热休克蛋白72在皮肤鳞着细胞癌中的表达

应用免疫组织化学（ＳＡＢＣ法）和酸原位杂交技术检测了正常人皮肤、皮肤鳞状细胞癌中热休克蛋白７２的表达情况。结果显示，ＨＳＰ７２在正常皮肤的表怪层及毛囊外根鞘即有构成性表达，可能是一种自身保护功能。ＨＳＰ７２在皮肤鳞癌中高表达，可能通过影响细胞生长调控而在肿瘤发生、发展过程中具有重要作用。     

热休克蛋白72在皮肤鳞状细胞癌中的表达

应用免疫组织化学（ＳＡＢＣ法） 和核酸原位杂交技术检测了正常人皮肤、皮肤鳞状细胞癌中热休克蛋白７２ 的表达情况。结果显示，ＨＳＰ７２ 在正常皮肤的表皮层及毛囊外根鞘即有构成性表达， 可能是一种自身保护功能。ＨＳＰ７２ 在皮肤鳞癌中高表达， 可能通过影响细胞生长调控而在肿瘤发生、发展过程中具有重要作用。     

16型人乳头瘤病毒E6/E7诱导角质形成细胞表达热休克蛋白70

目的 了解１６型人乳头瘤病毒（ＨＰＶ１６）Ｅ６／Ｅ７、ＨＳＰ７０与角质形成细胞永生化及转化之间的关系。方法 Ｌｉｐｏｆｅｃｔａｍｉｎｅ介导法进行转染,以Ｇ４１８筛选阳性克隆,免疫印迹分析及免疫荧光双标记染色结合激光扫描共聚焦显微镜对ＨＳＰ７０和ＨＰＶ１６Ｅ６／Ｅ７在转染然质形成细胞中的表达进行了观察。结果 免疫印迹分析显示正常负擀形成细胞和转染角质形成细胞均在相对分子质量７００００处出现一条染色带     

皮肤科学基础

２００１０６６３β－溶血型链球菌与角质形成细胞增殖和凋亡研究／张峻岭（天津长征医院皮肤科）…／／中国皮肤性病学杂志．－２０００，１４（５）．－３０２～３０４ 为探讨β－溶血型链球菌诱发银屑病的机制，应用３Ｈ－ＴｄＲ掺入法测定角质形成细胞增殖反应，以流式细胞仪测定亚二倍体含量．行ＤＮＡ片段化分析，再应用 Ａｎｎｅｘｉｎ Ｖ法测定磷脂酰丝氨酸（ＰＳ）“膜外化”细胞含量，观察细胞凋亡的早期表现。结果链球菌抗原悬液（ＳＰ）活化的Ｔ淋巴细胞上清液作用于角质形成细胞的Ｃｏｌｏ－１６细胞４８ｈ后，细胞的刺激指数…     

红斑狼疮皮损中浸润细胞免疫表型和角质形成细胞HLA-DR抗原表达的研究

应用抗ＨＬＡＤＲ、ＣＤ３、ＣＤ４、ＣＤ８、ＣＤ２０的单克隆抗体和ｓｔｒｅｐｔｒａｖｉｄｉｎｐｅｒｏｘｉｄａｓｅｓｔａｉｎｉｎｇ（ＳＰ）技术对１０名正常人皮肤,１６例ＳＬＥ皮损和１９例ＤＬＥ皮损进行了免疫组化研究。观察到正常人皮肤角质形成细胞未见ＨＬＡＤＲ抗原表达,而ＳＬＥ（６／１６）,ＤＬＥ（８／１９）皮损处角质形成细胞可以表达ＨＬＡＤＲ抗原。在ＳＬＥ、ＤＬＥ真皮内浸润细胞主要为Ｔ淋巴细胞（ＣＤ３＋浸润细胞）,且以ＴＨ细胞（ＣＤ４＋浸润细胞）占优势。另外,还发现在两种ＬＥ表皮角质形成细胞表达ＨＬＡＤＲ抗原处,真皮内可见ＣＤ３＋浸润细胞和激活的Ｔ淋巴细胞（ＨＬＡＤＲ＋浸润细胞）。讨论了ＬＥ皮损角质形成细胞ＨＬＡＤＲ抗原表达及其与病损内浸润细胞免疫表型的关系。ＬＥ皮损处ＨＬＡＤＲ＋角质形成细胞可能具有抗原递呈作用,而角质形成细胞异常表达ＨＬＡＤＲ抗原则可能与真皮内浸润单个核细胞或淋巴细胞释放的ＩＦＮα,ＴＮＦγ等有关。     

不同肤色个体表皮角质形成细胞Caspase14表达的研究

目的 探讨不同肤色人群角质形成细胞中Caspase14表达变化,明确黑素细胞和（或）黑素对其表达的影响。 方法 以Western印迹检测不同肤色个体原代角质形成细胞Caspase14的表达。取体外培养不同肤色的2代角质形成细胞,分别加入不同肤色的黑素细胞在分化培养基下共同培养24 h,以不加黑素细胞组为对照,Western 印迹检测Caspase14的表达。结果 浅、深肤色个体包皮原代角质形成细胞Caspase14的表达存在显著差异,深肤色者（Fitzpatrick Ⅳ/Ⅴ）原代角质形成细胞中Caspase14的表达显著高于浅肤色者（FitzpatrickⅠ/Ⅱ）（P ＜ 0.01）;深、浅肤色个体的角质形成细胞在与不同肤色个体的黑素细胞在分化培养基中共培养后24 h后,检测发现,与对照组相比,浅肤色个体角质形成细胞Caspase14表达的增加,差异无统计学意义（P ＞ 0.05）,而深肤色个体角质形成细胞Caspase14的表达则显著上调（P ＜ 0.05）。结论 深肤色个体角质形成细胞Caspase14表达显著高于浅肤色个体者。黑素细胞显著增加深肤色者角质形成细胞中Caspase14水平的表达。     

人类乳头瘤病毒16全基因在体外培养的正常人角质形成细胞 …

目的 利用含有人类乳头瘤病毒（ＨＰＶ）１６全基因的质粒转染正常人角质形成细胞，观察ＨＰＶ１６ｍＲＮＡ在转染细胞的表达。方法 用ＦｕＧＥＮＥ＾ＴＭ６转染试剂，将携带ＨＰＶ１６全基因的质粒ｐＳＶ２－ｎｅｏ／１６转染体外培养的正常人角质形成细胞，在转染后２４ｈ提取细胞总ＲＮＡ和ＤＮＡ，进行ＲＴ－ＰＣＲ和Ｓｏｕｔｈｅｒｎ印迹分析，结果 ２４ｈ后，ＲＴ－ＰＣＲ成功地扩增出１１０ｂｐ的片段，转染细胞中已出现Ｈ     

氧化苦参碱诱导角质形成细胞凋亡研究

目的 探讨苦参治疗银屑病的机制。方法 采用流式细胞仪测定亚二倍体细胞含量、ＤＮＡ片段化分析和ＡｎｎｅｘＶ（膜联蛋白Ｖ）凋亡检测法，观察氧化苦参碱９ＯＭＴ）对角质形成细胞凋亡的影响。结果 ＯＭＴ使角质形成细胞的亚二倍体细胞、ＤＮＡ片段化率及磷脂酰丝氨酸（ＰＳ）膜外化细胞含量明显升高（Ｐ〈０．０１），首次发现一定浓度的ＯＭＴ可诱导角质形成细胞凋亡。结论 ＯＭＴ诱发角质形成细胞凋亡，这可能是苦参治疗银屑病的主要机制之一。     

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。     

银屑病患者皮损中细胞凋亡情况的评价

目的 探讨细胞恨与银屑病的关系。方法 用免疫组化ＳＰ法检测银屑病皮损与非皮损中Ｂａｘ、Ｆａｓ、Ｆａｓ－１的表达,并用ＴＵＮＥＬ（末端脱氧核苷酰转移介导的ｄ－ＵＴＰ－生物素缺口本端标记）技术检测细胞凋亡情况。结果 与正常人相比,银屑病表皮角质形成细胞,真皮炎症细胞及炎症细胞及血管内皮均存在不同程度的上述蛋白的过度表达,伴细胞凋亡增多,部分在治疗后末完全恢复正常。结论 推测银屑病皮损中角质形成对细胞过     

Microinjection of an antibody against HSP 72 in keratinocytes to study acute UV injury.

First, a method of microinjection of antibodies in primary human keratinocytes in culture was established. Second, in acute UV irradiation, the physiological role of heat shock protein (HSP) 72 in keratinocytes was studied with this method. Primary human keratinocytes in culture were injected with "controls" as fluorescent dyes, phosphate buffered saline (PBS), an irrelevant secondary antibody and an antibody against a protein with known protective function in UV erythema, HSP 72. UV irradiation was applied and survival, colony forming and immunohistochemistry for injected and non-injected keratinocytes were evaluated in a time course. Puncturing the plasma membrane with injections of "controls" as FITC, PBS and the IgG anti-mouse antibody did not result in reflux of injected material or any alteration in morphology or colony-forming ability for 24 h. Keratinocytes injected with an mAb to HSP 72 without UV irradiation survived microinjection for up to 12 days, while surprisingly, more than double of injected and irradiated ones died after 12 h compared to not injected and irradiated ones. Moreover, microinjection of the antibody to HSP 72 in the nucleus resulted in a loss of the immunohistochemical labeling for HSP 72 in these cells after 12 h. Microinjection of the "controls" did not harm the survival, forming of colonies and expression of HSP 72 in keratinocytes for 24 h. In contrast, microinjection of an mAb against HSP 72 led to an increase in cell death after UV irradiation, confirming that HSP 72 is important for UV protection. Microinjection of antibodies in human keratinocytes in culture might allow the study of the physiological role of some proteins.     

Hair cycle-dependent expression of heat shock proteins in hair follicle epithelium

Background Heat shock proteins (HSPs) have a physiologic function in unstressed cells, which is believed to include a role as a “molecular chaperone.” The hair cycle is characterized by rhythmic tissue remodeling processes, and is an intriguing model for studying the relation between keratinocyte differentiation and HSP expression under physiologic circumstances. We have therefore studied, by immunofluorescence, the expression of selected HSPs during the murine hair cycle. Methods The association between hair follicle cycling and the expression of three selected HSPs (HSPs 27, 60, and 72) was examined by immunofluorescence, using the depilation-induced hair cycle of C57BL76 mice. Results HSP expression was absent from telogen follicles, and was restricted predominantly to keratinocytes in the bulge and the cycling epithelial portion of the hair follicle during anagen and catagen. Immunoreactivity for HSPs 27, 60, and 72 in the hair bulb increased significantly during anagen VI and the catagen transformation of the follicle, and decreased again abruptly with completion of the catagen-telogen transformation. The expression pattern of HSPs 60 and 72 in situ was cytoplasmic, whereas that of HSP 27 was both cytoplasmic and nuclear. Conclusions These observations suggest that the synthesis of HSPs by hair bulb keratinocytes is related to the anagen-catagen transformation of the follicle, possibly reflecting keratinocyte apoptosis and/or terminal differentiation in the regressing, cycling portion of the follicle. In addition, the rather proximal localization of HSP expression makes it unlikely that the HSPs examined interact with the more distally located intrafollicular γ/δ T-cell receptor-positive lymphocytes.     

Heat shock proteins in cultured human keratinocytes and fibroblasts

Heat shock induces in cells the synthesis of specific proteins called heat-shock proteins. We have compared the induction of these proteins in human keratinocytes, skin fibroblasts, and a human epithelial tumor cell line following exposure to weak and strong inducing agents (heat, cadmium sulphate, and sodium arsenite). The induction of heat shock proteins was measured in cells by one-dimensional gel electrophoresis of [35S] methionine-labeled proteins and by immunofluorescence using a specific HSP72 monoclonal antibody. Both HSP90 and HSP116 were constitutively expressed in these cell types. Exposure of these cells to weak inducing agents such as heat or cadmium sulphate resulted in the synthesis of HSP72 and HSP90, whereas HSP28 and HSP116 synthesis was detected in keratinocytes and fibroblasts following exposure to the strong inducing agent sodium arsenite. In addition, sodium arsenite induced the synthesis of HSP46 in human keratinocytes. Immunofluorescence demonstrated a rapid and reversible accumulation of the 72-kD heat shock protein within the nucleolus of heat-stressed human keratinocytes and fibroblasts.     

银屑病表皮热休克蛋白27、70、60的表达

目的：探讨热休克蛋白（HSP）在银屑病发病中的作用。方法：通过免疫组化和图像分析检测了25例银屑病患者治疗前后皮损、非皮损区表皮组织中HSP27、HSP70和HSP60的表达，并与6例正常人作对照。结果：HSP27、HSP70在非皮损、正常人表皮中呈基础表达，在银屑病患者皮损中的表达很弱，治愈后表皮后HSP27、HSP70的表达又恢复；HSP60的表达在银屑病皮损组表皮中均为阳性，而银屑病非皮损组、治愈后组表皮中及正常人对照表皮组织中HSP60的表达均阴性。结论：热休克蛋白在银屑病应激保护机制中可能发挥一定的作用。     

Effects of thermal shocks on interleukin-1 levels and heat shock protein 72 (HSP72) expression in normal human keratinocytes

Interleukin-1 expression is reported to be modified under a number of cell conditions including physiological stress, injury and activation. We report the effects of the physiological stresses cold and heat shock on IL-1 levels in keratinocytes. Having observed that normal human skin obtained from plastic surgery, usually stored at 4C for a few hours, highly expressed HSP72, a constant feature of stressed human keratinocytes, we wondered whether this induction could be linked to a cold shock and to modification of IL-1 levels in keratinocytes. Cultured keratinocytes were incubated at 4, 37, 40 and 43C for 1.5, 4, 8 and 16 h in a defined medium. HSP72 expression was studied by immunohistochemistry and immunoblot and IL-1 was quantified using specific and sensitive radio-immunoassay. Our findings showed that intracellular IL-1 and IL-1 levels are not significantly modified by thermal shock. HSP72 is only induced after cell exposure to 43C and is not a cold-shock protein. These results demonstrate that thermal stress is not an inductive signal for IL-1 modification in keratinocytes.     

Expression of Heat Shock Protein 70 in Human Skin Cells as a Photoprotective Function after UV Exposure

BackgroundHuman skin is exposed to various environmental stresses, such as heat, cold, and ultraviolet (UV) radiation. Heat shock proteins (HSPs) induced by temperature elevations, as a physiologic response to mediate repair mechanisms and reduce cellular damage.ObjectiveThe purpose of this study was to investigate the induction of HSPs in human skin cells after UV exposure.MethodsWe performed immunoblotting using a specific monoclonal antibody to the HSP70 family, one of the best-conserved stress proteins in humans, with cultured normal human keratinocytes, A431 cells, human melanocytes, SK30 cells, and human dermal fibroblasts (HDF).ResultsOur results indicated that high expression of HSP70 in the unstressed state was noted in epidermal cells, including normal human keratinocytes, A431 cells, human melanocytes, and SK30 cells, but epidermal cells showed no additional up-regulation of HSP70 after UV irradiation. On the other hand, HDF expressed very small amounts of HSP70 at baseline, but significantly higher amounts of HSP70 after UV exposure.ConclusionThese findings suggest that constitutive expression of HSP70 in epidermal cells may be an important mechanism for protection of the human epidermis from environmental stresses, such as sunlight exposure.     

Heat shock protein 27 is expressed in normal and malignant human melanocytes in vivo

BACKGROUND: Heat shock proteins (HSPs) are a family of highly conserved proteins found ubiquitously in mammalian cells, believed to be regulators of normal cell physiology and the cellular stress response. In addition, the small 27-kDa heat shock protein (HSP27) has previously been found to be a differentiation marker for keratinocytes and a prognostic marker associated with increased survival in certain cancerous tumors. METHODS: Using immunohistochemistry on routinely processed paraffin sections, we examined skin biopsies from 15 invasive melanomas, 13 intradermal nevi, and two compound nevi immunostained with a mouse monoclonal antibody to HSP27. In addition, cultured melanocytes were heat stressed at 45 degrees C for 1 h and then fixed and immunostained in order to localize HSP27 expression intracellularly. RESULTS: We found cytoplasmic and strong perinuclear staining of HSP27 in melanocytes in normal skin, in melanomas, and in nevi. Nuclear reactivity was absent. In addition, in cultured non-malignant melanocytes, HSP27 expression relocated from the cytoplasm to the nucleus with heat stress. CONCLUSIONS: To our knowledge, this investigation is the first to demonstrate that HSP27 is expressed in melanocytes in normal skin, in nevi, and in non-malignant cultured melanocytes.     

Keratinocyte conditioned medium stimulates type IV collagenase synthesis in cultured human keratinocytes and fibroblasts

We have previously shown that conditioned medium from cultured human keratinocytes stimulates proliferation of a variety of cell types involved in wound healing. as well as re-epithelization of wounds in human skin in vitro. We now present evidence for an autocrine/paracrine control of the synthesis of type IV collagenases in human keratinocytes and fibroblasts. During wound healing, keratinocytes migrate over the wound bed, an activity coupled with lysis of basement membranes, and hence requiring the presence of collagenases. Collagenases are also needed for the production and remodelling of the granulation tissue. In order to study the autocrine/paracrine control of collagenase production in keratinocytes and fibroblasts, we stimulated these cells in culture with conditioned medium from cultured keratinocytes. Protease synthesis was determined by affinity labelling with ³H-diisopropylfluorophosphoridate (DFP) and by zymography. Keratinocyte-conditioned medium was found to increase the expression of 72 and 92 kDa type IV collagenase in human keratinocytes, and the 72 kDa collagenase in human fibroblasts. indicating that an autocrine/paracrine control mechanism is involved in collagenase production in these cell types during wound healing. This increased expression of collagenases could he partly responsible for the stimulated healing seen in wounds treated with sheets of cultured keratinocytes.