毛囊无黑素性黑素细胞的分离和培养

目的：分离和培养毛囊无黑素性黑素细胞，探讨其生物学特性。方法：采用胶原酶V游离毛囊，0．5％分离酶消化婴幼儿包皮。0．05％胰酶和0．53mmol／L乙二胺四乙酸(EDTA)消化游离的毛囊和来自包皮的表皮，分别制备单细胞悬液进行培养。传至第3代，待细胞爬上盖玻片后，分别以NKI／heteb和HMB-45单抗染色。结果：毛囊的培养细胞，绝大多数为双极细胞，增殖较快，被NKI／beteb单抗识别，而HMB-45染色阴性，包皮的黑素细胞染色结果则与之相反．结论：毛囊的外根鞘中存在功能和形态上不成熟的无黑素性黑素细胞(AMMC)、AMMC长期培养的成功，有利于研究AMMC在毛囊生物学中所起的作用。     

甲氧沙林对体外培养的人毛囊外根鞘无色素黑素细胞的激活作用

目的:观察甲氧沙林(8-MOP)对体外培养的人毛囊外根鞘无色素黑素细胞黑素合成相关酶的影响。方法:3种浓度(10、50、100μmol/L)的8-MOP作用于体外培养的人毛囊外根鞘无色素黑素细胞,经免疫荧光染色后,采用激光共聚焦显微镜观察不同浓度、不同作用时间的8-MOP对无色素黑素细胞形态的影响,荧光定量分析酪氨酸酶、酪氨酸相关蛋白(TRP)1、TRP2表达量的变化。结果:50、100μmol/L8-MOP作用于人毛囊外根鞘无色素黑素细胞3d能显著促进酪氨酸酶的表达(P<0.01)熏作用7d后TRP1、TRP2的表达也增加(P<0.05)。结论:8-MOP在体外能直接刺激毛囊外根鞘无色素黑素细胞的活化。     

人头皮毛囊外根鞘无色素黑素细胞的分离和纯化培养进一步研究

目的：从人头皮毛囊外根鞘分离纯化培养无色素黑素细胞。方法：采用两步酶法(0．50％分离酶和0．50％胶原酶V)获得游离的毛囊，高浓度0．50％，胰酶30min消化游离的毛囊外根鞘细胞，并以优化的培养基进行细胞培养。用HMB-45和NKI／beteb单克隆抗体免疫组化染色、透射电镜对培养物进行鉴定。结果：培养物中初始贴壁细胞大多数为无色素黑素细胞，仅有少量的角质形成细胞，无成纤维细胞污染。经二次传代差别胰酶处理很容易去除角质形成细胞。取培养3周的细胞经免疫组化和透射电镜鉴定证实为无色素黑素细胞。传3代后细胞得到完全纯化。结论：两步酶法结合高浓度的胰蛋白酶处理细胞可彻底清除成纤维细胞，获得无色素黑素细胞的纯培养。     

Improved method of differentiation,selection and amplification of human melanocytes from the hair follicle cell pool

Hair root harbours a complex cell pool with an immense developmental potential. Several lineages, including skin, can be differentiated from the multipotent to pluripotent cells of outer root sheath (ORS) of hair follicle. Outer root sheath presents the most opulent non‐invasively gained adult stem cell source known. For the purposes of cultivating melanocytes designated for graft‐based treatments of depigmentation disorders, we have established an ex vivo/in vitro cultivation method by introducing several methodological improvements to the ORS explant method of Dieckmann. As a result, we gained a higher, purer yield of differentiated melanocytes in half the time (at least 10⁶ of 95% pure cells in 4 weeks). This reliable cultivation procedure begins with the epilation of 60 hairs and yields high numbers of ORS melanocytes that could be used for grafting applications. The procedure not only utilises the developmental potential of hair root cell pool and favors differentiation into melanocytes, but also contributes to the general trend of minimal‐to‐non‐invasive strategies for regenerative medicine.     

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.     

人表皮黑素细胞、毛囊无色素黑素细胞和鼠黑素瘤细胞的原子力显微镜观察

目的：观察培养的人表皮黑素细胞、毛囊无色素黑素细胞和S91鼠黑素瘤细胞的形态结构。方法：培养并纯化来自正常人包皮的黑素细胞以及来自毛囊的无色素黑素细胞，同时复苏S91细胞株，传代后接种到内置云母片的培养板中，细胞贴附到云母片上后固定，用原子力显微镜扫描观察。结果：正常人表皮黑素细胞有3级分支，在主干及分支的顶端和侧缘可见膨出的球形结构。鼠黑素瘤细胞仅有很短的2级分支，在2级树突近端可见黑素小体。毛囊无色素黑素细胞只有1级树突，并只在树突近端有少数黑素小体。结论：表皮黑素细胞在形态上比黑素瘤细胞、毛囊无色素黑素细胞更成熟，有更多的黑素颗粒从树突的顶端和侧缘以胞吐的形式被输出。     

毛囊外根鞘NKI/beteb和HMB-45免疫组化及胰酶消化后细胞的电镜观察

目的：研究头皮毛囊外根鞘中无活性黑素细胞的存在部位以及毛囊经胰酶消化的细胞组成。方法：拔取正常头发，琼脂预先包埋后，切片行NKI／beteb和。HMB-45单抗染色。另取毛囊用胰酶消化，收集细胞，在透射镜下进行观察。结果：拔取的正常毛囊外根鞘中存在NKL／beteb阳性细胞，而HMB-45染色阴性。在毛母质中有大量的HMB-45阳性细胞。毛囊经胰酶消化的细胞为有活性的黑素细胞(MC)、无色素性黑素细胞(AMMC)、毛皮质细胞和成纤维细胞。结论：毛囊外根鞘中存在功能和形态不成熟的AMMC。     

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。     

内皮素-1对毛囊外根鞘无色素黑素细胞黏附和移行及其细胞骨架形态的影响

目的：体外研究内皮素-1（ET-1）对毛囊外根鞘无色素黑素细胞（AMMC）黏附和移行的作用,并观察其对细胞骨架蛋白微丝、微管的形态影响。方法：3种浓度的ET-1作用体外纯化培养的人AMMC,观察其在细胞外基质蛋白如纤维连接蛋白、层黏连蛋白、Ⅳ型胶原包被培养板上的黏附,以及通过Boyden小室微孔滤膜的情况。采用免疫荧光双重染色法对AMMC分别进行罗丹明（红色）和异硫氰酸（绿色）标记纤维型-肌动蛋白（F-actin）、β微管蛋白,激光共聚焦显微镜观察3种浓度ET-1处理前后AMMC纤维型-肌动蛋白、B微管蛋白形态的变化。结果：与空白对照组相比,ET-1促进了AMMC在纤维连接蛋白上的黏附,20ng/mL ET-1作用最明显（P〈0．01）,而200ng/mL时对细胞的黏附不继续增加。但对层黏连蛋白和Ⅳ型胶原上的黏附无明显影响（P〉0．05）。另外,ET-1呈浓度依赖性地促进AMMC通过微孔滤膜。激光共聚焦显微镜观察显示,〉20ng/mL ET-1可明显促进AMMC胞质中束状应力纤维形成,并集中分布于细胞膜内侧,但对微管结构无明显影响。结论：ET-1在体外可以促进AMMC的黏附和移行．其作用可能与诱导束状应力纤维形成和促进其向细胞膜内侧分布有关。     

干细胞因子结合基质蛋白对毛囊无色素黑素细胞黏附与移行的调节作用

目的:研究干细胞因子(stemcellfactor,SCF)结合基质蛋白对毛囊无色素黑素细胞(amelanoticmelanocytes,AMMC)黏附与移行的调节作用。方法:四甲基偶氮唑蓝(MTT)法测定细胞的黏附率。48孔细胞趋化小室测定细胞移行。使用罗丹明标记的鬼笔环肽标记肌动蛋白(F-actin),共聚焦显微镜观察SCF对细胞骨架的调节作用。结果:纤黏连蛋白(FN)、板层素(LN)和Ⅳ型胶原(CⅣ)均以浓度依赖方式促进了AMMC的黏附,其中FN和LN对AMMC促黏附作用相近。SCF减少了AMMC对FN和CⅣ的黏附,对FN的作用更为明显,而对LN起促黏附作用。FN和CⅣ对AMMC有趋化作用,LN无明显作用。SCF作用后可明显使FN对AMMC的趋化作用增强。SCF单独对AMMC有趋化作用,结合FN后,作用明显加强。未黏附在FN上的AMMC胞质内无明显应力纤维;黏附在FN上以后,胞质内可见F-actin排列规则并聚集成束,形成束状应力纤维;SCF作用后,胞体内应力纤维更加致密。结论:SCF调节了AMMC在基质蛋白FN、LN和CⅣ上的黏附与移行,尤其是SCF与FN具有协同促移行作用这可能是白癜风复色过程中AMMC从外毛根鞘向表皮移行机制的一部分。     

An improved and rapid method to construct skin equivalents from human hair follicles and fibroblasts

To produce sufficient amounts of high quality skin equivalents (SE), either allogenic for dermatopharmacological and dermatotoxicological studies or autologous for transplantation purposes, we established a rapid, easy and cost effective three-dimensional SE model on the basis of human dermal fibroblasts, collagen and freshly plucked hair follicles. Acidic liquid collagen was polymerized with sodium hydroxide in the presence of fibroblasts to form a dermal equivalent (DE) resembling normal human dermis. At 24 h later, freshly plucked hair follicles were implanted into the surface of these DEs after cutting their bulbs off. Another 48 h later, the surface of the SEs was lifted to the air-liquid interface. Fourteen days after implantation, outgrowing keratinocytes from the outer root sheath of the hair follicles completely covered the surface of the SE and built a fully developed, multi-layered and cornified epidermis. Histology and immunofluorescence studies with specific antibodies directed against components of keratinocytes, fibroblasts, cell-adhesion molecules, different extracellular matrix and basement membrane proteins revealed the similarity of our three-dimensional SEs to the in vivo situation in normal human skin. Using autologous cell sources and cell culture media enriched with serum from the respective cell donor, it will be possible to use these SEs for autologous transplantation, thereby reducing the risk of transplant rejection.     

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。     

人毛囊无色素黑素细胞培养的纯化和培养基成分对细胞影响的实验研究

目的研究geneticin在毛囊黑素细胞培养过程中对成纤维细胞的去除作用,研究12-O-十四烷佛波酯-13-醋酸酯TPA和含牛垂体提取物BPE角质形成细胞无血清培养基K-SFM对毛囊无色素黑素细胞AMMC形态和增殖的影响。方法通过胶原酶法培养毛囊黑素细胞,观察不同浓度geneticin对污染的成纤维细胞的去除。同时观察不同浓度TPA和含BPE的K-SFM对AMMC形态和促增殖的影响。结果采用100μg/mLgeneticin处理2d后,剩余细胞主要是黑素细胞,其中部分成纤维细胞呈死亡状态,继续培养至第7天后,黑素细胞的纯度达到90%以上。50ng/mLTPA可以促进细胞增殖,与100ng/mLTPA比较差异无显著性(P>0.05),但对细胞形态影响不大;100ng/mLTPA明显促进黑素细胞的树突增加。含20%、40%和80%的K-SFM含BPE培养基中,浓度为80%时促增殖作用最明显。结论100μg/mLgeneticin作用2d去除黑素细胞培养中污染的成纤维细胞是一种简单有效的方法,并可重复使用。在毛囊黑素细胞培养中,TPA以50ng/mL的浓度即可明显促进增殖,而不影响细胞的形态。含BPE的K-SFM可以浓度依赖性地促进AMMC增殖。     

人毛囊内无色素黑素细胞的原子力显微镜观察

目的用原子力显微镜观察人头发毛囊内无色素黑素细胞的表面结构。方法分离酶Ⅱ消化带毛囊的头皮，游离单个毛囊，以胰酶／EDTA消化之，获取细胞悬液，以添加黑素细胞生长物质的254培养基重悬细胞，常规培养。传第3代后接种到内置云母片的6孔培养板中，细胞贴附到云母片上后，固定．原子力显微镜常温常压下，触摸式扫描。结果毛囊无色素性黑素细胞多呈梭形，有2个突起，少数有3个树突。每个树突无明显的二级分枝，个别在树突侧缘有隆起。在一级树突的顶端可见丝状伪足。结论毛囊内无色素性黑素细胞形态不成熟，不能转运黑素颗粒到角质形成细胞，但它有黑素小体运动的物质基础——丝状伪足。     

维A酸促毛囊外毛根鞘无色素黑素细胞分化的实验研究

目的：研究维A酸(all-transretinoic acid，ATA)对毛囊无色素黑素细胞（famelanotic melangeytes，AMMC)的激活作用。方法：以高、中、低3种不同浓度的ATA作用于培养的人毛囊外毛根鞘AMMC，倒置显微镜观察细胞形态的变化，细胞计数法测定ATA对AMMC增殖率的影响，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(TRP-2)表达的变化。结果：ATA能抑制AMMC的增殖，并能促进AMMC表达TYR和TRP-1，但对TRP-2的表达没有影响。结论：ATA能够促进AMMC的分化，同时抑制增殖，其抑制机制可能与凋亡有关。     

BMP-4对人毛囊外根鞘细胞增殖和迁移的影响

目的观察BMP-4对体外培养的人毛囊外根鞘细胞增殖和迁移的影响。方法用含不同浓度BMP-4的MSCM培养基培养毛囊外根鞘细胞48小时后行MTT检测毛乳头细胞的增殖情况;将铺满90%培养皿底的毛囊外根鞘细胞划痕后以含不同浓度BMP-4的MSCM培养基培养,分别于0h,8h,24h和48h测量毛囊外根鞘细胞爬行的距离。结果与其他浓度相比,10μg/mlBMP-4明显促进毛囊外根鞘细胞的增殖(P<0.05),不同浓度BMP-4均促进毛囊外根鞘细胞的迁移,48小时10μg/ml浓度BMP-4的创面愈合指数达到100%(P<0.05)。结论 BMP-4对于毛囊形成及发育具有重要意义。     

Restorative effect of hair follicular dermal cells on injured human hair follicles in a mouse model

No model is available for examining whether in vivo‐damaged human hair follicles (hu‐HFs) are rescued by transplanting cultured hu‐HF dermal cells (dermal papilla and dermal sheath cells). Such a model might be valuable for examining whether in vivo‐damaged hu‐HFs such as miniaturized hu‐HFs in androgenic alopecia are improvable by auto‐transplanting hu‐HF dermal cells. In this study, we first developed mice with humanized skin composed of hu‐keratinocytes and hu‐dermal fibroblasts. Then, a ‘humanized scalp model mouse’ was generated by transplanting hu‐scalp HFs into the humanized skin. To demonstrate the usability of the model, the lower halves of the hu‐HFs in the model were amputated in situ, and cultured hu‐HF dermal cells were injected around the amputated area. The results demonstrated that the transplanted cells contributed to the restoration of the damaged HFs. This model could be used to explore clinically effective technologies for hair restoration therapy by autologous cell transplantation.     

Simple and rapid method to isolate and culture follicular papillae from human scalp hair follicles

Study of the involvement of the hair follicle papilla in hair growth regulation was greatly facilitated by the isolation and cultivation of this tiny cluster of fibroblast-like cells in the rat vibrissae and in the human hair follicle. While isolation of the hair follicle papilla from the former is relatively straightforward, the current method to isolate the much smaller human hair follicle requires significant skill. Thus, the routine initiation of primary cultures of human scalp hair follicle papilla cells requires significant training, time, and commitment. In an attempt to simplify hair follicle papilla cell culture methodology for new laboratory personnel, we have made significant refinements to the current method. Our method requires only two simple manipulations to isolate hair follicle papilla from intact isolated hair follicles. This very rapid and easy method isolates clean and intact hair follicle papillae. Together with their attachment via scratching to the growth surface, the isolation and cultivation of this important hair follicle component can now be achieved easily by the laboratory newcomer. The method relies for its simplicity on the removal of the hair follicle papilla from the outside of the intact hair follicle rather than via internal manipulations from within the hair follicle.     

The calcium-binding protein calretinin is a marker of the companion cell layer of the human hair follicle

BACKGROUND: Ultrastructural studies of the hair follicle show that the outer root sheath (ORS) does not consist of a homogeneous cell population. The innermost cell layer of the ORS, also called the companion layer, is a single cell layer closely associated with the Henle layer of the inner root sheath. OBJECTIVES: To describe the immunohistochemical expression of calretinin, a calcium-binding protein, in the human hair follicle. METHODS: Immunohistochemical studies using two different antisera to calretinin were performed in paraffin-embedded and in frozen scalp specimens using standard techniques. RESULTS: Calretinin immunostaining was consistently and specifically found in the companion cell layer of hair follicles. CONCLUSIONS: These findings provide further evidence to support the notion that the companion layer is not only morphologically, but also immunohistochemically, different from the other cells of the ORS.