Immunohistochemical detection of p27 and p21 proteins in canine hair follicle and epidermal neoplasms

Trichoblastomas, trichoepitheliomas, and squamous cell carcinomas in the skin of dogs were analysed by immunohistochemistry for the nuclear expression of p27, p21 and proliferating cell nuclear antigen (PCNA). High levels of p27 were present in trichoepitheliomas and trichoblastomas compared with squamous cell carcinomas. Detectable p21 was found in trichoepitheliomas and squamous cell carcinomas, but trichoblastomas had low level of p21 nuclear reactivity. Low levels of PCNA were detected in trichoepitheliomas and trichoblastomas compared with squamous cell carcinomas. The results suggested that nuclear p27 acts as a cyclin-dependent kinase (CDK) inhibitor in trichoepitheliomas and trichoblastomas. Nuclear p21 expression is involved in the induction of epithelial differentiation and seems to be unrelated to CDK inhibition.     

Epithelial‐to‐mesenchymal transition: immunohistochemical investigation of related molecules in canine cutaneous epithelial tumours

Background –  Epithelial‐to‐mesenchymal transition (EMT) is a multistep process, important in tumour invasion and metastasis, characterized by loss of epithelial markers, redistribution of β‐catenin and gain of mesenchymal markers. Hyposthesis/Objectives –  Our aim was to investigate the immunohistochemical aberrant expression of cytokeratin, vimentin, survivin and heat shock protein 72 (Hsp72) in canine cutaneous epithelial tumours, to understand the association of expression of these molecules with features of malignancy and their role in the EMT phenotype. Methods –  Ten canine squamous cell carcinomas (SCCs; one with lymph node metastasis), 30 canine hair follicle tumours (six pilomatricomas, eight infundibular keratinizing acanthomas, six trichoepitheliomas and 10 trichoblastomas) and five normal skin samples were investigated by immunohistochemistry using specific anti‐vimentin, ‐cytokeratin, ‐survivin and ‐Hsp72 antibodies. A semi‐quantitative method was used to analyse the results, as follows: 0 to <5%; ≥5 to <10%; ≥10 to <25%; and ≥25% of positive cells. Immunofluorescence was performed to investigate survivin–vimentin and survivin–Hsp72 colocalization in selected SCCs. Results –  In malignant hair follicle tumours and SCCs, a reduced intensity of cytokeratin and increased survivin and Hsp72 expression were observed. In SCCs, loss of cytokeratin expression and vimentin immunolabelling, suggestive of the EMT phenotype, were evident in <5% of neoplastic cells in the front of tumour invasion. In the same areas, strong nuclear survivin and cytoplasmic Hsp72 staining was evident, often colocalizing. Only a few neoplastic cells in the front of tumour invasion showed vimentin–survivin colocalization. Conclusions and clinical importance –  A possible simultaneous involvement of survivin and Hsp72 in tumour invasion and the multistep process of EMT of cutaneous epithelial tumours of dogs is suggested.     

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。     

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.     

CD34 glycoprotein identifies putative stem cells located in the isthmic region of canine hair follicles

It is widely documented that a pool of multipotent stem cells located in humans and mice hair follicle outer root sheath (bulge region) is involved in the restoration of the whole follicular unit during each anagen phase. To the authors' knowledge, data regarding the location and characterization of hair follicle stem compartment in dogs have not been reported in the recent relevant literature. In this study, we investigated the haematopoietic stem and progenitor cell antigen CD34 as a marker of putative stem cells located in a bulge-like region of canine hair follicles. The presence of CD34 mRNA and glycoprotein was assessed on formalin-fixed, paraffin-embedded canine skin samples by in situ hybridization technique and by standard immunohistochemistry, respectively. A strong expression of CD34 mRNA and glycoprotein was observed in a well-defined area of the hair follicle isthmic region and appeared uniformly concentrated at the level of the basal layer of the outer root sheath. These findings provide compelling support to the hypothesis that in dogs, a subpopulation of basal keratinocytes located in the hair follicle isthmic region and characterized by the selective expression of CD34 is potentially associated with the stem cell compartment of this skin appendage.     

Immunohistochemical studies on cytokeratin 8 and 18 expressions in canine cutaneous adnexus and their tumors

The expressions of cytokeratin 8 and 18 (CK8 and CK18) in the normal canine skin (2 cases) and cutaneous adnexal tumors (127 cases) were investigated immunohistochemically. In the normal skin, co-expression of CK8/18 was found in the glandular epithelium of apocrine sweat glands, and single CK8-immunoreactivity was detected occasionally in the external root sheath at the isthmus and suprabulbar regions of the hair follicles. Neoplastic glandular epithelial cells in all apocrine gland tumors (21/21 cases, 100%) had co-expression of CK8/18. In trichoblastomas (27/28 cases, 96%), most neoplastic cells were diffusely positive for CK8, but those were negative for CK18. Single CK8-expression was also observed in basaloid neoplastic cells in several cases of trichoepitheliomas (7/19 cases, 37%) and pilomatricoma (1/7 cases, 14%). In several cases of trichoblastomas (4/28 cases, 14%) and trichoepitheliomas (2/19 cases, 11%), tumor cells forming glandular structures had co-expression of CK8/18. There were no positive reactions for both CK8 and 18 in infundibular keratinizing acanthomas, and sebaceous and hepatoid gland tumors. The present findings indicate that co-expression of CK8/18 is a specific feature of apocrine sweat glands and single CK8-expression represents the natures of external root sheath or pluripotential stem cells. Thus, the combination of CK8- and 18-immunostainings may have the utility to confirm the directions of differentiation in canine cutaneous adnexal tumors providing a reliable hallmark for histopathological diagnoses.     

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.     

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.     

Pyridoxine regulates hair follicle development via the PI3K/Akt,Wnt and Notch signalling pathways in rex rabbits

This study was conducted to evaluate the effect of pyridoxine on the development of hair follicles in Rex rabbits and the underlying molecular mechanism. Two hundred 3-month-old Rex rabbits were randomly divided into 5 groups and fed diets supplemented with 0, 5, 10, 20, or 40 mg/kg pyridoxine. The hair follicle density on the dorsal skin and the gene and protein expression levels of components of the phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB or Akt), Wnt, Notch and bone morphogenetic protein (BMP) signalling pathways were measured. In addition, free hair follicles were isolated from Rex rabbits and cultured with pyridoxine in vitro to measure hair shaft growth. Furthermore, dermal papilla cells (DPC) were isolated from the skin of Rex rabbits and cultured with pyridoxine in vitro to measure the gene and protein expression levels of components of the PI3K/Akt, Wnt, Notch and BMP signalling pathways. The results showed that the addition of dietary pyridoxine significantly increased the total follicle density, secondary follicle density, and secondary-to-primary ratio (S/P, P < 0.05), that the growth ratio of hair stems was promoted by pyridoxine in basic culture medium, and that the growth length of tentacle hair follicles cultured in the pyridoxine group was longer than that in the control group (P < 0.05). In addition, pyridoxine changed the DPC cycle progression and promoted cell proliferation, and appropriate concentrations of pyridoxine (10 and 20 μmol/L) significantly inhibited cell apoptosis (P < 0.05). Pyridoxine significantly affected the gene expression of components of the PI3K/Akt, Wnt and Notch signalling pathways in the skin and DPC of Rex rabbits (P < 0.05), increased the levels of phosphorylated catenin beta 1 (CTNNB1) and Akt, and decreased the level of phosphorylated glycogen synthase kinase 3 beta (GSK-3β) (P < 0.05). Therefore, the molecular mechanism by which pyridoxine promotes hair follicle density in Rex rabbits probably occurs through activation of the PI3K/Akt, Wnt and Notch signalling pathways, prolonging hair follicle growth and delaying the onset of telogen.     

毛囊的周期性变化和分子调控及其在绒山羊上的研究进展

毛囊是一个形态和结构较为复杂的皮肤附属器官,它控制着毛发的生长,具有自我更新和周期性生长的特点。毛囊的周期性变化依靠毛囊上皮细胞和真皮间充质细胞的相互作用,先是毛囊长出毛干为生长期,接下来是凋亡驱动的退行期,然后进入休止期。其变化过程是一系列信号分子相互作用形成的,包括启动信号、维持毛囊生长的信号及抑制毛囊生长的信号等。绒山羊初级毛囊和次级毛囊在出生后也表现周期性变化,次级毛囊由于光周期的影响而呈很强的季节性的周期变化,而初级毛囊则变化不明显。山羊绒是绒山羊次级毛囊的衍生物,次级毛囊的生长发育直接影响山羊绒的产量和品质。因此,研究皮肤毛囊周期性变化的规律及它们的分子调控机理不仅可揭示毛囊的发育规律而且对绒山羊的育种具有重要指导意义。     

细毛羊毛囊干细胞分离培养方法比较研究

试验旨在对细毛羊毛囊干细胞的分离培养方法进行初步研究,建立细毛羊毛囊干细胞体外培养体系。采用两步酶消化法、机械分离法和两步酶消化法+机械分离法3种方法分离培养细毛羊毛囊干细胞,通过测定毛囊干细胞的数量、克隆形成率及毛囊干细胞的增殖能力等综合评估3种培养方法的优劣,寻求既能方便获得大量毛囊干细胞,又能减少其分化的理想培养条件。倒置显微镜下观察3种培养方法获得的细胞均为铺路石状,均表达角蛋白K19和整合素β1,表明成功获得细毛羊毛囊干细胞,建立体外培养体系。采用"两步酶消化法和机械分离法"相结合的方法获得毛囊干细胞体外培养效果最优。     

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。     

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.     

羊驼毛囊干细胞分离、培养与鉴定

采用0．2％中性蛋白酶Ⅱ和0．25％胰酶：0．02％EDTA（1：1）“两步酶”消化法从羊驼背部皮肤分离得到羊驼毛囊干细胞。用无血清角质细胞培养基（K-SFM）培养进行形态学观察、细胞生长曲线克隆形成率及免疫组化染色检测。结果显示，细胞生长曲线表明，不同代次毛囊干细胞接种前3d生长缓慢，4～6d进入倍增期，有无限增殖的趋势，所分离的羊驼毛囊干细胞具备毛囊干细胞特征（体积小、立体感强），呈现未分化特征；CK19、CK15、81-integrin和CD34免疫组化染色阳性；第3、5、7、9代克隆形成率分别为（32．7±2．27）％、（47．0±3．46）％、（46．3±3．18）％和（43．3±3．76）％。结果表明，用“两步酶”消化法成功分离获得羊驼毛囊干细胞，无血清角质细胞培养基（K—SFM）可使羊驼毛囊干细胞体外维持未分化状态并传代至12代。     

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。     

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.     

毛囊发育与周期性生长的调控信号通路研究进展

毛囊是皮肤的重要附属结构,也是控制哺乳动物被毛生长的重要器官。哺乳动物出生后,毛囊具有终生呈周期性生长的特性,毛囊干细胞、毛乳头细胞、毛母质细胞及脂肪细胞等参与了毛囊周期性生长,Wnt、BMP、Notch等信号通路与毛囊生长发育密切相关。本文从哺乳动物毛囊的结构、周期性生长特征以及参与毛囊周期性生长调控的相关信号通路等进行了详细阐述,为深入了解哺乳动物毛囊的周期性生长调控机制,以及为今后指导绒山羊、绵羊、长毛兔等毛用动物和獭兔、水貂、狐狸、貉等皮用动物选育和提高生产性能提供理论参考。     

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。     

绒山羊毛囊发育中非编码RNA的研究进展

非编码RNA(non-coding RNA,ncRNA)是指从基因组上转录而来,不编码蛋白质,但在RNA水平上能行使一定生物学功能的RNA。非编码RNA的种类较多,种类的不同造成功能的差异。毛囊是绒山羊皮肤特殊的附属物,位于皮肤的真皮层,发生于绒山羊胚胎期。由于真皮细胞和上皮细胞间的信号分子传递,使其发育呈周期性变化,历经生长期、退行期和休止期。近年来,有关ncRNA在绒山羊毛囊发育中作用报道较多。文章就其中的微小RNA(miRNA)、长链非编码RNA(lncRNA)及环状RNA(circRNA)的生物发生及其在绒山羊毛囊发育中的作用机制进行了归纳、总结。miRNA是真核生物中存在的一种长18~25 nt的ncRNA分子,可通过与靶信使核糖核酸mRNA特异结合,抑制转录后基因表达。在绒山羊毛囊发育中,miRNA通过抑制相关基因及相关信号通路中关键分子的表达而调控毛囊的发育周期以及毛囊的再生能力。lncRNA是长度超过200 nt的ncRNA,经过剪接,具有polyA尾巴与启动子结构,在绒山羊毛囊发育中能促进毛囊细胞增殖与分化,通过调控基因的靶向表达与多种信号通路互作调节毛囊发育及绒毛生长的周期活动。circRNA是与传统的线性RNA不同的封闭环状RNA,含有许多miRNA结合位点,在绒山羊毛囊发育与绒毛生长中起miRNA分子海绵的作用,即通过miRNA的介导,调控靶基因的表达,促进毛囊干细胞向次级毛囊分化,进而解除与毛囊发育与绒毛生长相关的miRNA对其靶基因的抑制作用提高靶基因的表达水平。笔者通过对miRNA、lncRNA及circRNA的研究进行综述,以期为构建毛囊生长发育的分子调控网络及深入探讨绒山羊毛囊发育的调控机制提供借鉴,为今后更好地利用现代分子生物学技术改善绒毛品质提供理论依据。     

山羊毛乳头细胞分离鉴定及TGF-β/smad通路相关基因的表达

试验旨在分离山羊毛乳头细胞,并探索其中TGF-β/smad通路相关基因的表达,为体外开展山羊毛囊发育的相关机制研究提供良好模型。采用中性蛋白酶与胶原酶两步消化法对山羊背部皮肤做处理,在体视显微镜下分离毛乳头细胞并进行纯化。细胞免疫荧光和Western blotting验证平滑肌肌动蛋白（α-smooth muscle actin,α-SMA）和波形蛋白（vimentin,VIM）在体外培养的毛乳头细胞中的表达,并检测TGF-β/smad通路中相关基因在山羊毛乳头细胞中的表达情况。结果显示,分离后进行贴壁培养的毛乳头细胞生长较慢,在分离15 d后具有成熟形态,可进行传代培养。细胞免疫荧光和Western blotting结果表明,α-SMA和VIM在体外培养的毛乳头细胞中均表达,TGF-β/smad通路中smad4、smad5基因的表达水平显著高于对照组（P<0.05）,smad2、smad6、smad7基因的表达量极显著高于对照组（P<0.01）,这些与毛囊发育相关的关键基因均呈现高表达,表明毛乳头细胞可能通过TGF-β/smad通路的调控从而影响毛囊的发育。本研究成功分离出山羊毛乳头细胞,为体外研究山羊毛囊发育机制奠定良好的理论基础和细胞模型。