体外重建皮肤与正常人皮肤基本特征的对比研究

目的 用正常人的皮肤角质形成细胞和黑素细胞在体外重建皮肤,为将来用于临床、生物及药理学研究奠定基础.方法 从正常儿童的包皮分离、培养角质形成细胞、黑素细胞和成纤维细胞.用成纤维细胞和I型鼠尾胶原制备真皮类似物.将角质形成细胞和黑素细胞接种到真皮类似物表面进行培养,制备表皮类似物.将重建的皮肤类似物和正常人皮肤进行对比研究.结果 皮肤类似物的组织形态包括基底层、基底上层、角质层与正常人皮肤相似.与基底膜形成有关的标志,在皮肤类似物和正常人皮肤中的表达是相似的.角蛋白10、泛角蛋白在皮肤类似物和正常人皮肤中的表达也是相似的.结论 组织形态和所检测的各种标志的表达显示,该皮肤类似物与正常人皮肤相似.     

Characterization of ultraviolet radiation-induced damage to keratinocytes in a skin equivalent in vitro

The human skin equivalent (HSE) provides a convenient model for studying the dermatological effects of exposure to ultraviolet (UV) radiation. HSEs, constructed by overlaying a collagen-fibroblast matrix with epidermal cells, were maintained submerged for 1 week after the addition of epidermal cells and then raised to the air-liquid interface for an additional 3 weeks. HSEs were exposed to sublethal doses of UV radiation ranging from 0 to 500 J/m², incubated up to 48 h in medium containing ³H-thymidine and fixed for ultrastructural and autoradiographic analysis. Exposure to radiation doses greater than 50 J/m² led to vacuolation of the cornified envelopes and enlargement of intercellular spaces. These doses also led to the formation of dense cytoplasmic bodies, and separation and vesiculation of the nuclear envelope in the basal cells. DNA synthesis in the basal cells was analyzed autoradiographically. Maximal numbers of labeled basal cells were observed 24 h after exposure to UV radiation at 50 J/m². Although the proportions of labeled cells varied among different epidermal donors, the maximal responses and time-course of ³H-thymidine incorporation remained consistent, supporting the usefulness of the HSE in studying the effects of UV irradiation on human skin.     

Differential response of basal keratinocytes in a human skin equivalent to ultraviolet irradiation

Abstract The human skin equivalent (HSE) provides a convenient model system for studying the cellular responses of basal keratinocytes to UV irradiation. HSEs, constructed by overlaying a collagen-fibroblast matrix with epidermal cells, were raised to an air-liquid interface to promote epidermal differentiation. HSEs were exposed to ultraviolet radiation from a 500-W Hot Quartz Hanovia therapeutic sunlamp, at a total dose of 100 J/m². The HSEs were then frozen every 4 h over a 48-h period and cryosectioned. For each time period, the expression of β₁ integrin and cyclin E, p53, or Bcl-2 were quantified using dual immunolocalization. Basal cells expressing β₁ integrin were divided into two subpopulations, denoted β₁ ^high or β₁ ^low. The proportion of β₁ ^high keratinocytes expressing Bcl-2 and cyclin E increased significantly 4 and 8 h, respectively, after exposure to UV; during the subsequent 16 h, this basal cell subpopulation expressed p53. By contrast, significant numbers of β₁ ^low basal keratinocytes expressed p53, but not Bcl-2. These results suggest that β₁ ^high and β₁ ^low populations of basal epidermal cells in HSEs respond differently to UV irradiation. Received: 30 October 1997     

Nerve growth factor in normal and psoriatic skin equivalent models

Abstract Our objective was to determine the pattern and time course of nerve growth factor expression in an established skin equivalent model that we have used in the past to study wound healing and psoriasis phenotypes. Skin equivalents were constructed in triplicate using normal neonatal foreskin keratinocytes plated on collagen gels containing fibroblast lines. These lines were derived from five specimens of psoriatic lesions, three specimens of normal skin from patients with psoriasis, and three specimens of eyelid skin from normal donors. Immunohistochemistry and a monoclonal nerve growth factor-b antibody were used to determine the pattern of protein staining over 2 weeks. We looked at the wound healing phenotype using the skin equivalent model for 7–14 days. When keratinocytes invaginate into the dermis of skin equivalents (beginning at around 7 days of growth), dark staining of nerve growth factor was seen under the basal membrane zone, suggesting that nerve growth factor serves in the development of the basal membrane zone and the epidermis, and may influence the migration of nerves through the basal membrane zone into the regenerated skin. Received: 17 July 1997 / Received after revision: 17 February 1998 / Accepted: 10 July 1998     

Keratinocytes modulate the biosynthetic phenotype of dermal fibroblasts at a pretranslational level in a human skin equivalent

In this study we investigated the influence of keratinocytes on the phenotype of fibroblasts in an in vitro human skin equivalent. Keratinocytes were seeded at the surface of fibroblast-populated mechanically restrained type I collagen gels (lattices). Lattices without keratinocytes were handled in parallel as controls. After 2 and 4 days in culture, the keratinocyte layer was removed and the steady-state level of the mRNA for the main extracellular matrix macromolecules and interstitial collagenase produced by the fibroblasts was measured by Northern and dot blot analysis. A 50% decrease in the amount of procollagen type I and type III mRNAs was observed after 2 and 4 days of coculture while collagenase gene expression was upregulated by 300% when compared with control lattices. No significant modulation of type IV and type VI collagen, elastin or laminin B1 mRNA levels was found. Fibronectin mRNA levels in fibroblasts were significantly increased only on day 4. All the observed changes could be reproduced using a conditioned medium collected from a lattice covered with keratinocytes added to a lattice containing fibroblasts alone. These results indicate that in an in vitro reconstituted skin, keratinocytes are able to modulate the biosynthetic phenotype of fibroblasts at a pretranslational level through a paracrine signalling pathway.     

Reconstruction of basement membrane in skin equivalent; role of laminin-1

Abstract To reconstruct the basement membrane in a skin equivalent, the epidermodermal interface was coated with porcine type IV collagen and mouse laminin-1 at various ratios before keratinocyte seeding. Laminin-1, a component of the basement membrane, induced massive infiltration of keratinocytes into the dermal equivalent, while type IV collagen induced discrete demarcation between dermal and epidermal compartments without any infiltrating cells. Immunohistochemical staining indicated that the laminin-induced infiltrating cells expressed endogenous type IV collagens at the cell periphery, which were not incorporated into the basement membrane structure. The infiltrating cells did not express fibronectin receptor α₅β₁ integrin but showed MMP-9 secretion and cell surface associated MMP-2. However, when laminin-1 was preincubated with type IV collagen, laminin-1-induced keratinocyte infiltration as well as MMP-9 induction were almost completely suppressed to basal levels. Therefore, replenishment of the type IV collagen lattice seemed to cause laminin-stimulated cells to anchor to the lattice, in a similar manner to the basal cells on the basement membrane of normal skin. Our study suggests that the molar ratio of basement membrane components may determine the behavior of basal cells within the wound healing microenvironment, which is probably regulated either by extracellular matrix deposition or degradation. Received: 24 October 2000 / Revised: 9 February 2001 / Accepted: 31 March 2001     

皮肤屏障与银屑病研究进展

皮肤作为人体的第一道防线,对抵御外界有害因素的损伤以及维持人体内环境的稳态有着至关重要的作用。皮肤屏障的结构和功能与一些皮肤病的发生发展有着重要的联系。研究发现皮肤屏障功能受损可能是银屑病发生的重要诱因,然而皮肤屏障缺陷与银屑病发生的确切机制仍不清楚。该文主要对近年来皮肤屏障与银屑病的研究进展作一综述。     

Mitogenic effects of sera from normal and psoriatic subjects on human skin fibroblasts

Summary Sera from normal and psoriatic subjects had similar mitogenic effects when added to monolayer cultures of human skin fibroblasts. Fibroblasts derived from a psoriasis patient proliferated faster in both types of sera than fibroblasts from a normal subject, despite similar rates of proliferation in foetal calf serum. The psoriatic fibroblasts appeared to be inherently capable of a greater response to human serum mitogens than the normal fibroblasts.     

Development and characterization of a canine skin equivalent

The development of a complex cellular model, which incorporates the basic cell components of the dog skin, would be a useful tool to investigate the biology and pathology of canine skin and also to replace animal testing partially. The aim of the present study was to develop and characterize a canine skin equivalent. Epidermal keratinocytes and dermal fibroblasts were freshly isolated from skin biopsies from healthy dogs. Fibroblasts were embedded into a bio-matrix from collagen type I matrix protein; this built the scaffold where the keratinocytes were seeded, at air exposed conditions. At 3, 7, 15 and 21 days of culture in special growth media, skin equivalents were analysed by histological, immunohistochemical and electron microscopical techniques. At 15 days, keratinocytes underwent differentiation to a multilayer epidermis with stratum basal, stratum spinosum, stratum granulosum and stratum corneum. Expression of epidermal cytokeratins in keratinocytes was detected by immunhistochemistry, and followed the same pattern than in the normal canine epidermis. Fibroblasts from the skin equivalent expressed vimentin as dermal fibroblasts do. A basement membrane (BM) was observed underneath the epidermis; ultrastructurally, it was similar to the normal canine BM and collagen IV and laminin 5 were detected immunohistochemically as major components of this structure. Skin equivalents developed from canine cutaneous cells presented a similar morphological structure than healthy canine skin. Moreover, the immunohistochemical analysis revealed the expression of the major markers of the epidermis (keratins), dermis (vimentin) and BM (collagen type IV, laminin 5).     

长链非编码RNA在皮肤病的研究进展

长链非编码RNA是一组内源性、长度超过200个核苷酸、缺少特异完整的开放阅读框、无或很少有蛋白编码功能的RNA分子,能在表观遗传学、转录和转录后水平等多个层面调控基因表达,广泛参与机体生理和病理过程.近年研究发现,长链非编码RNA在多种皮肤病的发生发展过程中发挥作用,如黑素瘤、非黑素瘤皮肤癌、银屑病等,对长链非编码RNA的研究将为这些疾病的预测、诊断和治疗以及预后评估提供有价值的信息.     

Human umbilical cord blood cells form epidermis in the skin equivalent model

Please cite this paper as: Human umbilical cord blood cells form epidermis in the skin equivalent model. Experimental Dermatology 2010. Abstract: Recently, human embryonic stem cells have been differentiated in vitro into functional epidermal keratinocytes. Here, we demonstrated that these cells can be generated also from non‐embryonic, human umbilical cord blood (hUCB) cells that have the potential to differentiate into cells of non‐hematopoietic lineage. Human UCB mono‐nucleated cells were cultivated in monolayer and in three‐dimensional skin equivalent cultures and assayed for the presence of phenotype‐specific markers. Our results determined that after one month of culturing in serum containing medium, the hUCB cells produced morphologically homogeneous colonies of epithelial cells expressing keratinocyte‐specific markers. They also formed stratified epidermis in organ cultures that contained sporadic CD1a‐positive cells within the accurate strata. We concluded that hUCB cells have the capacity to differentiate into functional epidermal keratinocytes and may serve as a source of high‐quality keratinocytes for clinical applications.     

皮肤中存在全功能下丘脑-垂体-肾上腺皮质轴类似单位

下丘脑-垂体-肾上腺皮质轴是生物体对体内外各种应激刺激做出快速反应所必需的神经内分泌负反馈调节系统.研究表明,除中枢神经系统存在经典下丘脑-垂体-肾上腺皮质轴以外,皮肤及毛囊附属器也存在着与经典下丘脑-垂体-肾上腺皮质功能类似的外周单位,参与皮肤对局部应激做出快速反应的调节,如改变皮肤及毛囊黑素单元的功能,增强皮肤对紫外线照射的抵抗和维持或重建毛囊的免疫赦免.     

Epidermal growth factor receptors in different skin tumors

Specific binding of 125I-labeled epidermal growth factor (EGF) was measured in 62 skin tumors of different severity. Within a group of 28 benign tumors, 11 of 15 condylomata acuminata were receptor positive, whereas the investigated mesenchymal tumors and normal skin as a control were receptor negative. 6 of 18 basal cell epitheliomas bound EGF specifically. In the group of precancerous and malignant skin tumors, 7 of 8 squamous cell carcinomas had the highest number of EGF binding sites and a high affinity state, whereas 5 malignant melanomas were receptor negative. The clinical relevance of these findings is not yet clear due to the short follow-up of the patients.     

Repeated tape stripping of normal skin: a histological assessment and comparison with events seen in psoriasis

The aim of the present study was to investigate the response of normal human skin to repeated courses of Sellotape stripping. The skin of healthy volunteers was stripped five times at 24-h intervals. Skin biopsies were taken before stripping (day 0) and on days 2, 4, 7 and 10. The responses were studied using H & E staining and an immunohistochemical analysis of several aspects of epidermal proliferation and keratinization. Although increased proliferation (nuclear binding to Ki-67 binding), acanthosis and parakeratosis were observed, the overall histological picture did not resemble psoriatic histology completely: no micropustules of Kogoj and no thinning of the suprapapillary plate were observed. Involucrin staining followed the recruitment of cycling epidermal cells showing a statistically significant elevation of positive cell layers from day 2 onwards. Filaggrin expression showed an increase from day 2 onwards, which was statistically significant on day 7 and day 10. Using the anti-keratin antibodies KS8.12 (K13 and K16) and RKSE60 (K10) we observed a fast induction of K13/K16 expression, while the staining of keratin 10 showed the same overall intensity at different time intervals. In conclusion, the response to repeated courses of tape stripping provides an adequate model for studies on epidermal proliferation, hypergranulosis and hyperkeratosis. This approach causes a more prolonged induction of these phenomena than a single course of stripping. In contrast to the situation following a single course of stripping, repeated tape stripping induced the expression of filaggrin. Therefore the repeated tape stripping model is less compatible with psoriasis than a single course of stripping.     

Characterization of activity of alkaline phosphatase (AAP) in capillary endothelium of normal and psoriatic skin

Summary The activity of alkaline phosphatase (AAP) in papillary capillaries of normal and psoriatic skin was characterized by enzyme inhibition studies. Quantitatively, there were pronounced differences between normal and psoriatic skin, i.e., increase of AAP, as determined by a grading system, and assimilation of the strength of AAP in venous and arterial side of the capillary loop in psoriasis.Qualitatively, the inhibition studies with different acting inhibitors revealed no difference between AAP in normal and psoriatic skin or between initial, fully developed and healed psoriatic lesions as well as noninvolved skin of psoriatics. Thus, the physiologic AAP seems to be stimulated in psoriasis.Generally, AAP of dermal capillaries is highly sensitive to cysteine inhibition.Dedicated to Professor Dr. W. Schneider on his 70th anniversary     

表皮生长因子受体在皮肤良恶性肿瘤中的表达

为探讨表皮生长因子受体（EGFR）与皮肤肿瘤的关系。作者用流式细胞免疫荧光技术，检测了33例皮肤良恶性病变。结果显示：EGFR的相对含量荧光指数（FI）在恶性肿中的表达明显高于良性肿瘤（P＜0．001）。提示EGFR在肿瘤的发生，发展中起着重要的作用。     

闭合蛋白表达异常与相关皮肤病

皮肤屏障由角质层、细胞间质和紧密连接组成.闭合蛋白是构成紧密连接的主要蛋白之一,存在于哺乳动物的皮肤、脑、神经系统和内脏组织中,在生理状态下影响紧密连接的整体性及其功能,是构成皮肤栅栏和屏障的主要功能蛋白之一.在人类组织中已发现27种闭合蛋白亚型,在上皮细胞间形成对电解质及溶质分子的屏障结构.研究表明,皮肤屏障功能受损是多种皮肤病如银屑病、特应性皮炎、家族性良性天疱疮、光线性角化病等重要的发病因素,这与闭合蛋白功能和结构的异常密切相关.闭合蛋白家族中不同成员在不同皮肤病中的表达各异.