New immunodeficient (nude-scid, beige-scid) mice as excellent recipients of human skin grafts containing intraepidermal neoplasms

Engraftment of normal or lesional human skin onto nude or SCID (severe combined immunodeficiency) mice has been used as an in vivo experimental model. However, this model has some limitations, such as shrinkage and loss of the grafted skin over time. To improve the experimental model, we have produced two new SCID-lineage mouse strains, BALB/cA-nude-scid ( nu/nu , scid/scid ) and BALB/cA-beige-scid ( bg/bg , scid/scid ) mice, by the method of cross intercross. Intraepidermal neoplastic lesions such as Bowen’s disease were grafted onto the back of the mice of these strains. The rate of reduction in the size of the grafts was lower on nude-scid and beige-scid mice than on SCID mice. Rates of survival of neoplastic cells in the grafts were higher in nude-scid mice than in SCID and beige-scid mice (SCID mice 38%, nude-scid mice 55%, beige-scid mice 38%). Neoplastic cells of Bowen’s disease grafted onto a beige-scid mouse proliferated and invaded the dermis during 233 days of observation, confirming the progression to invasive squamous cell carcinoma from carcinoma in situ. The present study revealed that nude-scid and beige-scide mice newly produced by us provide a very useful in vivo experimental model for the investigation of carcinogenesis and tumor progression in human skin. Received: 10 April 1996     

Hyperpigmentation of human skin grafted on to athymic nude mice: immunohistochemical study

Human skin grafted on to athymic nude mice (BALB/C-nu/nu) spontaneously hyperpigments. We wished to identify the morphological and molecular bases for the hyperpigmentation for this phenomenon. We present data on the relationship of healing, regeneration of melanocytes and production of some melanogenic stimuli. Biopsies were taken at preset times post-graft and studied by histological and immunohistochemical methods. DOPA-positive melanocytes first became visible 120 h post-graft and melanin deposition became visible along the basal cell layer 2 weeks post-graft and increased in quantify with time. By immunochemical stains the quantity of three melanocyte specific enzymes, i.e. tyrosinase, tyrosinase-related protein-1 (TRP-1) and DOPA-chrome tautomerase (TRP-2), was markedly enhanced 1 week after grafting and persisted until 4 weeks post-graft. α-Melanocyte-stimulating hormone and adrenocorticotrophic hormone were clearly detected in the epidermis soon after grafting. They were still strongly detected in the epidermis and in the dermis 2–4 weeks post-graft. We conclude that hyperpigmentation in the grafted skin accompanies a marked increase in the quantity of melanogenic enzymes and melanogenic peptides. The neouropeptides might be one of many factors which stimulate melanogenesis.     

Autoradiographic studies of cell dynamics in immunogenic granulomas transplanted into the skin of athymic nude mice

Summary Schistosome egg granulomas in the livers of thymus-intact (nu/+) mice are large and contain eosinophils and mast cells, while those in nude athymic (nu/nu) mice are small and devoid of eosinophils or mast cells. To investigate the cell sources and cell kinetics of hepatic granulomas of nu/+ mice isolated and grafted into the skin of nu/nu mice, biopsies taken after grafting were examined by light and electron microscopy and autoradiography after ³H-thymidine (TdR) injection of either the donor or recipient mice. At 1 week, the grafted granulomas appeared to be amorphous and were surrounded by leukocytes, and the ³H-TdR-labeled donor cells had disappeared. After 2 weeks, repopulation with macrophages began and by 3–5 weeks, the granulomas morphologically resembled hepatic lesions of nu/+ mice. Injection of recipients with ³H-TdR before grafting, showed that labeled macrophages, eosinophils, and mast cells repopulated in granulomas. No granulomas were seen when nu/nu mice were grafted with schistosome eggs alone, and organ culture of nu/+ granulomas before grafting reduced the number of repopulated granulomas. These findings indicate that nu/+cells in grafted granulomas are replaced by nu/nu cells. Granulomatous reaction of the grafted sites in nu/ nu mice is influenced by a substance in nu/+ granulomas, and cells of nunu mice locally acquire a nu/+ type response.     

Wound healing of human skin transplanted onto the nude mouse after a superficial excisional injury: human dermal reconstruction is achieved in several steps by two different fibroblast subpopulations

Abstract It has been established that human skin grafted onto the nude mouse is able to regenerate after being subjected to a full-thickness wound. In the present work, we sought to determine the cells involved in the connective tissue repair process following superficial wounding. Two months after transplantation, superficial wounds were made at the center of the graft using mechanical dermabrasion. At various times thereafter, ranging from 2 days to 6 weeks, healing grafts were harvested and processed for immunohistological study with species-specific and cross-reacting antibodies directed against human or mouse antigens. The grafted human skin regenerated according to the following series of events. First, the human dermis underneath the scab became devoid of human fibroblasts while the surrounding human dermis preserved its own characteristics. The TUNEL reaction on early-phase healing wounds indicated that apoptosis occurred steadily within this area and could be the mechanism by which cells disappeared. Moreover, cell death was reduced when the wound was covered with an occlusive dressing. The human dermis beneath the wound was then invaded by mouse cells which deposited type I collagen on the human extracellular matrix and produced mouse granulation tissue at the surface above it. Human keratinocytes migrated over the mouse granulation tissue to reconstruct the epidermis. Eventually, the mouse granulation tissue was progressively invaded by human fibroblasts, which formed a human neodermis. The overall process appeared to depend upon several successive epithelial-mesenchymal interactions, which were not species-specific. This suggests that myofibroblasts arise from a specific subpopulation of fibroblasts, probably located at the interface between the dermis and adipose tissue, and that the granulation tissue is eventually remodeled by another population of fibroblasts present in the human dermis. Received: 31 March 1999 / Accepted after revision: 15 July 1999 / Accepted: 13 August 1999     

Genetically modified mice as a tool to study inflammatory skin diseases

Although mice have a long tradition as models for human skin diseases, they have recently received increasingly more attention. This is because of the rapid advancement of genetic engineering methods which made it possible to create mice with precisely defined genetic changes. Many of these mice develop impressive and sometimes unexpected, puzzling phenotypes. Their interpretation is a major challenge to the basic researcher and can often be ameliorated by the input of an experienced dermatologist. Together with recent examples of genetically modified mouse models of inflammatory skin diseases we give a short overview of the methods used to generate such mice, describe possible ways to analyse them, and discuss problems that arise in the interpretation of the findings.     

Morphology of the keratin filament network in palm and sole skin: Evidence for site-dependent features based on stereological analysis

Ridged or glabrous skin of palms and soles has a specialized function and can be preferentially involved volved in various disorders of keratinization. To better define the morphological features of ridged skin, we carried out a qualitative and quantitative (stereological) analysis of normal epidermis from the palm and sole of four subjects. Skin from the upper arm was examined for control purposes. The study focused on the appearance and arrangement of the keratin filament network in relation to epidermal differentiation. Whereas palm and sole epidermis was essentially similar both qualitatively and quantitatively, it differed markedly from the epidermis from the arm. The volume density of keratin filaments was significantly higher (P<0.03) in all subcorneal layers of the palm and sole compared with the arm. The volume density of the keratin filaments increased markedly from the basal to the upper spinous layer of ridged skin and they formed denser aggregates in the upper spinous and granular layers, providing an extensive matrix for the deposition of keratohyalin. The presence of dense keratin aggregates appeared to be a distinct ultrastructural feature of human ridged skin. Such keratin aggregates have not been described in normal skin from other sites, but showed some resemblance to the keratin clumps seen in non-ridged skin of patients with the Dowling-Meara form of epidermolysis bullosa simples.     

光动力疗法对人宫颈癌Caski细胞裸鼠移植瘤MIF表达的影响

目的:探讨光动力疗法对人宫颈癌Caski细胞裸鼠移植瘤巨噬细胞移动抑制因子(MIF)表达的影响。方法:选取BALB/c裸鼠,皮下注射人宫颈癌Caski细胞,10d后将达到荷瘤标准的40只裸鼠采用随机数字表法分为四组,即A组、B组、C组和D组,各10只。A组作为阴性对照、B组给予单次光动力疗法、C组给予多次光动力疗法和D组给予顺铂腹腔注射。对比治疗前和治疗2周后肿瘤体积,治疗前后裸鼠体质量、瘤体积、瘤质量变化及抑瘤率,并分别采用western-blot和免疫组化法对各组裸鼠肿瘤组织MIF表达情况进行检测,行对比分析。结果:B组、C组和D组体质量、瘤体积和瘤质量变化和A组比较差异均有统计学意义(P<0.05),且C组和D组上述指标及抑瘤率和B组比较差异均有统计学意义(P<0.05),而C组和D组间各项指标差异均无统计学意义(P>0.05);电镜分析结果显示各组肿瘤胞浆可见明显空泡,团间有明显坏死灶,并且随着光动力疗法剂量增高,坏死区域增大越明显。B组、C组和D组MIF蛋白IOD值均较A组显著降低(P<0.05),且C组和D组MIF蛋白相对表达量和IOD值均较B组显著降低(P<0.05),而C组和D组MIF蛋白相对表达量和IOD值比较差异均无统计学意义(P>0.05)。结论:采用多次光动力疗法对人宫颈癌Caski细胞裸鼠移植瘤效果和顺铂相近,且可显著降低MIF表达水平。     

A study on the delayed effect of tilapia skin collagen on skin aging for mice and its possible mechanism

Objective

Study the possible mechanism and delayed effect of tilapia skin collagen on skin aging for mice.

Materials and Methods

Kunming (KM) mice were randomly divided into the aging model group, the normal group, the positive control group (vitamin E) and the low, medium, high dose tilapia skin collagen groups (2.0, 4.0, 8.0 mg/g). The normal group was only injected with saline at the back and the neck. The other groups were injected subcutaneously with 5% D-galactose and ultraviolet light jointly to establish the aging model. After modeling, the positive control group was treated with a dose of 10% vitamin E once a day, and the low, medium, high dose tilapia skin collagen group was separately applied 2.0, 4.0, 8.0 mg/g of tilapia skin collagen for 40 days. The changes of skin tissue morphology, water content, hydroxyproline (Hyp) content, and superoxide dismutase (SOD) activity in mice were studied at the day 10, 20, 30, 40, 50.

Results

Compared with the normal group, the skin of mice in the aging model group was thinner, looser, and the skin moisture content, Hyp content, SOD activity were all decreased. For mice of the low, medium, high dose tilapia skin collagen group, the thickness of dermis increased, possessing close arrangement, and the moisture content, Hyp content, SOD activity were up-regulated significantly, which effectively alleviated the aging process of skin. The dose of tilapia skin collagen was directly proportional to the anti-aging effect.

Conclusions

Tilapia skin collagen has an obvious effect on improving skin aging.     

Effects of ultraviolet light irradiation on the skin of MRL/l mice

Summary MRL/Mp-lpr/lpr (MRL/l) and MRL/Mp-+/+ mice were irradiated with middle-wavelength ultraviolet light (UVB), and the development of skin lesions, skin lupus band test (LBT), anti-DNA antibodies in sera, and the extent of glomerulonephritis of the kidney were examined. Long-term exposure to low doses of UVB irradiation accelerated the development of skin lesions and enhanced the intensity of the positive skin lupus band in MRL/l mice. The contents of anti-DNA antibodies in sera, the incidence of positive findings in LBT, and the extent of glomerulonephritis were not influenced by the UVB irradiation. The promotion of the development of the skin lesions in MRL/l mice by the UVB exposure was not considered to be associated with acceleration of systemic autoimmune phenomena.This is the twelfth part of a series of papers entitled Pathogenesis of Lupus Dermatoses in Autoimmune Mice     

人羊膜上皮细胞复合去表皮的真皮构建组织工程皮肤修复裸鼠皮肤缺损的实验研究

目的 探索由人羊膜上皮细胞(hAEC)、成纤维细胞与去表皮的真皮(DED)构建组织工程皮肤修复裸鼠全层皮肤缺损的可行性.方法 采用低浓度胰蛋白酶多步消化分离法提取hAEC,并用两步酶消化法处理健康小儿包皮,获得成纤维细胞悬液,传代培养.将体外扩增培养至第3～5代的成纤维细胞和第2代hAEC分别接种在DED真皮面和基底膜面,体外器官培养构建组织工程皮肤.取3～4周龄健康雄性裸鼠20只,抽签法随机分为2组,在所有小鼠背部正中制备全层皮肤缺损模型.组织工程组用构建的组织工程全层皮肤覆盖创面;对照组创面仅覆盖凡士林油纱.分别于术后第7、14、21、28天对裸鼠全身及移植部位大体观察,比较两组间创面愈合时间及创面愈合率,并对移植部位行组织学观察.结果 hAEC具有干细胞特征,免疫荧光显示其表达结合转录因子4(0CT-4)、阶段特异性胚胎抗原4(SSEA-4).器官培养2周后,体外构建的组织工程皮肤形成4～9层复层表皮,且表皮的组织结构形态与正常皮肤类似.移植组织工程皮肤至裸鼠创面后肉眼观察显示,在移植后第7、14、21天,组织工程组创面愈合率分别为57.49％±6.11％、92.80％±3.10％、98.83％±0.25％,均明显高于对照组(22.93％±4.26％、54.57％±7.94％、91.16％±4.79％),差异均有统计学意义(n=10,t值分别为27.36、32.23、11.80,均P＜0.001),组织工程组创面愈合时间[(21.51±1.51)d]明显短于对照组[(28.80±1.14)d](n=10,￡=42.23,P＜0.001),且在移植后第28天移植物颜色与自体皮肤颜色接近.组织学观察显示,组织工程组上皮层次清晰,角化明显,真皮层细胞生长良好;对照组移植区上皮较薄且有部分缺损,真皮层次欠佳,且可见炎症细胞浸润.结论 用hAEC、成纤维细胞复合DED构建的组织工程皮肤移植后能在裸鼠体内存活,且创面愈合更佳,可望成为一种较理想的组织工程皮肤.     

Expression of the human Cathepsin L inhibitor hurpin in mice: skin alterations and increased carcinogenesis

The serine protease inhibitor (serpin) hurpin (serpin B13) is a cross class-specific inhibitor of the cysteine protease Cathepsin (Cat) L. Cat L is involved in lysosomal protein degradation, hair follicle morphogenesis, epidermal differentiation and epitope generation of antigens. Hurpin is a 44 kDa protein which is expressed predominantly in epidermal cells. In psoriatic skin samples, hurpin was strongly overexpressed when compared with normal skin. Keratinocytes overexpressing hurpin showed increased resistance towards UVB-induced apoptosis. To further analyse the functional importance of this inhibitor, we have generated transgenic mice with deregulated Cat L activity by expressing human hurpin in addition to the endogenous mouse inhibitor. The three independent transgenic lines generated were characterized by identical effects excluding insertional phenotypes. Macroscopically, mice expressing human hurpin are characterized by abnormal abdominal fur. The number of apoptotic cells and caspase-3 positive cells was reduced after UV-irradiation in transgenic animals compared with wild-type mice. Interestingly, after chemical carcinogenesis, transgenic mice showed an increased susceptibility to develop skin cancer. Array analysis of gene expression revealed distinct differences between wild-type and hurpin-transgenic mice. Among others, differentially expressed genes are related to antigen presentation and angiogenesis. These results suggest an important role of Cat L regulation by hurpin which might be of clinical relevance in human skin diseases.     

Human skin cancer stem cells: a tale of mice and men

Carcinomas, cancers of epithelial tissues, are the commonest malignancies and cause the greatest cancer mortality worldwide. Among these, the incidence of keratinocyte-derived non-melanoma skin cancers (NMSC), by far the greatest, is increasing rapidly. Yet despite access to tumor tissue, acceptance of human NMSC as a model carcinoma has been hindered by the lack of a reliable xenograft model. Instead, we have relied on the murine two-step carcinogenesis protocol as a reproducible squamous cell carcinoma (SCC) model, but this differs from their human counterpart in cause, site, genetic basis and biological behaviour. By xeno-engraftment of primary human SCC, we were recently successful in demonstrating the presence of primary human SCC cancer stem cells or tumor-initiating cells. These findings once more align the study human SCC as the archetypal carcinoma model. In this review, we describe the evidence for the existence of tumor-initiating cells, with emphasis on skin cancer, limiting our discussions to primary human cancer studies where possible.     

皮肤鳞状细胞癌整体可视化动物模型实验研究

目的:建立整体可视化皮肤癌的模型,以期实时动态观察药物与肿瘤体积、血清指数等指标的量效关系,为研究皮肤癌临床治疗提供一种更好的动物模型.方法:利用pEGFP-N1表达质粒转染皮肤鳞状细胞癌(以下简称鳞癌)SCL-1细胞,经筛选后建立稳定表达绿色荧光蛋白(EGFP)的皮肤鳞癌细胞株pEGFP-SCL-1.细胞悬液接种于裸鼠皮下,借助整体荧光成像系统实时监测细胞在接种部位生长情况.结果:皮肤鳞癌细胞pEGFP-SCL-1稳定、高效表达EGFP,裸鼠皮下注射部位种植成瘤,1周左右肿瘤向周围组织浸润;常规组织病理学检查验证了可视化模型的可靠性.结论:利用稳定转染的皮肤鳞癌pEGFP-SCL-1细胞,在注射部位种植建立整体可视化皮肤鳞癌,为研究皮肤癌治疗提供有效的阶段性监测的实验模型.     

Preliminary study among truck drivers in Turkey: effects of ultraviolet light on some skin entities

In this study, we aimed to compare driver (left) and opposite (right) sides for some skin entities in drivers. We also evaluated the effect of occupational duration, age and skin type among this population. One hundred and forty-eight truck drivers were included in this study. Sun exposed areas were classified. Dermatological entities such as actinic keratosis, solar lentigo, seborrheic keratosis, melanocytic nevus, spider angioma, ephelid, basal cell carcinoma (BCC), squamous cell carcinoma, and melanoma were compared to driver and right sides. In addition, drivers were divided into two subgroups according to: (i) age (subgroups A1, 20-40 years, and A2, >40 years); (ii) occupational duration (subgroups O1, 1-10 years, and 02, >10 years); and (iii) skin type (subgroups S1, skin types I and II, and S2, skin types III and IV. Each of the two subgroups were compared with respect to the total prevalence (left and right sides) of lesions excluding the location. Seborrheic keratosis and lentigo solaris were found to be increased on the driver side of the face (P = 0.00) and ear (P = 0.013), respectively. Statistically significant increases of lesions on the driver side were determined according to the subgroups: melanocytic nevi were found to be increased on the driver side in A1 (P = 0.00), A2 (P = 0.00), O2 (P = 0.00), S1 (P = 0.00) and S2 (P = 0.006) groups; actinic keratoses were found to be significantly increased on the driver side in A2 and O2 groups (P = 0.03 for both).     

正常儿童和老年人前臂内外侧皮肤电阻值对比分析

目的了解正常儿童皮肤与老年人光老化皮肤电阻值水平差异。方法研制皮肤表面电阻值测定仪,对照检测41例12岁以下儿童与25例60岁以上的人群组前臂皮肤自然光暴露侧和非光暴露侧部位皮肤表面电阻值。结果前臂光暴露部位皮肤电阻值显著高于非光暴露部位电阻值(P<0.001),且老年组电阻值显著高于儿童组(P<0.001)。结论不同部位不同年龄皮肤电阻值不同,皮肤电阻值与年龄的增加呈正相关。     

A retrospective study on the clinical efficacy of the intense pulsed light source for photodamage and skin rejuvenation

Objective: The objective of this retrospective review is to investigate the long-term effect of skin rejuvenation by the intense pulsed light (IPL) source for the treatment of photoaging. Methods: From 5300 clinical cases that our department has treated with the IPL, the first 2534 were chosen for this study. Each patient received a minimum of 3 IPL treatments during this time—many were yearly treatments. Clinical photographs were taken on a yearly basis for up to 12 years and sent to a blinded independent panel to study the effects of continuous IPL treatments. Results: Results showed that the effective rate for the IPL was between 88.24% and 96.45%. Conclusions: IPL therapy is an effective treatment for photoaging and can truly have an effect on reversing the signs of photodamage on skin.     

Organotypic keratinocyte coculture using normal human serum: an immunomorphological study at light and electron microscopic levels

Organotypic human skin equivalents of keratinocytes and fibroblasts embedded in collagen matrix have been the subject of studies dealing with various culture conditions. Development of standardized living skin equivalents using defined culture media containing respective supplements can provide important instruments of investigation in skin biology. In addition, tissue engineering has created human skin substitutes for treatment of acute and chronic wounds. In our study, we generate a modified organotypic human skin equivalent using normal human serum instead of fetal calf serum (FCS). This living skin equivalent shows regular stratification of the epidermis and the dermal-epidermal junction zone at the light and electron microscopic level after 1 and 3 weeks of coculture. Indirect immunofluorescence reveals regular expression of differentiation antigens and the major structural proteins collagen IV, laminin 5 and the integrin chains alpha 6 and beta 4 at the dermo-epidermal junction zone. Immunoelectron microscopy demonstrates expression of collagen IV, alpha 6 and beta 4 integrin after 1 and 3 weeks of coculture. This organotypic skin model could be the basis for autologous skin grafting for acute or chronic wounds using autologous serum as well as patients' keratinocytes and fibroblasts, thus minimizing the risk of transmitting infectious agents.