UVA对人角质形成细胞的氧化损伤作用

①目的　探讨紫外线A(UVA)对人角质形成细胞氧化损伤的机制。②方法　用 5J/cm2 UVA照射角质形成细胞 ,酶生化法检测活性氧 (ROS)的含量及超氧化物歧化酶 (SOD)、谷胱甘肽过氧化物酶 (GSH PX)活性的变化 ,流式细胞仪测定紫外线对角质形成细胞凋亡的影响 ,原位杂交技术观察 p2 1mRNA的变化 ,并与非照射组比较。③结果　与对照组比较 ,UVA照射组ROS含量升高 (t =113.6 8,P <0 .0 0 1) ,SOD、GSH PX活性下降 (t =5 7.2 3、19.0 4 ,P <0 .0 0 1) ,细胞凋亡率升高 (t=5 3.2 8,P <0 .0 0 1) ,p2 1mRNA表达增强。④结论　UVA对人角质形成细胞的损伤与氧自由基生成增多及细胞抗氧化能力抑制有关     

Protein kinase C agonist and antagonist effects in normal human epidermal keratinocytes

Abstract Several lines of evidence implicate protein kinase C (PKC) in the development of basal cell and squamous cell carcinomas, tumors which originate from epidermal keratinocytes. To examine PKC in a model relevant to human skin, we exposed normal human epidermal keratinocytes (NHEK) in serum-free media to a variety of PKC agonists and antagonists. NHEK PKC activity increased up to 10-fold within the 1st hour of exposure to tetradecanoyl phorbol acetate (TPA), and gradually returned to control values within 72 h. TPA-induced PKC activity was enhanced by pretreatment of cultures with protein and RNA synthesis inhibitors. TPA-induced growth arrest and differentiation was antagonized by staurosporine. Down-regulation by bryostatin pretreatment blocked TPA-stimulated differentiation. Our overall conclusion is that activation of PKC in cultured human keratinocytes is required for differentiation. These results are crucial to the analysis of compounds suspected of promoting or inhibiting epidermal tumors.     

The mitogenic effect of KGF and the expression of its cell surface receptor on cultured normal and malignant human oral keratinocytes and on contiguous fibroblasts

This study examined the mitogenic response to keratinocyte growth factor (KGF) of normal and tumour-derived human oral keratinocytes in which the degree of cellular differentiation was known and in contiguous fibroblast cultures derived from the malignant epithelial cultures. Keratinocytes, but not fibroblasts, were stimulated by KGF. There by demonstrating epithelial target cell specificity of the ligand. KGF-induced stimulation of the tumour-derived keratinocytes cultured in the absence of the 3T3 fibroblast support broadly correlated with the degree of cellular differentiation; well-differentiated keratinocytes were stimulated more by KGF than their less differentiated counterparts. Malignant oral keratinocytes expressed KGF cell surface receptors (K_D 451-709 pM; receptors/cell 2306-413645), but KGF receptor mRNA did not correlate with either KGF-induced mitogenesis or the degree of epithelial cell differentiation. When the tumour-derived keratinocytes were cultured in the presence of 3T3 fibroblasts, the mitogenic response to KGF was comparable to normal epithelial cells. The results suggest that KGF-mediated growth stimulation may not be significant in providing a selective advantage for the growth of malignant keratinocytes.     

Expression of ɛBP, a β-galactoside-binding soluble lectin, in normal and neoplastic epidermis

Abstract The study of animal lectins and glycoconjugates has become an important area of research in biomedical sciences, as these molecules are believed to play important roles in a variety of biological processes. This report describes a study of the expression of an animal lectin, IgE-binding protein (?BP), also known as Mac-2 and CBP35, in human skin. We have analyzed cultured human keratinocytes as well as normal human skin and a number of epidermal neoplasms, by immunoblotting. immuno-fluorescence and immunohistochemistry. We showed that ?BP is expressed in human keratinocytes, hair follicles, sebaceous and sweat glands. We found that cBP expression retains in various epidermal neoplasms, including basal cell carcinoma, squamous cell carcinoma and keratoacan-thoma, although the level of expression appears to be reduced as compared to normal epidermis. The immunohistochemical analysis also suggests that the level of ?BP expression appears to be dependent on the degree of cellular differentiation of keratinocytes.     

Effect of gamma-interferon on IL-1 alpha, beta and receptor antagonist production by normal human keratinocytes

Abstract In inflammatory dermatoses. activated T cells produce inter-feron-gamma (IFN-γ), which interacts with keratinocytes and contributes to the direct activation of these cells by inducing, among other factors, the expression of HLA-DR antigens and intercellular adhesion molecule-1. However, the action of IFN-γ on epidermal cell cytokine production is not known. Our aim was to assess the effect of IFN-γ on the production of IL-1 by normal human keratinocytes cultured in low calcium medium (MCDB153). In comparison with controls, the addition of nontoxic IFN-yγ concentrations (50-500 U/ml) to cell cultures induced a significant increase of IL-1α and IL-1β production predominantly after 100 U/ml treatment in the cell extracts as well as in the supernatants at 24h and 48h. The production of the antagonist. IL-1RA, was also enhanced and the effect of the critical concentration (100 U/ml) was more evident. However, the absence of a characteristic dose response could not be explained by an antiproliferative effect of high IFN-γ concentrations (250 and 500 U/ml) on cultured keratinocytes or by the induction of the nuclear stress protein, Hsp72. two phenomena known to down-regulate IL-1 biosynthesis. In conclusion, the modifications in keratinocyte IL-1 production under IFN-γ stimulation can contribute to activate the epidermal cells and thus involve them in the local immune response.     

CD24 expression on human keratinocytes

Abstract: CD24 or Nectadrin is a cell surface glycoprotein expressed in pre-B lymphocytes, T lymphocytes, neurons, muscle cells and carcinoma cells. Its function is not completely known, but it has been suggested that it is involved in cell adhesion and signalling. CD24 has recently been identified as the human molecule homologous to the murine heat-stable antigen (HSA). HSA is expressed by murine keratinocytes and delivers costimulatory signals in T-cell activation. Long-term cultures of normal human keratinocytes (HKC) were obtained from skin of human female breast sections and either left untreated or were treated with phorbol-12-myristate-13-acetate (PMA) at 10–100 ng/ml, calcium 0.5–2 mM or IFN-γ 100–1000 U/ml, for 24–48 h. Using RT-PCR and flow cytometry we showed that HKC express low levels of CD24 even under basal conditions, and the treatment with calcium, PMA or IFN-γ increased levels of CD24 mRNA and protein. To the best of our knowledge, this is the first report to measure CD24 expression and production by cultured HKC in basal conditions and after stimulation. Further studies are needed to determine biological and therapeutical relevance of these findings.     

快速培养纯化猪角朊细胞及复合羊膜体外培养实验研究

目的研究快速培养和纯化猪角朊细胞的方法及观察角朊细胞在脱细胞羊膜上的生长状况,为组织工程皮肤研究提供实验依据.方法采用加10%胎牛血清的人无血清角朊细胞培养基(defined keratinocyte-SFM,DKSFM)培养原代猪角朊细胞,分别用DKSFM(A组)、加5%胎牛血清的DKSFM(B组)、加10%胎牛血清的DKSFM(C组)培养传代猪角朊细胞,于接种后1、3、5和7 d观察猪角朊细胞的形态及生长曲线.利用猪角朊细胞与培养瓶黏附牢固的特点,用0.02%乙二胺四乙酸(ethylene diamine tetraacetic acid,EDTA)和0.05%胰蛋白酶分步消化,纯化细胞.将第2代猪角朊细胞接种在冻干脱细胞羊膜上,直接苏木素染色、石蜡切片、免疫组织化学染色等方法观察猪角朊细胞在脱细胞羊膜上的生长状况.结果采用加10%胎牛血清的DKSFM培养原代猪角朊细胞,细胞生长速度快,形态好,培养5 d铺满培养瓶底60%～70%.B组培养的传代猪角朊细胞生长速度较C组培养猪角朊细胞生长速度快.A组培养传代的角朊细胞,细胞生长缓慢.用0.02?TA和0.05%胰蛋白酶分步消化的方法纯化猪角朊细胞,可获得纯度为95%以上的猪角朊细胞.将第2代猪角朊细胞接种在脱细胞羊膜后12 d,可形成单层细胞,呈多角形、铺路石样排列;14 d和16 d细胞进一步密集,16 d细胞出现老化.结论 10%胎牛血清的DKSFM培养原代猪角朊细胞,5%胎牛血清的DKSFM培养传代猪角朊细胞,细胞生长速度快.用0.02?TA和0.05%胰蛋白酶分步消化的方法纯化猪角朊细胞,可以获得高纯度猪角朊细胞.脱细胞羊膜为猪角朊细胞提供良好的支架,接种培养后12 d,细胞形态最好.     

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.