表皮生长因子受体及其配体与角质形成细胞

表皮生长因子受体是角质形成细胞自泌生长因子的主要受体 ,对其功能、功能调节、相关配体和活化后胞内信号转导的研究将有助于了解表皮的自身稳定机制和探讨有关疾病 (如银屑病 )的发病机制及治疗对策。     

人表皮生长因子基因体外转染人角质形成细胞的研究

基因治疗已开始用于多种疾病的治疗,而表皮作为人体最易获取的组织则显示了良好的应用前景 [1]。我们拟采用逆转录聚合酶链反应 (RT PCR)扩增人表皮生长因子 (hEGF)及其信号肽 cDNA,并构建到 pBK CMV穿梭质粒中,再经脂质体 LipofectAMINE介导转染培养的人角质形成细胞,以期获得真核表达的 hEGF蛋白,为表皮组织基因治疗的研究打下基础。 一、材料和方法 (一 )主要试剂：限制性内切酶、 T4 DNA连接酶及 PCR试剂盒等均为美国 Bio Rad公司产品;逆转录试剂盒为 Boehringer Mannheim公司产品; DMEM、 Opti MEM…     

表皮生长因子受体及其配体与角质形成细胞

表皮生长因子受体是角质形成细胞自泌生长因子的主要受体。对其功能、功能调节、相关配体和活化后胞内信号转导的研究将有助于了解表皮的自身稳定机制和探讨有关疾病（如银屑病）的发病机制及治疗对策。     

角质形成细胞生长因子与银屑病的关系

角质形成细胞生长因子是近年来发现的有着重要生物功能的生长因子,它属于成纤维细胞生长因子家族,由成纤维细胞产生。角质形成细胞生长因子可刺激角质形成细胞的增生、分化、移行,促进上皮细胞的再生、增厚,对银屑病的发病机制及其治疗可能有一定的启示。本文就角质形成细胞生长长因子和的生物学功能及其与角质形成细胞及银屑病的关系做一定综述。     

人角质形成细胞的体外改良培养及生物学特征

目的：探讨体外人角质形成细胞的改良培养方法并观察其生物学特征。方法：取健康人包皮环切术包皮,采用EDTA预处理和冷消化法分离表皮,制成细胞悬液,通过倒置相差显微镜、电镜观察其形态学特征,绘制细胞生长曲线;免疫组化（ABC法）测定抗角蛋白单克隆抗体鉴定细胞。结果：体外培养的人皮肤角质形成细胞经过1～2天的潜伏期,即进入指数增生期,持续约5天,然后进入平台期。第7天左右细胞完全融合连成片状,形态呈扁平不规则多边形,免疫组化显示胞浆被染成棕黄色,为角蛋白阳性。透射电镜下见角质形成细胞胞浆内有大量束状张力纤维呈典型角质形成细胞特征。结论：采用改良体外培养方法获得的人角质形成细胞保持正常的生物学特征,从而建立一种简单易行的人角质形成细胞优化培养技术。     

角质形成细胞体外培养技术及应用的进展

角质形成细胞具有广泛的生物学特征 ,对皮肤的结构和功能有着重要的影响。其体外培养技术的建立至今已有近 3 0年的历史。从液体浸没培养到空气 -液体交界面培养 ;从单层细胞培养到复层器官型培养及器官培养 ,形成了多种培养模型可供实验研究和临床应用。当今角质形成细胞的培养技术已广泛应用于皮肤生理学、病理学、药理学、药物毒理学、组织工程学和基因治疗等方面。介绍角质形成细胞体外培养的生长条件和环境、体外培养方法和应用三方面的最新进展。     

角质形成细胞生长因子与银屑病的关系

角质形成细胞生长因子是近年来发现的有着重要生物学功能的生长因子 ,它属于成纤维细胞生长因子家族 ,由成纤维细胞产生。角质形成细胞生长因子可刺激角质形成细胞的增生、分化、移行 ,促进上皮细胞的再生、增厚 ,对银屑病的发病机制及其治疗可能有一定的启示。本文就角质形成细胞生长因子的生物学功能及其与角质形成细胞及银屑病的关系做一综述。     

重组牛碱性成纤维细胞生长因子对体外培养人角质形成细胞保 …

表1 MTT测定牛碱性成纤维细胞生长因子对人角质形成细胞增殖的影响 表2 MTT测定牛碱性成纤维细胞生长因子对人角质形成细胞促增殖作用的最低有效浓度 碱性成纤维细胞生长因子 (bFGF)的主要作用是促进成纤维细胞的增殖。有文献报道 bFGF可以促进角质形成细胞（ KC）的生长和分化过程 [1],且在伤口愈合过程中出现 bFGF和角质形成细胞生长因（ KGF）的表达 [2,3]。噻唑蓝（ MTT）染色光吸收实验法是近年来在国内外广泛用于分析药物对细胞作用的方法,具有简便、准确和快速等优点 [4]。我们用体外培养正常人 KC, MTT法和细胞直接…     

角质形成细胞的体外培养及NF-κB的表达

目的:检测体外培养角质形成细胞的核因子(NF-κB)的表达,以探讨NF-κB在银屑病炎症反应中的作用.方法:采用无血清培养法获得角质形成细胞,与银屑病患者外周血单一核细胞(PBMC)混合培养,利用流式细胞仪检测NF-κB表达.结果:角质形成细胞在5天可见明显的集落,10天左右可长满单层.与正常对照组相比,银屑病患者混合培养细胞较对照角质形成细胞NF-κB表达显著增高.结论:NF-κB活性增强是银屑病炎症反应的重要因素之一.     

大黄素和大黄酸对角质形成细胞体外培养细胞周期的影响

研究大黄蒽醌衍生物对细胞增殖动力学的影响已有文献报告[1] ,但关于大黄蒽醌衍生物对细胞周期的影响 ,尚未见报告。本文通过体外培养细胞的方法 ,采用流式细胞分析仪检测细胞周期变化 ,观察大黄蒽醌衍生物 (大黄素和大黄酸 )对角质形成细胞株ｃｏｌｏ 16的细胞周期影响 ,进一步探讨大黄蒽醌衍生物的药理作用。1　材料和方法1 1　细胞和药物来源 :选用的人表皮角质形成细胞株ｃｏｌｏ 16 (属鳞癌细胞株 ) ,由美国康奈尔大学医学院Ｅｌｋｏｎ ＫＢ教授惠赠。大黄素和大黄酸 ,由北京医科大学药学院郑俊华教授提供的标准品 (纯度 10 0 % )。1 2…     

Effects of human recombinant tumor necrosis factor-alpha (TNF-alpha) on the proliferative potential of human keratinocytes cultured in serum-free medium

The effects of human recombinant tumor necrosis factor-alpha (TNF-alpha) on human keratinocytes cultured in a serum-free medium were investigated. TNF-alpha markedly suppressed cell growth. The growth-inhibitory effect was reversible and cytostatic at a concentration of 1-5 U/ml, but appeared to be irreversible and cytocidal at 10 U/ml. The growth suppressive effect was more marked when TNF-alpha was added in the late growth phase or preconfluent phase than when it was added in early or mid-growth phases. No effects of TNF-alpha on cell adhesion to the substrate were observed. These results indicate that TNF-alpha is a very potent anti-proliferative agent for human keratinocytes.     

Kinetics of withdrawal from the cell cycle in cultured human epidermal keratinocytes

In keratinizing epithelia one of the earliest changes in the process of terminal differentiation is cessation of replication or withdrawal from the cell cycle. In this report, we measured the loss of colony-forming ability, and confirmed that withdrawal from the cell cycle is a specific event that occurs during maturation of the keratinocyte in culture. In addition, the rate of withdrawal was assayed by labeling cultures for 24 h with [14C]dThd and then measuring the fraction of labeled cells that undergo repeated cycles of DNA synthesis. These additional cycles of replication were measured by feeding BrdUrd to the cultures and quantitating the distribution of 14C-labeled DNA in unsubstituted and BrdUrd-substituted DNA in CsCl density gradients. The results show that the fraction of 14C-labeled DNA undergoing replication decreases exponentially by 23% every 24 h. This cessation of replication could not be explained by a reduced level of replication in the entire culture since during each day of the experiment about 8% of the total DNA underwent replication. The exponential decrease in replication of 14C-labeled DNA represents withdrawal from the cell cycle. Since the doubling time for keratinocytes is approximately 24 h, these results suggest that following each cycle of replication, there is a probability of 0.23 that postreplicated cells will withdraw from the cell cycle.     

Regulation of epidermal growth factor receptor expression in normal and transformed keratinocytes

Summary Transformed keratinocytes (SCC-4, SCC-15, SCC-12F2, SVK14) or normal keratinocytes which differ in their differentiation programme were used to study the regulation of EGF-receptor expression. The capacity of the cells to differentiate was modulated by changing the extracellular calcium concentration. We were able to demonstrate that EGF-receptor expression in normal and transformed keratinocytes depends upon the cell type and one or more levels of regulatory control. At the DNA level, EGF-receptor gene amplification occurred in poorly differentiating cells. At the mRNA level, cells showing EGF-receptor gene amplification expressed elevated mRNA and protein levels when cultured under low Ca²⁺ conditions. Cells not exhibiting EGF-receptor gene amplification showed equal mRNA expression, regardless the Ca²⁺ concentration in the culture medium. At the protein level, EGF-receptor protein was decreased in cells exhibiting EGF-receptor gene amplification when extracellular Ca²⁺ was increased (to 1.6 mM) to stimulate differentiation, the decrease in protein being comparable to mRNA expression. Cells not exhibiting EGF-receptor gene amplification showed equal protein expression, regardless of the Ca²⁺ concentration in the culture medium. Under the same conditions, SV40 transformed keratinocytes showed equal mRNA but elevated protein expression in cells grown under low Ca²⁺ conditions. At the membrane level, normal keratinocytes and SCC-12F2 cells showed elevated numbers of cell surface exposed EGF-receptors in cells grown under low Ca²⁺ conditions, but equal mRNA and protein expression. These and previous findings demonstrate that EGF-receptor expression is regulated at the levels of DNA, mRNA and protein as well as by the plasma membrane composition, depending upon the cell type.     

N-Acetylcysteine downregulates vascular endothelial growth factor production by human keratinocytes in vitro

Abstract The present study was designed to evaluate the action of various antioxidants including N-acetylcysteine (NAC) and the flavonoids resveratrol and quercetin on the production of VEGF by human keratinocytes (HKC). NAC, resveratrol, and quercetin dose-dependently suppressed the incorporation of ³H-thymidine into HKC. Values of median inhibitory concentration for NAC, resveratrol, and quercetin were 10 mM, 55 μM, and 15 μM, respectively (P < 0.01). RT-PCR demonstrated VEGF 121 and VEGF 206 expression in all HKC samples. HKC showed baseline expression and a progressive gradual time-dependent increase in VEGF secretion (510 ± 75 pg/ml at 24 h), and EGF (2.5–100 ng/ml) enhanced the secretion of VEGF in a dose-dependent fashion. HKC were incubated with NAC (2.5–20 mM) for 2 h prior to the addition of EGF (5 ng/ml) or PMA (10 ng/ml), and a significant decrease (P < 0.01) was found after 24 h of incubation with 2.5 mM NAC. However, neither resveratrol nor quercetin reduced the synthesis of this cytokine. In summary we conclude that NAC and the flavonoid antioxidants resveratrol and quercetin inhibit HKC proliferation regardless of the stage of differentiation and that NAC significantly inhibits VEGF secretion in basal and EGF- or PMA-treated HKC. Received: 28 February 2000 / Revised: 21 July 2000 / Accepted: 11 October 2000     

UVB radiation induces phosphorylation of the epidermal growth factor receptor, decreases EGF binding and blocks EGF induction of ornithine decarboxylase gene expression in SV40-transformed human keratinocytes

Abstract We previously demonstrated that epidermal growth factor (EGF) induces a several-fold increase in ornithine decarboxylase (ODC) activity and the steady-state level of ODC mRNA in cultured SV40-transformed human keratinocytes (1). Pretreatment of cell cultures with ultraviolet B (UVB) radiation resulted in a reduction of EGF-induced ODC activity. To determine whether UVB inhibits the accumulation of ODC mRNA by EGF, cells were pretreated with 20 mJ/cm² UVB or sham-irradiated and then incubated with 100 ng/ml EGF. Northern blot analysis revealed that UVB irradiation entirely blocked the EGF induction of ODC mRNA. Since the binding of EGF to its plasma membrane receptor is the first step in initiating a biological response, the effect of UVB on EGF binding was evaluated. UVB treatment of cultured keratinocytes resulted in an immediate and dose-dependent reduction of EGF binding. Scatchard analysis revealed thai the reduction of EGF binding was due to a 52% decrease in the number of available receptors, from 6.2 × 10⁴/cell to 3.0 × 10⁴/cell. However, UVB decreased the EGF-binding affinity very little (Kd = 0.60 nM in control and Kd=0.75 nM in UVB-treated Z114 cells). In addition, UVB did not alter the rate of EGF internalization. These data suggest that UVB blocks the signal transduction pathway of EGF that is involved in regulation of ODC gene expression. Immunoblot analysis of extracts from irradiated cells showed that UVB induced tyro-sine phosphorylation of EGFR and that the quantity of EGFR protein was unaffected by UVB treatment. Phosphorylation of EGFR may be responsible for decreased binding of EGF to its receptor.     

Effects of ozone in normal human epidermal keratinocytes

Ozone is a tropospheric pollutant that can form at ground level as a result of an interaction between sunlight and hydrocarbon engine emissions. As ozone is an extremely oxidative reaction product, epidermal cells are in the outer layer of defense against ozone. We exposed normal human epidermal keratinocytes (NHEK) to concentrations of ozone that have been measured in cities and assayed for its effects. Hydrogen peroxide and IL‐1α levels both increased while ATP levels decreased. We found a decrease in the NAD‐dependent histone deacetylase, sirtuin 3. Lastly, we found that ozone increased DNA damage as evaluated by Comet assay. Taken together, our results show increased damage to NHEK that will ultimately impair normal cellular function as a result of an environmentally relevant ozone exposure.     

Regulation of epidermal growth factor receptor expression in human melanocytes

The epidermal growth factor receptor (EGFR) and its ligand, transforming growth factor alpha (TGFalpha), are reportedly involved in autocrine growth of melanoma cells. The signal pathway has also been implicated in early events of transformation, suggesting a function for EGFR in normal cells. This study reports the presence of EGFR in cultured melanocytes and examines some cellular responses to TGFalpha. Western analysis revealed 170 kDa bands in extracts of cultured neonatal human melanocytes, corresponding to the receptor Mr. Protein expression was more pronounced in cells during active growth. EGFR were less evident in cultures populated predominantly by melanized cells, indicating that receptor expression became reduced in differentiating cells. Immunocytochemistry confirmed these observations and also showed that EGFR reactivity was predominantly localized in the cell body but absent from dendrites. Addition of TGFalpha to early cultures induced a rapid increase in phosphotyrosine signal of the 170 kDa protein. Longer treatment (24-48 h) increased the intensity of the EGFR signal, suggesting that receptors had been upregulated. However, inclusion of TGFalpha in cultures did not result in an increase in cell numbers when compared to controls. The observations provide evidence of the existence of a receptor-mediated pathway in melanocytes which has transforming potential in vivo.     

Decreased expression of epidermal cytoplasmic antigens in cultured human keratinocytes

The expression of upper cytoplasmic (U-CYT) antigens which are expressed only in the superficial layers of the epidermis and are markers of epidermal cell differentiation in vivo and of basement zone (BMZ) antigens reacting with bullous pemphigoid serum was studied in keratinocytes in tissue culture. The cells were cultured at an acid pH (5.6-5.8) similar to that of skin and without feeder cells, dermal tissue, or collagen. It was found that the expression of U-CYT antigens decreased markedly in culture. These antigens were expressed in 45-65% of epidermal cells prepared from fresh skin, but in only 5-10% of cells which had been grown in primary culture over 1 mo, and in no cells in secondary or tertiary culture. By contrast, BMZ antigens continued to be expressed in culture. These antigens were expressed by 20-35% of epidermal cells prepared from fresh tissue and by 15-35% of keratinocytes in primary, secondary or tertiary culture. These findings indicate that U-CYT and BMZ antigens can be used to type subpopulations of human keratinocytes in suspension, and suggest that the differentiation of these cells in vitro differs from that which occurs in vivo.