NCSTN基因沉默对HaCaT细胞增殖分化的影响

【摘要】 目的 检测NCSTN基因稳定沉默的人永生化角质形成细胞（HaCaT）增殖活性及相关分化蛋白的改变,初步探索反常性痤疮发病的可能机制。方法 构建慢病毒介导shRNA沉默NCSTN基因的HaCaT细胞模型（shRNA组）,转染空载慢病毒的HaCaT细胞作为阴性对照组,实时定量PCR和Western印迹法检测NCSTN基因的沉默效率。CCK8法检测HaCaT细胞增殖活性,实时定量PCR和Western印迹法检测HaCaT细胞角蛋白（CK1、CK5、CK7、CK10、CK14、CK16、CK17、CK18、CK19、CK20）及其他分化分子（内披蛋白、兜甲蛋白）mRNA和蛋白的表达。两组计量资料的比较采用两独立样本t检验。结果 shRNA组NCSTN mRNA及蛋白表达（0.42 ± 0.19、0.30 ± 0.07）均显著低于阴性对照组（1.00 ± 0.34、1.00 ± 0.26;t = 5.196、2.637,P < 0.001、< 0.05）,基因沉默效率达70%。与阴性对照组相比,shRNA组HaCaT细胞明显增殖活跃,CK16、CK19蛋白表达显著下调（t = 3.787、3.817,P < 0.01、< 0.05）,终末分化分子内披蛋白表达明显降低（t = 2.904,P < 0.05）。结论 稳定沉默NCSTN基因表达会引起HaCaT细胞异常增殖分化,为后续探究NCSTN基因突变引起反常性痤疮提供新思路。     

降钙素基因相关肽通过ERK1/2信号通路对角质形成细胞增殖的影响

目的： 研究降钙素基因相关肽（CGRP）对角质形成细胞增殖活性的影响，并探讨其可能涉及的信号转导通路。方法： ①胸腺嘧啶掺入法（［3H］-TdR）观察CGRP诱导的角质形成细胞株HaCaT细胞增殖，及CGRP受体拮抗剂CGRP8-37、细胞外信号调节激酶ERK1/2特异性抑制剂PD98059对CGRP诱导的增殖活性的影响；②免疫印迹技术观察CGRP诱导后ERK1/2的磷酸化，及CGRP8-37、PD98059对ERK1/2磷酸化的影响。结果： ①CGRP在一定范围内可剂量依赖性地诱导HaCaT细胞增殖，该作用可被CGRP8-37和 PD98059阻断；②CGRP可时间依赖性地诱导HaCaT细胞ERK1/2的磷酸化，CGRP8-37和PD98059可减弱其作用。结论： CGRP可诱导HaCaT细胞增殖，CGRP受体及其相关的ERK1/2信号通路参与其调控机制。     

四硫代钼酸铵作为新型硫化氢供体的鉴定及其对皮肤细胞的保护作用

 目的: 本研究旨在检测金属络合物四硫代钼酸铵(ATTM)的硫化氢(H₂S)释放能力并进一步观察其减轻氧化应激诱导的皮肤细胞损伤的作用。方法:反应瓶法检测ATTM在细胞培养基中释放的H₂S,二氯二氢荧光素二乙酸酯或罗丹明123染色结合荧光显微镜照相术分别检测细胞内活性氧簇(ROS)的含量和线粒体膜电位(ΔΨ_m),用试剂盒检测细胞存活率和乳酸脱氢酶(LDH)的释放。结果:与H₂S供体GYY4137类似,ATTM在细胞培养基中可剂量依赖性地释放H₂S。在25~400 μmol/L 的浓度范围内,ATTM处理对人皮肤细胞(HaCaT细胞)的存活率无明显影响(P>0.05)。紫外线照射或外源性给予ROS供体(过氧化氢,H₂O₂)处理均可增加HaCaT细胞内ROS的含量。400 μmol/L H₂O₂处理可明显降低HaCaT细胞的存活率(P<0.01),而在H₂O₂处理前给予ATTM预处理,细胞存活率明显提高,其中在100和200 μmol/L浓度时ATTM具有最好的细胞保护作用(P<0.01)。400 μmol/L H₂O₂处理还可损害细胞的ΔΨ_m和细胞膜并使LDH释放增加(P<0.01)。而在细胞遭受损伤前给予200 μmol/L ATTM预处理可明显改善ΔΨ_m的水平(P<0.05)并抑制LDH从细胞内释放(P<0.01)。结论:ATTM具有释放H₂S的能力并可保护人皮肤细胞对抗氧化应激诱导的损伤效应。     

中波紫外线对人角质形成细胞MMP-9 mRNA的影响

目的：研究中波紫外线（UVB）诱导的永生化人角质形成细胞中MMP-9 mRNA的表达,探讨其与UVB照射所致的细胞损伤的关系。方法：绘制细胞生长曲线,采用MTT方法测定UVB照射后细胞的增殖活性,反转录-聚合酶链反应（RT-PCR）方法测定UVB照射后HaCaT细胞中MMP-9 mRNA的表达。结果：UVB照射后,HaCaT细胞的增殖活性受到抑制,MMP-9 mRNA表达增强;随UVB剂量增加,照射组的MMP-9 mRNA表达逐渐增多,与未照光组比较,差异有统计学意义（P〈0.01）。结论：UVB照射可引起HaCaT细胞中MMP-9 mRNA表达显著增加,且与UVB照射剂量呈正相关,与光老化的发生有一定的关系。     

人表皮黑素细胞与HaCaT细胞共培养体外模型的建立

目的：建立人表皮黑素细胞与HaCaT细胞共培养的体外模型,观察α-促黑素以及烟酰胺对黑素在两细胞间传递的影响。方法：分别培养人表皮黑素细胞和HaCaT细胞,接种黑素细胞48h后再将HaCaT细胞以1：2的比例与黑素细胞共培养,分别以不同浓度的α-促黑素和烟酰胺干预共培养的细胞9天,Fontana—Masson银染法观察共培养模型中黑素颗粒在两细胞间传递的情况。结果：培养3天即可观察到约20％HaCaT细胞核周围出现从邻近黑素细胞传递来的黑素颗粒,随着时间的延长阳性染色HaCaT细胞比例逐渐增多,第7天达到高峰（约80％）,α-促黑素以及烟酰胺分别以浓度依赖方式促进／抑制了黑素颗粒在两细胞间的传递。结论：成功建立了人表皮黑素细胞与HaCaT细胞共培养体外模型,为大规模筛选促／抑黑素传递的药物奠定了实验基础。     

Comparative evaluation of cytotoxicity and phototoxicity of mono and diacylglycerol amino acid-based surfactants

Extensive efforts have been made, recently, to find surfactants with lower irritancy potential than those presently commercially employed in pharmaceutical and cosmetic preparations. Cytotoxic and phototoxic effects of novel mono and diacylglycerol amino acid-based surfactants (glutamic acid, or arginine) were evaluated.All tested surfactants showed a clear concentration–response relationship to two immortalized cell lines, murine fibroblast cell line, 3T3, and one human keratinocyte cell line, HaCaT, demonstrated by and decrease of NR uptake. Concentrations resulting in 50% inhibition of NR uptake (IC₅₀) range from 30 to 300 μg mL⁻¹.The potential phototoxicity which could result in irritant products, was determined by modulated cytotoxicity via the resazurin reduction to resorufin and neutral red uptake (NRU) endpoints. Surfactants with two chains showed, in general, less cytotoxic but higher phototoxic effect than surfactants with only one chain.     

Preliminary approach to elucidate the role of pigment as a binding site for drugs and chemicals in anagen hair: differential uptake of <Superscript>3</Superscript>H-haloperidol by pigment-producing compared to non-pigment-producing cell lines

A striking difference was observed for cellular-bound drug in HaCaT and Sk-Mel-1 cells for a fixed drug exposure time of 72 h and varying ³H-haloperidol concentrations in the culture media. Drug uptake was dependent on drug concentration and linearly correlated for both the non-pigment- and the pigment-producing cells which however was different in magnitude. In an additional investigation the time course of drug uptake during ³H-haloperidol exposure (400 pmol/ml; 28 days) revealed increasing drug concentrations in the Sk-Mel-1 population, whereas drug concentrations in the keratinocytes reached a plateau within a short time period. In contrast to the HaCaT cells no tendency to saturation was observed for the pigment-producing cell line. At the end of the experiments ³H-haloperidol concentrations in Sk-Mel-1 were found to be approximately tenfold higher than in HaCaT. Received: 7 November 2000 / Accepted: 31 May 2001     

Lipid composition and synthesis of HaCaT cells, an immortalized human keratinocyte line, in comparison with normal human adult keratinocytes

Abstract Cultured keratinocytes are frequently employed for studies of epidermal lipid metabolism. Interpretation of experimental data may be complicated by donor to donor variability, the relatively short culture lifetime and variations between passages, problems thai are not encountered to the same extent with immortalized cell lines. The present study was undertaken to compare the lipid composition and synthesis of normal human adult keratinocytes (NHAK) with HaCaT cells, a long-lived. spontaneously immortalized human keratinocyte line, in relation to proliferation and differentiation. No differences between the two cell types were observed: a) in total lipid content; b) in the distribution of major lipid classes during growth at 50%, 75% and 100% confluence: c) in cultures grown at 0.6 mM calcium, at which differentiation is retarded, or at 1.6 mM calcium, at which some differentiation takes place; d) in the incorporation of [¹⁴C] acetate into cellular lipids at confluence, or e) in the fatty acid composition of major cellular lipid classes. At 100% confluence NHAK and HaCaT cells differ in their cholesterol metabolism. At all stages of growth, cholesterol synthesis in HaCaT cells is more LDL-dependent than in NHAK. Furthermore, NHAK become less LDL-dependent at confluence whereas HaCaT cells do not. HaCaT cells also revealed a significantly larger fraction of phosphatidyl-ethanolamine, -serine and -inositol at 0.6 mM calcium concentration than NHAK. These findings suggest that HaCaT cells do not differentiate as well as NHAK in vitro and may therefore serve as a model for the study of lipid metabolism in cells defective in terminal differentiation.