白癜风皮损黑素细胞HMB-45、酪氨酸酶及其相关蛋白1的免疫组化研究

目的:研究白癜风白斑处黑素细胞分布及酪氨酸酶(TYR)等标记分子的表达情况。方法:对吸疱法所取白斑皮肤以免疫组化染色识别TYR、酪氨酸酶相关蛋白1(TRP1)和HMB-45,以Mias99彩色图像分析软件定量图像分析阳性表达的强弱。结果:白斑处黑素细胞数目明显低于正常对照(P<0.01),患者非皮损处与正常人皮肤比较差异无显著性(P>0.05)。皮损处黑素细胞树突减少,平均为3.4个,低于对照组(P<0.05)。残留黑素细胞各抗体呈强阳性染色。白斑毛囊之间可见黑素细胞散在、呈围管样分布。白斑处阳性细胞HMB-45图像灰度值低于正常着色皮肤(P<0.05),而TRP1高于正常皮肤(P<0.01),TYR与正常皮肤无差异(P>0.05)。结论:白斑处仍然有黑素细胞。残存黑素细胞树突回缩、数目变少,而且黑素细胞中黑素细胞标记分子表达异常。     

倍他米松对毛囊外毛根鞘无色素黑素细胞激活的试验研究

目的：研究倍他米松（betamethasone,BT）对毛囊无色素黑素细胞（amelanotic melanocytes,AMMC）的激活作用。方法：以高、中、低3种不同浓度的BT作用于培养的人毛囊外毛根鞘AMMC,测定药物作用前后酪氨酸酶（tyrosinase,TYR）活性和黑素生成的变化。通过间接免疫荧光法结合激光共聚焦显微镜半定量分析药物作用前、后AMMC TYR、酪氨酸酶相关蛋白（tyrosinase related protein,TRP）-1和TRP-2表达的变化,透射电镜分析黑素小体在药物作用前后的变化。结果：BT促进了AMMC表达TYR和TRP-1,并且以剂量依赖方式促进TYR活性,诱导黑素生成。AMMC主要含有Ⅰ、Ⅱ和Ⅲ期黑素小体,但是BT作用后AMMC含有大量Ⅲ或Ⅳ期黑素小体,并且以Ⅳ期黑素小体为主。结论：BT能够激活AMMC,这可能部分解释了糖皮质激素治疗白癜风的机制。     

人类毛囊黑素细胞的研究现况

毛囊黑素细胞在形态分布，抗原表达及功能作用上与表皮黑素细胞不同；其生物学特性受角朊 细胞，激素和紫外线等机体内，外多种因素的调控。     

人类毛囊黑素细胞的研究现况

毛囊黑素细胞在形态分布、抗原表达及功能作用上与表皮黑素细胞不同;其生物学特性受角朊细胞、激素和紫外线等机体内、外多种因素的调控。白癜风皮损中表皮黑素细胞缺失,而毛囊黑素细胞成为其色素恢复的再生来源;相反,斑秃时毛囊黑素细胞受累,而表皮黑素细胞不受影响。研究影响毛囊黑素细胞生存和病变的因素及其机制将对了解白癜风和斑秃的病因、发病机理和开发新的治疗方法具有重要意义。     

6味中药乙醇提取物对B16黑素瘤细胞酪氨酸酶及其相关蛋白-1、-2 mRNA表达的影响

目的 研究墨旱莲(旱莲草)等6味用于治疗白癜风中药的乙醇提取物对小鼠B16黑素瘤细胞酪氨酸酶及酪氨酸酶相关蛋白-1、-2(TRP-1、-2)mRNA表达的影响.方法 半定量逆转录-聚合酶链反应(RT-PCR)测定酪氨酸酶及TRP-1、-2 mRNA的表达,并与阳性对照甲氧沙林(8-MOP)进行比较.结果 山慈姑(毛慈姑)、苦参、黑芝麻和墨旱莲的乙醇提取物显著上调细胞酪氨酸酶mRNA的表达(P<0.05);除苦参对TRP-2 mRNA有明显上调作用外(P<0.05),其他中药对TRP-1和TRP-2 mRNA的表达无明显作用(P>0.05).结论 山慈姑、苦参、黑芝麻和墨旱莲有上调酪氨酸酶mRNA的作用,苦参还可上调TRP-2 mRNA的表达.     

从大容量随机噬菌体抗体库筛选人源性抗酪氨酸酶相关蛋白1抗体

目的从大容量随机噬菌体抗体库中筛选人源性抗酪氨酸酶相关蛋白1(TRP1)单链抗体并进行鉴定及基因测序分析。方法以原核表达并纯化的TRP1为抗原,通过“吸附洗脱扩增”过程从大容量随机噬菌体抗体库中筛选特异性抗TRP1蛋白的单链抗体,分别通过ELISA和基因测序的方法对其抗原结合活性和序列进行分析鉴定。结果经过4轮筛选,获得18个能与TRP1蛋白结合的阳性克隆,其中3个克隆的噬菌体抗体能与TRP1特异性结合;基因测序分析表明,所获抗体分别属于不同的抗体克隆。结论利用噬菌体抗体库技术可以不经免疫过程制备出高特异性的人源性抗TRP1抗体。     

TRP-1反义核酸转染黑素细胞 黑素瘤细胞的光镜及电镜观察

目的　观察TRP 1反义核酸真核表达载体转染的黑素细胞及黑素瘤细胞形态学改变, 探讨TRP 1功能。方法　将TRP 1反义核酸真核表达载体转染黑素细胞及黑素瘤细胞,以倒置光学显微镜及透射电镜分别观察细胞显微结构及超微结构的变化。结果　光镜下可见TRP 1反义核酸转染的黑素细胞及黑素瘤细胞树突变短粗,细胞胞体增大,呈上皮样细胞改变;电镜下可见线粒体变大,电子密度降低,溶酶体、脂滴、糖原颗粒增多,细胞核肿胀及凋亡细胞。结论　TRP 1在黑素细胞及黑素瘤细胞形态学改变中发挥重要作用,本研究为阐明TRP 1基因的功能提供较为全面的形态学证据。     

大黄对人表皮黑素细胞的增殖及对酪氨酸酶的双向调节作用

为了研究大黄对人表皮黑素细胞增殖的影响及对酪氨酸酶的双向调节作用,我们采用体外黑素细胞培养的方法,观察了大黄三种不同有效成分对表皮黑素细胞、酪氨酸酶的调节作用,现将结果报道如下.     

加味桃红四物汤对黑素细胞酪氨酸酶和酪氨酸激酶受体基因蛋白表达的影响

目的研究加味桃红四物汤对体外培养的人表皮黑素细胞(MC)可溶性总蛋白以及酪氨酸酶(Tyr)基因和酪氨酸激酶受体基因(c—kit)表达的作用。方法采用免疫印迹法检测了加味桃红四物汤对酪氨酸酶和酪氨酸激酶受体蛋白含量的影响；595nm比色标准曲线测定黑素细胞总蛋白含量；采用细胞数统计和显微镜观察的方法测定加味桃红四物汤对细胞毒性和对细胞增殖的影响。结果加味桃红四物汤对黑素细胞可溶性总蛋白和酪氨酸酶的合成有促进作用，分别为4．76％和1．79％；对酪氨酸激酶受体蛋白的合成具有显著促进作用，高达79．41％。结论加味桃红四物汤治疗白癜风的作用机制之一是与酪氨酸激酶受体蛋白含量的提高相关，而与黑素细胞增殖和酪氨酸酶相关蛋白的含量无明显关系。     

人毛囊无色素黑素细胞培养方法的改良及色素生成相关酶表达的研究

目的进一步改良人毛囊无色素黑素细胞（amelanotic melanocytes，AMMC）培养的方法，并研究了AMMC内黑素生成相关酶的表达：酪氨酸酶（tyrosinase,TYR）、酪氨酸酶相关蛋白-1（tyrosinase related protein-1，TRP—1）和酪氨酸酶相关蛋白-2（tyrosinase related protein-1，TRP-2）。方法采用1％分离酶（dispase）消化分离毛囊，选用含干细胞因子（stem cell factor，SCF）、内皮素-3（endothelin-3，ET-3）、碱性成纤维细胞生长因子（basic fibroblast grow factor，bFGF）和霍乱毒素（cholera toxin，CT）的成黑素细胞培养基培养AMMC，同时培养表皮黑素细胞（melanocytes，MC）作为对照，采用免疫组化、多巴染色和透射电镜对细胞进行鉴定，蛋白印记法分析TYR、TRP-1和TRP-2的表达。结果1％分离酶消化24h可以容易获得游离的毛囊，结合我们所用的培养基完全排除了成纤维细胞的污染。所用成黑素细胞培养基促AMMC增殖明显，细胞可传5代以上。免疫组化结果显示，AMMC表达gp100、酪氨酸酶（tyrosinase，TYR）、酪氨酸酶相关蛋白-1（tyrosinase related proteifl-1，TRP-1）和酪氨酸酶相关蛋白-2（tyrosinase related protein-1，TRP-2），证明培养的细胞为黑素细胞。AMMC的多巴染色呈阴性，并且透射电镜发现AMMC胞质中仅含大量的Ⅰ期、Ⅱ期黑素小体，未见Ⅲ、Ⅳ期黑素小体，因此说明AMMC处于未分化状态。TYR和TRP-1在MC中的表达明显强于AMMC。TRP-2的表达在两种细胞间没有明显差别，进一步说明AMMC与MC具有异质性。结论我们成功培养了完全纯化、快速增殖的AMMC，并且培养的细胞不能生成黑素，生物学性状更接近活体中的状态，其与表皮黑素细胞具有异质性。     

Oligonucleotide treatment increases eumelanogenesis, hair pigmentation and melanocortin-1 receptor expression in the hair follicle

It was previously reported that telomere homologue oligonucleotides (T-oligos) can induce a variety of cellular responses in skin including increased melanogenesis. To assess the effects of T-oligos on hair pigmentation, we administered thymidine dinucleotide (pTT), one-third of the TTAGGG telomere repeat sequence, intradermally at distinct time points of the depilation-induced hair cycle in C3H/HeJ mice. Penetration of T-oligos into the hair follicle (HF) was monitored by using FITC-labelled pTT and confocal microscopy. pTT treatment on days 1-5 after depilation, during early anagen, did not significantly alter the number and proliferation of melanocytes (Trp-2-positive cells), compared with vehicle-treated controls. However, pTT treatment on days 5-12 after depilation, during mid- to late anagen, resulted in the formation of darker hairs, that showed a significantly increased eumelanin/total melanin ratio in their sub-apical agouti band region, compared with vehicle-treated controls (P < 0.05). By RT-PCR and western blot, full thickness skin of pTT-treated mice showed increases in Trp-1, Trp-2 and tyrosinase mRNA and protein levels, compared with control mice. Western blot analyses of two receptors that positively regulate eumelanogenesis, melanocortin type 1 receptor (MC-1R) and kit, showed increased expression of MC-1R protein in pTT-treated versus control skin, while the levels of c-kit receptor remained unchanged. These data demonstrate that pTT treatment increases eumelanogenesis in HFs, associated with increased tyrosinase, TRP-1 and MC-1R expression. These data also raise the possibility of using T-oligos to modulate hair pigmentation.     

胎儿头皮毛囊的黑素细胞定位及精细结构观察

目的 探讨胎儿毛囊黑素细胞的定位及精细结构。方法 6个月胎儿因宫内发育畸形而引产、死亡后,取其带毛头皮,一部分常规包埋切片,分别用NKI/beteb、HMB-45、酪氨酸酶、酪氨酸酶相关蛋白1（TRP1）单抗染色。另一部分无菌处理后,0.1 g/L的胶原酶Ⅱ和胰酶消化获得毛囊细胞,培养并传代后,透射电镜和原子力显微镜观察。结果 胎儿头皮毛囊NKI/beteb阳性细胞位于外根鞘,而在毛球内许多细胞HMB-45、酪氨酸酶、TRP1单抗染色阳性。在毛囊细胞的体外培养中,除去成纤维细胞和角质形成细胞后,可见两种黑素细胞,一种数目极少,色素很多,传代后消失;另一种数目较多,开始无色素,但增殖很快。传第3代后,几乎所有细胞NKI/beteb染色阳性。扫描电镜和原子力显微镜下,多数细胞为双极梭形,偶尔有3个树突。细胞体呈圆形或卵圆形,极突上无明显的分支,其内有少数散在的黑素体。结论 胎儿头皮毛囊外根鞘的黑素细胞推测为成黑素细胞和（或）其子代细胞。在早期的体外培养中,细胞增殖很快,但形态及功能上不成熟。     

The 'melanocyte-keratin' mystery revisited: neither normal human epidermal nor hair follicle melanocytes express keratin 16 or keratin 6 in situ

Background  Keratin family proteins are generally accepted as being restricted to epithelial cells. However, several studies have challenged this paradigm by reporting, for example, that melanoma cells can express keratins and that normal human epidermal melanocytes, which derive from the neural crest, express keratin 16 (K16) in situ .
Objectives  We wished to confirm or refute that K16 and/or its intermediate filament partner, keratin 6 (K6), are expressed in normal human epidermal and/or hair follicle melanocytes in situ .
Methods  Cryosections of normal human scalp skin were subjected to highly sensitive double immunohistochemistry with specific antibodies against K16 or K6 and against the melanocyte-specific marker NKI/beteb (gp100). Immunoreactivity (IR) was visualized by conventional light microscopy and confocal fluorescence microscopy.
Results  Despite the use of different, high-sensitivity immunostaining methods, stringent positive and negative controls, and monospecific, well-characterized antikeratin antibodies, we could detect neither K16 nor K6 IR within intraepidermal or intrafollicular pigment cells of normal human scalp skin. Instead, NKI/beteb+ cells were found to be intimately embedded in foci of K16+ and/or K6+ keratinocytes, which might create the illusion of keratin expression by these cells.
Conclusions  Human epidermal or hair follicle melanocytes do not express K16 and/or K6 while residing in their natural habitat.     

Human melanocytes can be isolated,propagated and expanded from plucked anagen hair follicles

Please cite this paper as: Human melanocytes can be isolated, propagated and expanded from plucked anagen hair follicles. Experimental Dermatology 2010; 19: 543–545. Abstract: Herein, we report a technically simple method for isolation and culture of human follicular melanocytes based on explant cultures of epilated hair follicles. This technique does not require any surgical intervention and allows the isolation and cultivation of follicular melanocytes from a comparatively small amount of raw material. Generally, 30–60 human anagen hair follicles have been plucked from the scalp of healthy donors and cultivated under low oxygen pressure (5%). After a short period of time cells of various types were growing out from the outer root sheath (ORS) of the hair follicles. Under the selected culture conditions, most of the cells other than melanocytes have been eliminated and a nearly 100% pure population of melanocytes has been achieved, as confirmed by immunohistochemical analyses for melanocyte‐specific markers, for example, Tyrosinase‐1, S‐100 and premelanosomal antigens. These melanocytes derived from the ORS were proliferating for up to 2 months.     

Human hair greying is linked to a specific depletion of hair follicle melanocytes affecting both the bulb and the outer root sheath

BACKGROUND: Although hair greying is a very common phenomenon characterized by loss of pigment in the hair shaft, the events that cause and control natural hair whitening with age in humans are still unclear. OBJECTIVES: To decipher the origin of natural hair whitening. METHODS: Human hair melanocytes were immunohistochemically characterized at different stages of whitening. RESULTS: Loss of hair shaft melanin was found to be associated with a decrease in both bulb melanin content and bulb melanocyte population. Although few melanocytes were present in the bulbs of grey hair, they still expressed tyrosinase and tyrosinase-related protein-1, synthesized and transferred melanins to cortical keratinocytes as seen by the presence of melanin granules. In white hair bulbs, no melanocytes could be detected either with pMel-17 or vimentin labelling. Pigmented hair follicles are known to contain inactive melanocytes in the outer root sheath (ORS), and grey and white hairs were also found to contain some of these quiescent melanocytes. However, their population was decreased compared with pigmented hair follicles, ranging from small to nil. This depletion of melanocytes in the different areas of white hairs was detected throughout the hair cycle, namely at telogen and early anagen stages. In contrast, the infundibulum and sebaceous gland of both pigmented and white hairs showed a similar distribution of melanocytes. Furthermore, other distinct cell populations located in the ORS, namely putative stem cells, Merkel cells and Langerhans cells were equivalently identified in pigmented and white hairs. CONCLUSIONS: Thus, hair greying appears to be a consequence of an overall and specific depletion of bulb and ORS melanocytes of human hair.     

Extracellular histones inhibit hair shaft elongation in cultured human hair follicles and promote regression of hair follicles in mice

Release of histone H4 in rat vibrissa dermal papilla (DP) cells exposed to sub‐toxic dose of colchicines has been recently reported. In addition, exposure to histone H4 has been reported to result in inhibited proliferation and reduced alkaline phosphatase (ALP) activity of cultured vibrissa DP cells. These findings prompted us to investigate the role of extracellular histones in hair growth using cultured human hair follicles and hair cycling using back skin of mice. We report here that exposure of cultured hair follicles to histone H4 and H2A resulted in significant inhibition of elongation of hair shafts, decreased expression of IGF‐1 and decreased expression and activity of ALP. Injection of histones into hypodermis of mice during anagen resulted in premature onset of catagen. Findings of the current study provide strong evidence suggesting the inhibitory role of extracellular histones in hair growth.     

Using human hair follicle‐derived keratinocytes and melanocytes for constructing pigmented tissue‐engineered skin

Background: Traditional tissue‐engineered skin does not produce a satisfactory long‐term result because it lacks natural skin pigmentation and leads to discolored cosmetically unpleasing skin that only functions to cover the body of patients. Additionally, the cell sources for tissue‐engineered skin are generally derived from normal skin, which is often limited in patients with skin defects. Methods: In this study, hair follicle melanocytes and keratinocytes were isolated from human scalp. The melanocytes were co‐cultured with keratinocytes until the second passage and then purified. Purified melanocytes and keratinocytes were seeded onto the chitosan–gelatin membrane for 1 week to construct pigmented tissue‐engineered skin. The pigmented skin equivalent was used to resurface the skin defect in nude mice. Four weeks after grafting, skin biopsies were harvested to take hematoxylin and eosin staining and immunohistochemistry staining of Melan‐A and HLA‐ABC. Results: Large quantities of purified melanocytes can be obtained with co‐culture method. The hematoxylin and eosin staining of repaired skin biopsy demonstrated that the tissue‐engineered skin can repair skin defects successfully. Engineered skin contained pigmentation and stained positive for Melan‐A and HLA‐ABC, which confirmed the presence of melanocytes and its sources were of human origin. Conclusion: This study demonstrated the possibility of constructing pigmented tissue‐engineered skin with human hair follicle‐derived keratinocytes and melanocytes, which brings a promising method to make up for the deficiency of traditional tissue‐engineered skin and provides an alternative treatment for depigmentation diseases.