分化,并會進一步誘發(fā)機體針對自身黑素細胞的免疫反應(yīng),導(dǎo)致黑素細胞出現(xiàn)不可逆性損傷。既往研究發(fā)現(xiàn)白癜風(fēng)患者黑素細胞較正常人更易受到氧化氧化應(yīng)激,但其中的具體機制尚未闡明。因此,明確白癜風(fēng)患者黑素細胞易受氧化應(yīng)激的具體機制對于闡明白癜風(fēng)發(fā)病的具體分子機制、提供針對性的臨床精準治療靶點具有極其重要的意義。細胞自噬是真核生物細胞內(nèi)降解聚集蛋白、受損細胞器及外源微生物等的一種保守的生物學(xué)現(xiàn)象。既往研究指出細胞處于氧化應(yīng)激時胞內(nèi)增高的ROS可以通過多種信號途徑激活細胞自噬,保護細胞免受氧化損傷。Nrf2-ARE通路已被證實是黑素細胞在氧化應(yīng)激時被激活的關(guān)鍵抗氧化通路,氧化應(yīng)激時胞內(nèi)增高的ROS可促進蛋白Nrf2轉(zhuǎn)移入核,與細胞內(nèi)多種II相解毒酶和抗氧化分子基因啟動子區(qū)域的ARE結(jié)合,從而轉(zhuǎn)錄促進其表達以發(fā)揮對抗氧化損傷的作用。新近研究指出:自噬過程中關(guān)鍵蛋白p62啟動子區(qū)的同樣有ARE結(jié)合位點,Nrf2可入核與p62啟動子區(qū)域ARE結(jié)合,上調(diào)其表達,從而參與細胞自噬完成。我們課題組既往已經(jīng)證實白癜風(fēng)黑素細胞存在Nrf2-ARE通路異常,由此,我們提出如下的假說:氧化應(yīng)激狀態(tài)下,正常黑素細胞內(nèi)ROS可通過激活Nrf2-P62通路促進細胞自噬,保護黑素細胞抵抗氧化損傷。而白癜風(fēng)黑素細胞存在ROS激活Nrf2-P62通路異常,導(dǎo)致細胞自噬水平激活障礙,使白癜風(fēng)黑素細胞易受氧化損傷。目的:1.明確氧化應(yīng)激時細胞自噬在正常人黑素細胞抗氧化損傷中的作用;2.分析氧化應(yīng)激時白癜風(fēng)黑素細胞自噬水平與正常人黑素細胞間的差異及其作用;3.揭示氧化應(yīng)激時Nrf2-p62通路對正常黑素細胞及白癜風(fēng)黑素細胞自噬的調(diào)控差異及其機制。方法:1.以不同濃度H_2O_2處理正常人黑素細胞系PIG1,CCK8法檢測細胞活性,確定最佳的體外黑素細胞氧化應(yīng)激模型;2.H_2O_2處理細胞不同時間,以免疫印跡、透射電鏡及激光共聚焦顯微鏡檢測正常人原代黑素細胞、正常人黑素細胞系PIG1、白癜風(fēng)患者黑素細胞系PIG3V細胞自噬的水平;3.H_2O_2、自噬抑制劑及自噬促進劑處理細胞后,以流式細胞技術(shù)檢測正常人原代黑素細胞、正常人黑素細胞系PIG1、白癜風(fēng)患者黑素細胞系PIG3V細胞凋亡、線粒體膜電位及胞內(nèi)ROS的變化情況。4.分別以Nrf2、p62干涉片段或過表達質(zhì)粒轉(zhuǎn)染正常人黑素細胞系PIG1及白癜風(fēng)患者黑素細胞系PIG3V,再予以H_2O_2處理細胞后,以免疫印跡、Real-time PCR和激光共聚焦顯微鏡檢測細胞自噬水平變化。5.分別以Nrf2干涉片段及p62過表達質(zhì)粒轉(zhuǎn)染白癜風(fēng)患者黑素細胞系PIG3V,再予以H_2O_2處理細胞后,以流式細胞技術(shù)、免疫印跡和激光共聚焦顯微鏡檢測細胞凋亡、線粒體膜電位、胞內(nèi)ROS及細胞自噬水平變化。結(jié)果:1.體外培養(yǎng)的正常人原代黑素細胞及正常人黑素細胞系PIG1在H_2O_2作用下細胞活性呈濃度依賴性降低,0.5 mM H_2O_2處理細胞24 h可建立最佳的體外黑素細胞氧化應(yīng)激模型;2.H_2O_2作用細胞一段時間內(nèi)可呈時間依賴性顯著刺激正常人原代黑素細胞及正常人黑素細胞系PIG1中自噬相關(guān)蛋白LC3II/I表達比值升高,細胞自噬溶酶體數(shù)量增多;3.自噬促進劑雷帕霉素預(yù)處理正常人原代黑素細胞及正常人黑素細胞系PIG1細胞后,可顯著降低兩種細胞在H_2O_2作用后的細胞凋亡比例及胞內(nèi)ROS水平,顯著提高兩種細胞在H_2O_2作用后的細胞線粒體膜電位水平。自噬抑制劑氯喹預(yù)處理正常人原代黑素細胞及正常人黑素細胞系PIG1細胞后,可顯著增高兩種細胞在H_2O_2作用后的細胞凋亡比例及胞內(nèi)ROS水平,顯著降低兩種細胞在H_2O_2作用后的細胞線粒體膜電位水平;4.H_2O_2作用正常人黑素細胞系PIG1一定時間后,細胞內(nèi)自噬相關(guān)蛋白LC3II/I表達比值及自噬溶酶體數(shù)量顯著高于H_2O_2作用白癜風(fēng)患者黑素細胞系PIG3V后。同時H_2O_2作用正常人黑素細胞系PIG1一定時間后,細胞凋亡比例及胞內(nèi)ROS水平顯著低于H_2O_2作用白癜風(fēng)患者黑素細胞系PIG3V后,細胞線粒體膜電位水平顯著高于H_2O_2作用白癜風(fēng)患者黑素細胞系PIG3V后;5.敲低正常人黑素細胞系PIG1中蛋白Nrf2表達后,H_2O_2誘導(dǎo)的細胞內(nèi)自噬相關(guān)蛋白LC3II/I表達比值、蛋白p62水平及自噬溶酶體數(shù)量顯著低于轉(zhuǎn)染空白對照組。敲低正常人黑素細胞系PIG1中蛋白p62表達后,H_2O_2誘導(dǎo)的細胞內(nèi)自噬相關(guān)蛋白LC3II/I表達比值及自噬溶酶體數(shù)量顯著低于轉(zhuǎn)染空白對照組。在敲低蛋白Nrf2的正常人黑素細胞系PIG1中過表達蛋白p62,H_2O_2誘導(dǎo)的細胞內(nèi)自噬相關(guān)蛋白LC3II/I表達比值及自噬溶酶體數(shù)量顯著高于單純敲低蛋白Nrf2的正常人黑素細胞系PIG1組;6.以氯喹預(yù)處理細胞以抑制胞內(nèi)蛋白經(jīng)自噬降解后,H_2O_2作用細胞不同時間段,正常人黑素細胞系PIG1細胞內(nèi)蛋白Nrf2及P62的表達顯著高于白癜風(fēng)患者黑素細胞系PIG3V,正常人黑素細胞系PIG1細胞核內(nèi)蛋白Nrf2表達顯著高于白癜風(fēng)患者黑素細胞系PIG3V;7.在白癜風(fēng)患者黑素細胞系PIG3V中過表達蛋白p62,H_2O_2誘導(dǎo)的細胞內(nèi)自噬相關(guān)蛋白LC3II/I比值及自噬溶酶體數(shù)量顯著高于白癜風(fēng)患者黑素細胞系PIG3V的轉(zhuǎn)染空白對照組。在白癜風(fēng)患者黑素細胞系PIG3V中過表達蛋白p62,H_2O_2作用后的細胞凋亡比例及胞內(nèi)ROS水平顯著低于白癜風(fēng)患者黑素細胞系PIG3V的轉(zhuǎn)染空白對照組,細胞線粒體膜電位水平顯著高于白癜風(fēng)患者黑素細胞系PIG3V的轉(zhuǎn)染空白對照組。結(jié)論:通過本研究,我們首次明確了氧化應(yīng)激可促進正常人黑素細胞中細胞自噬水平升高,氧化應(yīng)激誘導(dǎo)的細胞自噬水平升高可以保護黑素細胞抵抗氧化損傷。在此基礎(chǔ)上,我們還進一步解析了Nrf2-p62信號通路調(diào)控氧化應(yīng)激誘導(dǎo)的正常人黑素細胞自噬水平變化的機制,揭示了Nrf2-p62信號通路的激活障礙是白癜風(fēng)患者黑素細胞更易出現(xiàn)氧化損傷的原因。本研究進一步完善了白癜風(fēng)氧化應(yīng)激發(fā)病機制的具體環(huán)節(jié),并且將可能為白癜風(fēng)的臨床治療提供新的思路。
[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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R758.41
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本文編號：2007966