【摘要】：目的:黑素細(xì)胞氧化損傷涉及多種色素相關(guān)性皮膚病的發(fā)病機(jī)制,如白癜風(fēng)、黑色素瘤等。黑素細(xì)胞由于其特殊的功能,以及所處的位置,極易受到內(nèi)外環(huán)境誘導(dǎo)的氧化應(yīng)激產(chǎn)生的過量活性氧簇造成的氧化損傷,活性氧簇能攻擊黑素細(xì)胞,干擾黑素細(xì)胞正常的代謝、增殖及分化,造成細(xì)胞線粒體功能受損、DNA破壞、蛋白修飾改變等,最終可導(dǎo)致黑素細(xì)胞惡性轉(zhuǎn)變或凋亡。細(xì)胞自噬是一種普遍存在于真核生物細(xì)胞內(nèi)的,高度保守的依賴溶酶體的降解途徑,能降解大分子蛋白質(zhì)、損傷的細(xì)胞器及外來微生物等。近來大量研究證實(shí)細(xì)胞自噬與氧化應(yīng)激關(guān)系密切,且發(fā)現(xiàn)多種氧化應(yīng)激相關(guān)疾病的發(fā)病過程與自噬功能障礙密切相關(guān),如心臟疾病、神經(jīng)退行性疾病阿爾茨海默病、帕金森病等。但目前國內(nèi)外目前對(duì)于細(xì)胞自噬與黑素細(xì)胞氧化損傷之間的關(guān)系還沒有研究透徹。因此本研究旨在通過檢測(cè)正常人黑素細(xì)胞氧化損傷時(shí)細(xì)胞自噬的變化,以及檢測(cè)細(xì)胞自噬對(duì)黑素細(xì)胞氧化損傷相關(guān)指標(biāo)的影響,來初步探討自噬在黑素細(xì)胞氧化應(yīng)激中扮演的角色。方法:1、從年齡10周歲的兒童包皮組織上分離正常人原代黑素細(xì)胞,將其與人黑素細(xì)胞系PIG1進(jìn)行培養(yǎng):人原代黑素細(xì)胞、正常人黑素細(xì)胞系PIG1分別培養(yǎng)于254培養(yǎng)基中,放入37℃、含5%CO2的孵箱中常規(guī)培養(yǎng),根據(jù)培養(yǎng)基及漂浮細(xì)胞情況,一般隔2日或3日換液,待細(xì)胞貼壁生長達(dá)70%-80%時(shí),以0.25%的胰酶消化傳代,用于后續(xù)實(shí)驗(yàn)。2、構(gòu)建黑素細(xì)胞氧化應(yīng)激模型:分別用1.0mM的H_2O_2處理正常人原代黑素細(xì)胞和人黑素細(xì)胞系PIG1,處理24小時(shí)后,用MTS法檢測(cè)細(xì)胞活性。3、通過免疫印跡檢測(cè)黑素細(xì)胞氧化損傷下自噬相關(guān)蛋白P62、LC3的表達(dá)情況,并用激光共聚焦顯微鏡觀察氧化應(yīng)激下黑素細(xì)胞內(nèi)自噬小體的形成情況。4、分別用自噬促進(jìn)劑及自噬抑制劑預(yù)處理黑素細(xì)胞后,以流式細(xì)胞技術(shù)檢測(cè)黑素細(xì)胞氧化應(yīng)激下細(xì)胞凋亡水平、細(xì)胞內(nèi)ROS水平及線粒體膜電位的變化。結(jié)果:1、原代黑素細(xì)胞在1.0mM H_2O_2處理24h后,細(xì)胞活性降低且差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。2、1.0mM H_2O_2處理黑素細(xì)胞后,黑素細(xì)胞自噬相關(guān)蛋白LC3II表達(dá)逐漸增高,LC3I表達(dá)逐漸下降,蛋白P62表達(dá)逐漸下降,自噬小體數(shù)量明顯增多。抑制黑素細(xì)胞自噬后,黑素細(xì)胞內(nèi)ROS水平、細(xì)胞凋亡水平高于對(duì)照組,線粒體膜電位下降(P0.05);促進(jìn)黑素細(xì)胞自噬后,黑素細(xì)胞內(nèi)ROS水平、細(xì)胞凋亡水平低于對(duì)照組,線粒體膜電位增高(P0.05)。結(jié)論:1、1.0mM H_2O_2可導(dǎo)致黑素細(xì)胞出現(xiàn)氧化應(yīng)激并造成細(xì)胞出現(xiàn)氧化損傷。2、氧化應(yīng)激可誘導(dǎo)黑素細(xì)胞自噬水平增高。3、氧化應(yīng)激下,細(xì)胞自噬可保護(hù)黑素細(xì)胞免受氧化損傷。
[Abstract]:Aim: melanocyte oxidative damage is involved in the pathogenesis of pigmented skin diseases such as vitiligo melanoma and so on. Due to its special function and location, melanocytes are vulnerable to oxidative damage caused by excessive active oxygen clusters induced by oxidative stress induced by internal and external environment. Active oxygen clusters can attack melanocytes and interfere with normal metabolism of melanocytes. Proliferation and differentiation, resulting in damage to mitochondrial function, DNA damage, protein modification changes and so on, can eventually lead to malignant transformation or apoptosis of melanocytes. Autophagy is a highly conserved lysosome-dependent degradation pathway commonly found in eukaryotes, which can degrade macromolecular proteins, damaged organelles and foreign microorganisms. Recently, a large number of studies have confirmed that autophagy is closely related to oxidative stress, and that the pathogenesis of various oxidative stress-related diseases is closely related to autophagy dysfunction, such as heart disease, neurodegenerative disease, Alzheimer's disease, Parkinson's disease, etc. However, at present, the relationship between autophagy and melanocyte oxidative damage has not been thoroughly studied at home and abroad. Therefore, the aim of this study was to detect the changes of autophagy in normal human melanocytes during oxidative injury and the effect of autophagy on the oxidative damage of melanocytes. To explore the role of autophagy in melanocyte oxidative stress. Methods: 1. Normal human primary melanocytes were isolated from 10-year-old children's foreskin and cultured with human melanocyte cell line PIG1: human primary melanocyte and normal human melanocyte cell line PIG1 were cultured in 254medium, respectively. Put in 37 鈩，

本文編號(hào)：2468066