The contribution of stem cells to epidermal and hair follicle tumours in the dog

Background –  Although cutaneous stem cells have been implicated in skin tumourigenesis in humans, no studies have been conducted to elucidate the presence and the possible role of stem cells in hair follicle tumours in the dog. Hypothesis –  Stem cell markers are expressed in canine epidermal and follicular tumours and can be used to better understand the biology and origin of these tumours. Animals and Methods –  In the present study, normal skin sections and 44 follicular tumours were retrospectively investigated for the immunohistochemical expression of keratin 15 (K15) and nestin. In addition, 30 squamous cell carcinomas were evaluated for K15 expression. Results –  In normal skin, K15 and nestin were expressed in the outer root sheath cells of the isthmic portion of the hair follicle (bulge region), and K15 expression was also scattered in the basal cell layer of the epidermis. Infundibular keratinizing acanthomas, pilomatricomas and squamous cell carcinomas were mostly negative for K15, trichoblastomas were moderately to strongly positive, tricholemmomas were either negative or strongly positive, and trichoepitheliomas had heterogeneous staining. Nestin expression was generally faint in all follicular tumours. Conclusions and clinical importance –  Our results show that K15 can be a reliable marker for investigating the role of stem cells in hair follicle tumours of the dog, while nestin was judged to be a nonoptimal marker. Furthermore, our study suggests that hair follicle stem cells are present in the bulge region of hair follicles and could possibly play a role in tumourigenesis of canine tumours originating from this portion of the follicle, namely trichoblastomas, tricholemmomas and trichoepitheliomas. The loss of K15 expression in squamous cell carcinomas compared with normal skin suggests that this event could be important in the malignant transformation.     

Immunohistochemical localization of fibroblast growth factor 18 in hair follicles of healthy beagle dogs

Increasing emphasis is being placed on the role of fibroblast growth factors (FGFs) in hair follicle cycling. In mice, expression of FGF18 mRNA peaks during the late telogen phase, leading to the hypothesis that FGF plays a role in anagen induction. There are no data on the presence of FGF18 in dogs. The main objective of this study was to identify and locate FGF18 in the canine hair follicle. The second objective was to assess potential differences in FGF18 concentration between biopsies taken in winter and summer, shoulder and flank regions, and between different sexes. Skin tissue from 10 healthy beagle dogs (three intact females, three spayed females and four intact males) was collected from the shoulder and flank. The biopsies were collected in February and August on day 0, after which the dogs were clipped and biopsies collected again from the shoulder and flank on days 1, 3, 7 and 17. Paraffin sections (4 μm thick) of the biopsies were stained with an anti-FGF18 antibody. The FGF18-positive cells were counted in the hair follicle epithelium from seven follicular units of each biopsy. Fibroblast growth factor 18 was detected as granular cytoplasmatic staining in follicles at the level of the inner root sheath, and rarely in the outer root sheath and dermal papilla. It was also detected in the apocrine glands, in arrector pili muscles and in vascular endothelial cells. There was no statistical difference in the number of FGF18-positive cells or follicles between sexes, different anatomical locations, seasons or the consecutive days of sampling.     

Epidermal structure created by canine hair follicle keratinocytes enriched with bulge cells in a three‐dimensional skin equivalent model in vitro: implications for regenerative therapy of canine epidermis

Background – Keratinocytes in the hair follicle bulge region have a high proliferative capacity, with characteristics of epithelial stem cells. This cell population might thus be an ideal source for generating the interfollicular epidermis in a canine skin equivalent. Hypothesis/Objectives – This study was designed to determine the ability of canine hair follicle bulge cell‐enriched keratinocytes to construct canine living skin equivalents with interfollicular epidermis in vitro. Animals – Four healthy beagle dogs from a research colony. Methods – Bulge cell‐enriched keratinocytes showing keratin 15 immunoreactivity were isolated from canine hair follicles and cultured on dermal equivalent containing canine fibroblasts. Skin equivalents were subjected to histological, immunohistochemical, western blot and RT‐PCR analyses after 10–14 days of culture at the air–liquid interface. Results – The keratinocyte sheets showed an interfollicular epidermal structure comprising four to five living cell layers covered with a horny layer. Immunoreactivities for keratin 14 and desmoglein 3 were detected in the basal and immediate suprabasilar layers of the epidermis, while keratin 10 and desmoglein 1 occurred in more superficial layers. Claudin 1 immunoreactivity was seen in the suprabasalar layer of the constructed epidermis, and filaggrin monomers and loricrin were detected in the uppermost layer. Basal keratinocytes in the skin equivalent demonstrated immunoreactivity to antibodies against basement membrane zone molecules. Conclusions and clinical importance – A bulge stem cell‐enriched population from canine hair follicles formed interfollicular epidermis within 2 weeks in vitro, and thus represents a promising model for regenerative therapy of canine skin.     

内蒙古绒山羊Homeoboxc9基因在皮肤毛囊中的表达

试验通过原位杂交检测Homeoboxc9（Hoxc9）基因在皮肤毛囊中的表达情况,结果显示Hoxc9在胚胎期75 d的上皮细胞中表达,伴随着毛囊的生长发育,Hoxc9基因分别在初级毛囊的毛母质、毛乳头、内根鞘、外根鞘和毛干几个部位表达;在次级毛囊的内根鞘和绒干表达。在这个过程中毛囊角质化细胞大量增殖,表明在毛囊发育中Hoxc9基因对于角质化细胞的增殖可能起一定作用。     

水貂毛皮成熟期皮肤中EGF、IGF-Ⅰ及IGF-ⅠR基因的表达

为探讨水貂皮肤和毛囊发育机理,试验运用组织学和免疫组化方法对水貂毛皮成熟期皮肤及毛囊中表皮生长因子（epidermal growth factor, EGF）和胰岛素样生长因子-Ⅰ（insulin-like growth factor-Ⅰ,IGF-Ⅰ）及其受体（insulin-like growth factor-Ⅰreceptor,IGF-ⅠR）进行了研究。结果表明,水貂皮肤表皮细胞和毛囊外根鞘细胞中都有IGF-Ⅰ、IGF-ⅠR和EGF基因的强阳性表达,3种基因主要分布于细胞质中,细胞核呈空泡状未见到阳性染色,呈苏木精复染的蓝色,皱褶区域和表皮下胶原结缔组织出现了阳性染色为非特异性阳性结果。IGF-Ⅰ、IGF-ⅠR和EGF基因在水貂皮肤表皮和毛囊外根鞘细胞中广泛表达,直接参与调控皮肤和毛囊的发育。表明EGF、IGF-Ⅰ和IGF-ⅠR基因在水貂皮肤及其衍生结构的发育中发挥着重要作用。     

幼年牦牛不同部位皮肤毛囊的组织学结构及TGF-β2与HIF-1α差异性表达分析

旨在探究幼年牦牛皮肤毛囊的组织学结构和TGF-β2及HIF-1α对幼年牦牛皮肤毛囊生长发育的影响，选取10头健康幼年牦牛，采集其颈部、背部、胸部、腹部、小腿部、腋下及阴囊皮肤组织，制作石蜡切片后，采用HE和Sacpic染色，对各部位皮肤组织中毛囊进行观察和计数，并筛选出多毛及少毛部位。利用qRT-PCR、Western blot及免疫组织化学法对TGF-β2和HIF-1α在幼年牦牛多毛皮肤和少毛皮肤进行定位与定量的初步研究。结果表明，幼年牦牛皮肤的毛囊分布于真皮层，常与汗腺及皮脂腺伴行，毛髓质及内根鞘结构不完整。Sacpic染色可见毛囊内根鞘为红色，外根鞘为苍绿色，结缔组织鞘为蓝绿色。腹部皮肤的毛囊数量最多[（2 085±15）个·cm^-2]，阴囊数量最少[（158±15）个·cm^-2]。免疫组织化学结果显示，TGF-β2及HIF-1α主要表达在表皮层、毛囊外根鞘、皮脂腺及汗腺。TGF-β2在阴囊和腋下的mRNA转录水平和蛋白质表达水平均显著高于腹部和背部。HIF-1α在腹部的mRNA转录水平和蛋白质表达水平均显著高于其他三个部位。幼年牦牛的毛囊处于退行期，在腹部数量最多，背部次之，阴囊最少；TGF-β2和HIF-1α在不同部位皮肤中的表达水平存在显著差异，为进一步研究TGF-β2和HIF-1α对牦牛皮肤毛囊生长发育的影响提供理论依据。     

Immunohistochemical localization of androgen and oestrogen receptors in canine hair follicles

Skin biopsies from seven different body sites were obtained from 21 dogs of different breeds (short, normal, long hair), which were presented for euthanasia. Commercially available polyclonal antibodies were used for the immunohistochemical detection of androgen and oestrogen receptors. Both receptors showed a similar distribution in canine skin, with specific intranuclear staining. In the epidermis, the percentage of androgen receptor (AR)-positive cells, but not that of oestrogen receptor (ER)-positive cells, was significantly higher in samples from the thorax and the flank. In the dermal papilla, the percentage of ER-positive (but not AR-positive) cells was significantly lower in biopsies from the flank. No significant difference was found for both receptors between the locations in the outer root sheath, among the three different hair types, between sex and between intact and castrated dogs.     

不同水平肌醇对獭兔皮肤中β-连环蛋白和激素敏感脂酶表达的影响

本试验旨在研究饲粮中添加不同水平的肌醇对獭兔皮肤中β-连环蛋白(β-catenin)和激素敏感脂酶(HSL)表达的影响。选取(40±1)日龄的同期断奶獭兔120只(公母各占1/2),随机分成4组,每组30只。在4组獭兔的饲粮中分别添加0、25、50、75 mg/kg的肌醇,试验期为3个月。在试验的第30天(2月龄)、第60天(3月龄)和第90天(4月龄),分别取腹部、背中部、臀部皮肤,采用免疫组织化学和蛋白质印迹(Western blot)法对β-catenin和HSL的蛋白质表达和定位进行检测。结果表明:β-catenin在毛囊中广泛表达,在毛根鞘细胞和毛乳头均有棕黄色阳性反应细胞。HSL在毛根鞘细胞,尤其是在内根鞘细胞内,呈现非常明显的棕黑色强阳性表达。饲粮中添加50 mg/kg肌醇可以极显著增加2~4月龄獭兔背中部皮肤毛囊中β-catenin和HSL阳性表达细胞的平均灰度值(P0.01),极显著增加4月龄獭兔背中部、腹部和臀部皮肤毛囊中β-catenin和HSL阳性表达细胞的平均灰度值(P0.01)。结果提示,肌醇能够通过上调4月龄獭兔毛囊中β-catenin和HSL的表达来促进毛囊的发育,在本试验中,饲粮肌醇水平达到50 mg/kg时效果最佳。     

Skin morphology of thyroidectomized rats

We induced hypothyroidism in rats by conducting a thyroidectomy (TD) and investigated subsequent changes in the morphology of the skin, especially that of the epidermis and hair follicles. The 6 rats in the TD group seemed less active than the 3 rats in the control group and had cold, dry paws. All of the rats in the TD group exhibited retarded hair growth 12 weeks after surgery. Histologically, all of the rats in the TD group exhibited epidermal thinning from 12 weeks after surgery. Many hair follicles were in the telogen phase: the bulbs and papillae were involuted and had migrated towards the epidermis. Hair follicle atrophy involving thinning of the outer root sheath and the inner root sheath was often observed. The immunoreactivities of antithyroid hormone receptors alpha and beta in the outer root sheaths of 5 of the TD rats were weaker than those of control rats. Cell proliferation in hair follicles of TD rats was weaker than in follicles of control rats 4 weeks after surgery. It is suggested that decreased expression of TRs and decreased cell proliferation activity in the hair follicles of rats is associated with a lack of thyroid hormone and results in retardation of hair growth.     

Effects of Photoperiod on Stem Cells Activation in Cashmere Goat Hair Follicles

In order to extend the anagen of cashmere goat hair follicles and increase the production of cashmere,this study was performed with artificially shorten the daylight time among Arbas White cashmere goats. Skin tissue sections from cashmere goats were collected to compare the morphologic changes between artificial daylight and natural daylight,and immunohistochemical method was used to study the hair follicle cell proliferation and important protein expression in related signaling pathways. The results showed that strong cell proliferation occurred in cashmere goat hair follicle cells during artificial daylight,plenty of cytokeratin 15 (K15) positive signals were distributed in the outer root sheath,β-catenin protein was actively expressed in hair matrix and root sheath, indicating that the hair follicles were in the anagen growth phase;Meanwhile,cashmere goat hair follicles under natural daylight were in telogen with weak signals. Above all prove that short photoperiod played an important role in promoting hair follicle growth,the artificial short photoperiod could change hair follicle growth cycle and make hair follicles earlier enter to the anagen growth phase,causing a variety of typical gene expressions during hair follicle growth.     

Cell proliferation kinetics in the hair root matrix of dogs with healthy skin and dogs with idiopathic seborrhea

Cell proliferation kinetic values were established for the hair root matrix of primary anagen follicles of 14 Beagles and 4 Cocker Spaniels with healthy skin and 9 Cocker Spaniels with primary idiopathic seborrhea. Indices were established by intradermal pulse labeling with tritiated thymidine, followed by cutaneous biopsy and autoradiography. The hair root matrix cell labeling index was 23.4 +/- 3.5% for Beagles, 24.4 +/- 4.0% for healthy Cocker Spaniels, and 24.9 +/- 4.3% for seborrheic Cocker Spaniels. These values indicate a rapidly proliferating cell population. Differences among these cell kinetic data for the 3 groups of dogs were not statistically significant. Although significant cell kinetic differences have been reported for other epidermal structures (interfollicular epithelium, upper hair follicle external root sheath, sebaceous glands) in seborrheic Cocker Spaniels, proliferation of hair root matrix cells apparently remains unaffected.     

光周期对于绒山羊毛囊干细胞激活的影响

为了延长绒山羊毛囊兴盛期,提高羊绒产量,本试验通过人工缩短内蒙古阿尔巴斯型绒山羊的日照时间,利用组织切片技术对比人工短光照和自然光照周期下绒山羊皮肤组织形态变化,利用免疫组织化学方法研究毛囊细胞增殖及相关信号通路重要蛋白的表达。结果显示,人工短光照周期下绒山羊毛囊细胞增殖强烈,干细胞标记角蛋白15（cytokeratin 15,K15）阳性信号大量分布于外根鞘,β-catenin信号活跃表达于毛母质及根鞘,毛囊提前进入兴盛期;而自然光照周期下绒山羊毛囊仍停留在休止期,相应蛋白信号表达较弱。综上表明,短日照对毛囊生长具有明显的促进作用,人工短光照周期可以提前激活毛囊干细胞,使其提前进入兴盛期,引起多种毛囊生长相关蛋白的表达。     

Proliferative and necrotizing otitis externa in four cats

Proliferative and necrotizing feline otitis externa is a rare disorder of unknown aetiology. This condition was diagnosed by skin biopsy in three adult domestic shorthair cats (3-5 years old) and one kitten (6 months old). The affected cats had large tan to dark brown-black coalescing plaques covering the concave surface of the pinnae and external ear canals. Friable material from the plaques and a thick exudate occluded the ear canals. The cats had a secondary bacterial and/or yeast otitis. Prior to the histopathological diagnosis, all cats received numerous otic preparations as well as oral antibiotics and corticosteroids without resolution. Histologically, all cases had strikingly similar changes; acanthosis with pronounced hair follicle outer root sheath hyperplasia and neutrophilic luminal folliculitis, follicular keratosis and individually necrotic keratinocytes in the outer root sheath of hair follicles. One case was documented via skin biopsy to have persisted for 4 years. The adult cats were treated with topical 0.1% tacrolimus and all showed marked improvement although one cat was lost to follow up. The lesions completely resolved with topical tacrolimus alone in one cat and topical tacrolimus in addition to oral prednisolone in another cat.     

不同日龄滩羊皮肤组织化学特征比较

为比较2、35日龄滩羊皮肤毛囊的发育特点与血管内皮生长因子（vascular endothelial growth factor，VEGF）和血管内皮生长因子受体2（vascular endothelial growth factor receptor 2，VEGFR2）的分布特征，探究出生后滩羊被毛生长发育的变化特点，试验应用常规HE染色及改良Masson胶原纤维染色、Gomori银氨法染色、磷钨酸染色等特殊染色观察2与35日龄滩羊皮肤组织结构特点；应用免疫组织化学法结合免疫荧光染色法观察VEGF及VEGFR2在2与35日龄滩羊皮肤组织中的分布定位，并用IPP图像分析软件进行定量分析。结果显示：与2日龄滩羊皮肤组织比较，35日龄滩羊表皮与真皮间界限更加清晰，毛囊结构发育完整；毛囊密度显著降低（P<0.05）；胶原纤维与弹性纤维含量增加，形成网格状分布。免疫组化及免疫荧光结果显示，VEGF及VEGFR2在滩羊皮肤表皮及毛囊外根鞘、皮脂腺上均有表达。统计表明，VEGF及VEGFR2在2日龄滩羊皮肤组织中的表达量均显著高于35日龄（P<0.05）。综合上述结果，滩羊毛囊发育过程中，胶原纤维和弹性纤维增加明显；VEGF与VEGFR2通路在毛囊角质形成中起直接调节作用。     

Clear cell trichoblastomas in two dogs

Two cases of clear cell trichoblastomas were diagnosed in young dogs. The tumour had ribbons of basaloid cells as seen in ribbon trichoblastomas, as well as differentiation to external root sheath of the hair follicle and few cells with sebaceous differentiation. This is the first report of clear cell trichoblastoma in dogs.     

Alopecia areata in C3H/HeJ mice involves leukocyte-mediated root sheath disruption in advance of overt hair loss

Alopecia areata (AA) can be induced in C3H/HeJ mice by grafting full-thickness AA-affected skin. An 8- to 12-week delay between surgery and overt hair loss onset provides an opportunity to examine disease pathogenesis. Normal haired C3H/HeJ mice were sham-grafted or grafted with AA-affected skin. Mice were euthanatized 2, 4, 6, 8, 10, and 12 weeks after surgery along with chronic AA-affected mice as a positive control. Until 6 weeks after grafting, inflammation was only evident around anagen-stage hair follicles in host skin adjacent to but not distant from the AA-affected graft. From 8 weeks on, AA-grafted but not sham-grafted mice exhibited a diffuse dermal inflammation at distant sites that progressively focused on anagen-stage hair follicles at 10 and 12 weeks. Perifollicular inflammation was primarily composed of CD4+ and CD8+ cells associated with follicular epithelium intercellular adhesion molecule -1 expression. Only CD8+ cells penetrated intrafollicularly by 12 weeks after surgery, although both CD4+ and CD8+ intrafollicular cells were observed in chronic AA-affected mice. Under electron microscopy, intrafollicular lymphocyte and macrophage infiltration associated with hair follicle dystrophy was prominent 10 weeks after surgery, primarily within the differentiating outer and inner root sheaths. This study shows that focal follicular inflammation develops some time in advance of overt hair loss and focuses on the differentiating root sheaths in C3H/HeJ mice. The severity of inflammation and the degree of hair follicle dystrophy induced by the infiltrate appear to reach a threshold level before overt hair loss occurs.     

26例犬毛囊肿瘤的组织病理学诊断和免疫组织化学分析

本研究旨在为犬毛囊肿瘤的诊断提供最优的免疫标记物,提高肿瘤诊断的准确性,缩短肿瘤的病理学诊断时间,为犬毛囊肿瘤病的临床精准治疗提供帮助。作者收集了26例临床犬毛囊肿瘤病例,分别用免疫标记物CK5/6、CK8/18、CK19、P63、CD34、CD10、Vimentin对肿瘤样本进行免疫组织化学标记（IHC）。26例犬毛囊肿瘤的IHC染色结果表明,CD34在86.9%的毛囊肿瘤中呈瘤细胞阳性;CD10和CK8/18在毛上皮瘤中呈瘤细胞阳性,在其他毛囊肿瘤中均为瘤细胞阴性;CK19在良性和恶性毛上皮瘤中分别为强阳性和中阳性,CD34在良性和恶性毛上皮瘤中分别为中阳性和弱阳性;CD10在毛母细胞瘤中呈周边间质特异性阳性;Vimentin在毛囊肿瘤中呈瘤细胞阴性,间质细胞阳性。结果显示,CK5/6、P63可用作本研究中所有毛囊肿瘤的阳性标记物;CD34可用作除毛母质瘤和毛囊瘤外的其他毛囊肿瘤的瘤细胞特异性阳性标记物;CD10和CK8/18可用作毛上皮瘤的瘤细胞阳性标记物;共同使用CK19和CD34可用于区分毛上皮瘤的良恶性;CD10可用作毛母细胞瘤周边间质特异性阳性标记物;Vimentin可用作毛囊肿瘤的瘤细胞阴性标记物。     

Equine basal cell tumors

Basal cell tumors from 11 horses were examined histologically. Distinct patterns identified in the order of frequency are adenoid (six), solid (three), and medusoid (two). A minor component of most of these neoplasms was a ribbon pattern and cystic spaces. Hyalinized connective tissue and clear cells reminiscent of hair follicle glassy membranes and outer root sheath were present in a solid and the medusoid tumors. Heavy melanin pigmentation was present in one solid basal cell tumor. In eight cases for which information was available, the tumors had not recurred 3 to 8 years after excision.     

云南半细毛羊毛囊干细胞表面标记物的鉴定

设计8对引物（CK14、CK15、CK19、CD34、CD271、CD200、nestin和β1）,分别扩增云南半细毛羊成纤维细胞和毛囊干细胞表面标记物.结果表明：在成纤维细胞中CK19和β1表现为阳性,而在毛囊干细胞中CK14、CK19和β1表现为阳性,其他标记物的表达都为阴性.因此,CK14可以作为鉴定云南半细毛羊毛囊干细胞的表面标记物之一.     

毛囊干细胞的分离方法及其应用

毛囊干细胞是皮肤组织工程理想的种子细胞,分离纯化是其研究的基础手段。毛囊干细胞能分化成毛囊、皮脂腺、表皮,在组织维持与更新及某些临床疾病的治疗中均起到重要的作用。作者介绍了近年来毛囊干细胞几种常用的分离纯化方法,包括组织块法和酶消化法等,为进一步研究毛囊干细胞的特性和应用提供参考。