细胞自噬在黑素细胞氧化应激中的作用
发布时间：2019-04-29 07:29
【摘要】：目的:黑素细胞氧化损伤涉及多种色素相关性皮肤病的发病机制,如白癜风、黑色素瘤等。黑素细胞由于其特殊的功能,以及所处的位置,极易受到内外环境诱导的氧化应激产生的过量活性氧簇造成的氧化损伤,活性氧簇能攻击黑素细胞,干扰黑素细胞正常的代谢、增殖及分化,造成细胞线粒体功能受损、DNA破坏、蛋白修饰改变等,最终可导致黑素细胞恶性转变或凋亡。细胞自噬是一种普遍存在于真核生物细胞内的,高度保守的依赖溶酶体的降解途径,能降解大分子蛋白质、损伤的细胞器及外来微生物等。近来大量研究证实细胞自噬与氧化应激关系密切,且发现多种氧化应激相关疾病的发病过程与自噬功能障碍密切相关,如心脏疾病、神经退行性疾病阿尔茨海默病、帕金森病等。但目前国内外目前对于细胞自噬与黑素细胞氧化损伤之间的关系还没有研究透彻。因此本研究旨在通过检测正常人黑素细胞氧化损伤时细胞自噬的变化,以及检测细胞自噬对黑素细胞氧化损伤相关指标的影响,来初步探讨自噬在黑素细胞氧化应激中扮演的角色。方法:1、从年龄10周岁的儿童包皮组织上分离正常人原代黑素细胞,将其与人黑素细胞系PIG1进行培养:人原代黑素细胞、正常人黑素细胞系PIG1分别培养于254培养基中,放入37℃、含5%CO2的孵箱中常规培养,根据培养基及漂浮细胞情况,一般隔2日或3日换液,待细胞贴壁生长达70%-80%时,以0.25%的胰酶消化传代,用于后续实验。2、构建黑素细胞氧化应激模型:分别用1.0mM的H_2O_2处理正常人原代黑素细胞和人黑素细胞系PIG1,处理24小时后,用MTS法检测细胞活性。3、通过免疫印迹检测黑素细胞氧化损伤下自噬相关蛋白P62、LC3的表达情况,并用激光共聚焦显微镜观察氧化应激下黑素细胞内自噬小体的形成情况。4、分别用自噬促进剂及自噬抑制剂预处理黑素细胞后,以流式细胞技术检测黑素细胞氧化应激下细胞凋亡水平、细胞内ROS水平及线粒体膜电位的变化。结果:1、原代黑素细胞在1.0mM H_2O_2处理24h后,细胞活性降低且差异具有统计学意义(P0.05)。2、1.0mM H_2O_2处理黑素细胞后,黑素细胞自噬相关蛋白LC3II表达逐渐增高,LC3I表达逐渐下降,蛋白P62表达逐渐下降,自噬小体数量明显增多。抑制黑素细胞自噬后,黑素细胞内ROS水平、细胞凋亡水平高于对照组,线粒体膜电位下降(P0.05);促进黑素细胞自噬后,黑素细胞内ROS水平、细胞凋亡水平低于对照组,线粒体膜电位增高(P0.05)。结论:1、1.0mM H_2O_2可导致黑素细胞出现氧化应激并造成细胞出现氧化损伤。2、氧化应激可诱导黑素细胞自噬水平增高。3、氧化应激下,细胞自噬可保护黑素细胞免受氧化损伤。
[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，
本文编号：2468066
[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，
本文编号：2468066
本文链接：https://www.wllwen.com/yixuelunwen/pifb/2468066.html
最近更新
教材专著
