肝细胞生长因子对体外培养的人外泌汗腺腺上皮细胞迁移、凋亡及p-Akt表达的影响

Objective To investigate the effect of hepatocyte growth factor (HGF) on migration and apoptosis of,as well as phosphorylated-Akt (p-Akt) expression in cultured human eccrine sweat gland epithehal cells (hESGc).Methods The first generation of hESGc were cultured in keratinocyte serum free medium (KSFM) and treated with various concentrations (2,20,40μg/L) of HGF for different durations.Then,cell scratch test was performed to detect cell migration,a double staining flow cytometry assay using annexin VFITC/propidium iodide to detect cell apoptosis.and Western blot to measure the expression of p-Akt.Results HGF of 2μg/L had no effect on the migration of hESGc,while that of 20 μg/L and 40μg/L could promote the migration of hESGc by 33.2% and 228.2%.respectively.The average number of cells migrating into the scrach zone was significantly lower in untreated cell group than that in 20 and 40μg/L HGF-treated cell group (17.3±5.5 vs 23.0±6.3 and 56.7±7.9,t=2.653, 15.858,P<0.05,0.01, respectively).The apoptosis rate was 14.76% in untreated cells,14.16%,13.5% and 8.87% in cells treated with HGF of 2,20 and 40μ/L, respectively;there was a significant difference between untreated cells and 40μg/L HGF-treated cells (t=7.852,P<0.01).HGF could activate the phosphorylation of Akt protein and increase the expression of p-Akt.Conclusion HGF could promote the migration of,inhibit the apoptosis of,and stimulate the p-Akt expression in.hESGc.     

Effects of tacrolimus on the expression of nuclear factor-κB and glucocorticoid receptor by HaCaT cells in vitro

Objective To investigate the effect of tacrolimus on the expression of nuclear factor-κB (NF-κB) in HaCaT cells stimulated by tumor necrosis factor-α(TNF-α),and on the expression of glucocorticoid receptor (GR)α and β in untreated HaCaT cells in vitro.Methods Cultured Ha CaT cells were treated with TNF-α(10μg/L) only,combination of TNF-α(10μg/L) and various concentrations (10-8mol/L, 10-7mol/L,10-6moL/L) of tacrolimus or tacrolimus of different concentrations only.After additional 12-,24-, 36- or 48-hour cnlture, Westem blot and immunofluorescenee-confocal laser scanning microscopy were used to detect the expressions of NF-κB,GRα and GRβ in HaCaT cells.Those untreated HaCaT cells served as the control.Results The relative protein expression level of NF-κB was increased in HaCaT cells after treatment with TNF-α for 24 and 48 hours zompared with untreated ceils (0.73±0.0316 and 0.8925±0.0171 vs 0.4988±0.03506,both P<0.05);however,the increase in NF-κB expression was inhibited by the combination treatment with tacrolimus,and the relative expression level of NF-κB protein was 0.6825±0.0263.0.6200±0.0163 and 0.5575±0.0299 in HaCaT cells treated with TNF-α plus tacrolimus of 10-8mol/L 10-7mol/L and 10-6mol/L,respectively;the difference was significant etween TNF-α-treated cells and those dealt with the combination of NF-α and tacrolimus of 10-7 or 10-6 mol/L (both P<0.05).No significant difference was observed in the expression of NF-κB by HaCaT cells between different time oints treated with tacrolimus of 10-8,10-7 or 10-6 mol/L.Also,there was no zignificant difference in the expression of GRα or GRβ between untreated HaCaT cells and those treated with tacrolimus of 10-8, 10-7 or 10-6 mol/L at any time point.Conclusions Tacrolimus ould inhibit the expression of NF-κB by TNF-α-stimulated HaCaT cells,but does not affect the expression of GRα or GRβ,in untreated HaCaT cells.     

Effects of tacrolimus on the expression of nuclear factor-κB and glucocorticoid receptor by HaCaT cells in vitro

Objective To investigate the effect of tacrolimus on the expression of nuclear factor-κB (NF-κB) in HaCaT cells stimulated by tumor necrosis factor-α(TNF-α),and on the expression of glucocorticoid receptor (GR)α and β in untreated HaCaT cells in vitro.Methods Cultured Ha CaT cells were treated with TNF-α(10μg/L) only,combination of TNF-α(10μg/L) and various concentrations (10-8mol/L, 10-7mol/L,10-6moL/L) of tacrolimus or tacrolimus of different concentrations only.After additional 12-,24-, 36- or 48-hour cnlture, Westem blot and immunofluorescenee-confocal laser scanning microscopy were used to detect the expressions of NF-κB,GRα and GRβ in HaCaT cells.Those untreated HaCaT cells served as the control.Results The relative protein expression level of NF-κB was increased in HaCaT cells after treatment with TNF-α for 24 and 48 hours zompared with untreated ceils (0.73±0.0316 and 0.8925±0.0171 vs 0.4988±0.03506,both P<0.05);however,the increase in NF-κB expression was inhibited by the combination treatment with tacrolimus,and the relative expression level of NF-κB protein was 0.6825±0.0263.0.6200±0.0163 and 0.5575±0.0299 in HaCaT cells treated with TNF-α plus tacrolimus of 10-8mol/L 10-7mol/L and 10-6mol/L,respectively;the difference was significant etween TNF-α-treated cells and those dealt with the combination of NF-α and tacrolimus of 10-7 or 10-6 mol/L (both P<0.05).No significant difference was observed in the expression of NF-κB by HaCaT cells between different time oints treated with tacrolimus of 10-8,10-7 or 10-6 mol/L.Also,there was no zignificant difference in the expression of GRα or GRβ between untreated HaCaT cells and those treated with tacrolimus of 10-8, 10-7 or 10-6 mol/L at any time point.Conclusions Tacrolimus ould inhibit the expression of NF-κB by TNF-α-stimulated HaCaT cells,but does not affect the expression of GRα or GRβ,in untreated HaCaT cells.     

IFN-γ联合全反式维A酸对人皮肤鳞状细胞癌SCL-1细胞生长凋亡的影响

目的 探讨IFN-γ联合全反式维A酸(ATRA)在体外对人皮肤鳞状细胞癌SCL-1细胞形态分化、增殖及生长凋亡的影响.方法 SCL-1细胞根据处理因素不同分为,①对照组;②1μmol/L ATRA组;③100 U/ml IFN-γ组;④500 U/ml IFN-γ组;⑤1000 U/ml IFN-γ组;⑥1000 U/ml IFN-γ+1μmol/LATRA组.用噻唑蓝(MTT)和流式细胞仪(FCM)分析细胞增殖及凋亡的变化.透射电镜、1%碘化丙啶染色及倒置相差显微镜观察细胞凋亡形态.结果 细胞的增殖抑制有明显IFN-γ浓度依赖性,其中1000 U/ml浓度抑制作用最明显.ATRA及IFN-γ能诱导SCL-1细胞凋亡,凋亡率分别是4.84%和11.96%,二者联合诱导凋亡率为18.71%;透射电镜及1%碘化丙啶染色可见1 μmol/L ATRA和1000 U/ml IFN-γ处理的SCL-1细胞发生一系列凋亡特征性的形态学改变,并见较多早期凋亡细胞.IFN-γ及ATRA诱导SCL-1细胞形态呈双极性改变,趋向良性分化.结论 IFN-γ可促进SCL-1细胞分化,抑制其增殖,在一定范围内有剂量依赖性,且与ATRA具有协同作用.

Abstract：

Objective To investigate the in vitro effect of interferon-γ (IFN-γ) and ATRA on the morphological transition, proliferation of and apoptosis in a human cutaneous squamous cell carcinoma cell line SCL-1. Methods Cultured SCL-1 cells were divided into 6 groups to be treated with ATRA of 1 μmol/L, various concentrations ( 100, 500, 1000 U/ml) of IFN-γ, the combination of ATRA of 1 μmol/L and IFN-y of 1000 U/ml,respectively, or to remain untreated. MTT assay and flow cytometry were performed to evaluate the cell proliferation and apoptosis. The morphological features of apoptotic cells were observed by a transmission electron microscope (TEM) and inverted phase contrast microscope after 1% propidium iodide staining. Results IFN-γ could inhibit the proliferation of SCL-1 cells in a dose-dependent manner, and the most pronounced inhibitory effect was observed at a dose of 1000 U/ml . ATRA and IFN-γ induced an apoptosis in SCL-1 cells, and the early apoptosis rate was 4.84%, 11.96% and 18.71% in SCL-1 cells after treated with ATRA of 1 μmol/L, IFN-γ of 1000 U/ml and their combination, respectively. A series of morphological changes characteristic of apoptosis,such as bipolar changes, were observed in SCL-1 cells treated with ATRA and IFN-γ, with the presence of many early apoptotic cells, which showed a trend towards benign differentiation. Conclusions Within a certain concentration range, IFN-γcan promote the differentiation, but inhibit the proliferation of SCL-1 cells in a dose-dependent manner, and ATRA could enhance the effects of IFN-γ.     

CpG ODN刺激的寻常性银屑病中性粒细胞培养上清液对HaCaT细胞增殖的影响

Objective To investigate the role of nentrophils in the pathogenesis of psoriasis vulgaris. Methods Neutrophils were isolated from venous blood samples of 25 patients with psoriasis vulgaris (including 13 cases of active psoriasis and 12 cases of inactive psoriasis) as well as 25 normal human con-trols, and cultured. Then, these neutrophils were grouped and treated with lipopolysaccharide (LPS, 100 g/L),CpG-A (50 mg/L), CpG-B (50 mg/L), and RPMI 1640 culture medium, respectively, for 24 hours followed by the collection of culture supematants. Human keratinocytes (HaCaT) were cultured in the presence of su-pematants of treated or untreated nentrophils for 72 hours followed by the detection of cell proliferation with MTT assay. To determine the role of proinflammatory factors, SOD/CAT and monoclonal antibody to IL-8 and TNF-alpha of 400 u/mL were used to pretreat HaCaT cells 1 hour prior to the stimulation with super-natants of neutrophils. Results Compared with culture medium, the supematant of unstimulated neutrophils from normal controls or patients with inactive psoriasis had no significant effect on the proliferation of HaCaT cells (P ＞ 0.05), but that from patients with active psoriasis markedly promoted the proliferation of HaCaT cells (t = 2.41, P < 0.05). ARe, stimulation by LPS, CpG-A and CpG-B, the supematant of active patient-derived neutrophils significantly promoted the proliferation of HaCaT cells compared with that of normal control-derived nentrophils (t = 3.11, 2.89, 2.29, respectively, all P < 0.05). In comparison with tmstimulated neutrophils, the supematant from LPS- and CpG-A stimulated nentrophiles significantly accelerated the pro-liferation of HaCaT cells. Furthermore, the proliferation of HaCaT cells induced by the supematants of LPS-,CpG-A-, CpG-B-stimulated neutrophils from psoriatic patients was statistically suppressed by the pretreat-ment with the monoclonal antibody to IL-8, TNF-alpha and SOD/CAT (all P < 0.05). Conclusions In patients with psoriasis vulgaris, there is an abnormal secretion of IL-8, TNF-alpha and superoxide by neutrophils in peripheral blood, and these proinflammatory factors could promote the proliferation of HaCaT cells.     

CpG ODN刺激的寻常性银屑病中性粒细胞培养上清液对HaCaT细胞增殖的影响

Objective To investigate the role of nentrophils in the pathogenesis of psoriasis vulgaris. Methods Neutrophils were isolated from venous blood samples of 25 patients with psoriasis vulgaris (including 13 cases of active psoriasis and 12 cases of inactive psoriasis) as well as 25 normal human con-trols, and cultured. Then, these neutrophils were grouped and treated with lipopolysaccharide (LPS, 100 g/L),CpG-A (50 mg/L), CpG-B (50 mg/L), and RPMI 1640 culture medium, respectively, for 24 hours followed by the collection of culture supematants. Human keratinocytes (HaCaT) were cultured in the presence of su-pematants of treated or untreated nentrophils for 72 hours followed by the detection of cell proliferation with MTT assay. To determine the role of proinflammatory factors, SOD/CAT and monoclonal antibody to IL-8 and TNF-alpha of 400 u/mL were used to pretreat HaCaT cells 1 hour prior to the stimulation with super-natants of neutrophils. Results Compared with culture medium, the supematant of unstimulated neutrophils from normal controls or patients with inactive psoriasis had no significant effect on the proliferation of HaCaT cells (P ＞ 0.05), but that from patients with active psoriasis markedly promoted the proliferation of HaCaT cells (t = 2.41, P < 0.05). ARe, stimulation by LPS, CpG-A and CpG-B, the supematant of active patient-derived neutrophils significantly promoted the proliferation of HaCaT cells compared with that of normal control-derived nentrophils (t = 3.11, 2.89, 2.29, respectively, all P < 0.05). In comparison with tmstimulated neutrophils, the supematant from LPS- and CpG-A stimulated nentrophiles significantly accelerated the pro-liferation of HaCaT cells. Furthermore, the proliferation of HaCaT cells induced by the supematants of LPS-,CpG-A-, CpG-B-stimulated neutrophils from psoriatic patients was statistically suppressed by the pretreat-ment with the monoclonal antibody to IL-8, TNF-alpha and SOD/CAT (all P < 0.05). Conclusions In patients with psoriasis vulgaris, there is an abnormal secretion of IL-8, TNF-alpha and superoxide by neutrophils in peripheral blood, and these proinflammatory factors could promote the proliferation of HaCaT cells.     

CpG ODN刺激的寻常性银屑病中性粒细胞培养上清液对HaCaT细胞增殖的影响

Objective To investigate the role of nentrophils in the pathogenesis of psoriasis vulgaris. Methods Neutrophils were isolated from venous blood samples of 25 patients with psoriasis vulgaris (including 13 cases of active psoriasis and 12 cases of inactive psoriasis) as well as 25 normal human con-trols, and cultured. Then, these neutrophils were grouped and treated with lipopolysaccharide (LPS, 100 g/L),CpG-A (50 mg/L), CpG-B (50 mg/L), and RPMI 1640 culture medium, respectively, for 24 hours followed by the collection of culture supematants. Human keratinocytes (HaCaT) were cultured in the presence of su-pematants of treated or untreated nentrophils for 72 hours followed by the detection of cell proliferation with MTT assay. To determine the role of proinflammatory factors, SOD/CAT and monoclonal antibody to IL-8 and TNF-alpha of 400 u/mL were used to pretreat HaCaT cells 1 hour prior to the stimulation with super-natants of neutrophils. Results Compared with culture medium, the supematant of unstimulated neutrophils from normal controls or patients with inactive psoriasis had no significant effect on the proliferation of HaCaT cells (P ＞ 0.05), but that from patients with active psoriasis markedly promoted the proliferation of HaCaT cells (t = 2.41, P < 0.05). ARe, stimulation by LPS, CpG-A and CpG-B, the supematant of active patient-derived neutrophils significantly promoted the proliferation of HaCaT cells compared with that of normal control-derived nentrophils (t = 3.11, 2.89, 2.29, respectively, all P < 0.05). In comparison with tmstimulated neutrophils, the supematant from LPS- and CpG-A stimulated nentrophiles significantly accelerated the pro-liferation of HaCaT cells. Furthermore, the proliferation of HaCaT cells induced by the supematants of LPS-,CpG-A-, CpG-B-stimulated neutrophils from psoriatic patients was statistically suppressed by the pretreat-ment with the monoclonal antibody to IL-8, TNF-alpha and SOD/CAT (all P < 0.05). Conclusions In patients with psoriasis vulgaris, there is an abnormal secretion of IL-8, TNF-alpha and superoxide by neutrophils in peripheral blood, and these proinflammatory factors could promote the proliferation of HaCaT cells.     

CpG ODN刺激的寻常性银屑病中性粒细胞培养上清液对HaCaT细胞增殖的影响

Objective To investigate the role of nentrophils in the pathogenesis of psoriasis vulgaris. Methods Neutrophils were isolated from venous blood samples of 25 patients with psoriasis vulgaris (including 13 cases of active psoriasis and 12 cases of inactive psoriasis) as well as 25 normal human con-trols, and cultured. Then, these neutrophils were grouped and treated with lipopolysaccharide (LPS, 100 g/L),CpG-A (50 mg/L), CpG-B (50 mg/L), and RPMI 1640 culture medium, respectively, for 24 hours followed by the collection of culture supematants. Human keratinocytes (HaCaT) were cultured in the presence of su-pematants of treated or untreated nentrophils for 72 hours followed by the detection of cell proliferation with MTT assay. To determine the role of proinflammatory factors, SOD/CAT and monoclonal antibody to IL-8 and TNF-alpha of 400 u/mL were used to pretreat HaCaT cells 1 hour prior to the stimulation with super-natants of neutrophils. Results Compared with culture medium, the supematant of unstimulated neutrophils from normal controls or patients with inactive psoriasis had no significant effect on the proliferation of HaCaT cells (P ＞ 0.05), but that from patients with active psoriasis markedly promoted the proliferation of HaCaT cells (t = 2.41, P < 0.05). ARe, stimulation by LPS, CpG-A and CpG-B, the supematant of active patient-derived neutrophils significantly promoted the proliferation of HaCaT cells compared with that of normal control-derived nentrophils (t = 3.11, 2.89, 2.29, respectively, all P < 0.05). In comparison with tmstimulated neutrophils, the supematant from LPS- and CpG-A stimulated nentrophiles significantly accelerated the pro-liferation of HaCaT cells. Furthermore, the proliferation of HaCaT cells induced by the supematants of LPS-,CpG-A-, CpG-B-stimulated neutrophils from psoriatic patients was statistically suppressed by the pretreat-ment with the monoclonal antibody to IL-8, TNF-alpha and SOD/CAT (all P < 0.05). Conclusions In patients with psoriasis vulgaris, there is an abnormal secretion of IL-8, TNF-alpha and superoxide by neutrophils in peripheral blood, and these proinflammatory factors could promote the proliferation of HaCaT cells.     

Novice identification of melanoma: not quite as straightforward as the ABCDs

The "ABCD" mnemonic to assist non-experts' diagnosis of melanoma is widely promoted; however, there are good reasons to be sceptical about public education strategies based on analytical, rule-based approaches--such as ABCD (i.e. Asymmetry, Border Irregularity, Colour Uniformity and Diameter). Evidence suggests that accurate diagnosis of skin lesions is achieved predominately through non-analytical pattern recognition (via training examples) and not by rule-based algorithms. If the ABCD are to function as a useful public education tool they must be used reliably by untrained novices, with low inter-observer and intra-diagnosis variation, but with maximal inter-diagnosis differences. The three subjective properties (the ABCs of the ABCD) were investigated experimentally: 33 laypersons scored 40 randomly selected lesions (10 lesions × 4 diagnoses: benign naevi, dysplastic naevi, melanomas, seborrhoeic keratoses) for the three properties on visual analogue scales. The results (n = 3,960) suggest that novices cannot use the ABCs reliably to discern benign from malignant lesions.     

Creatine supplementation normalizes mutagenesis of mitochondrial DNA as well as functional consequences

Mutations of mitochondrial (mt) DNA play a role in neurodegeneration, normal aging, premature aging of the skin (photoaging), and tumors. We and others could demonstrate that mtDNA mutations can be induced in skin cells in vitro and in normal human skin in vivo by repetitive, sublethal ultraviolet (UV)-A-irradiation. These mutations are mediated by singlet oxygen and persist in human skin as long-term biomarkers of UV exposure. Although mtDNA exclusively encodes for the respiratory chain, involvement of the energy metabolism in mtDNA mutagenesis and a protective role of the energy precursor creatine have thus far not been shown. We assessed the amount of a marker mutation of mtDNA, the so-called common deletion, by real-time PCR. Induction of the common deletion was paralleled by a measurable decrease of oxygen consumption, mitochondrial membrane potential, and ATP content, as well as an increase of matrix metalloproteinase-1. Mitochondrial mutagenesis as well as functional consequences could be normalized by increasing intracellular creatine levels. These data indicate that increase of the energy precursor creatine protects from functionally relevant, aging-associated mutations of mitochondrial DNA.     

Skin as a universal organ as reflected throughout history

In ancient medicine the skin was regarded as an organ in and of itself, as "soma organikon," and even as the largest organ of all. Keeping in mind the historical background, we examine: 1. skin as reflected by speech and proverbs; 2. skin as a world of its own, concerned with feeling, grasping, comprehension; 3. skin, according to tradition, as an organ reflecting almost every pathological and regenerative process. The paper is presented in the form of an essay and a historical survey is given; there is no intent to provide information on systematic or specific problems of dermatology.