毛囊外毛根鞘无活性黑素细胞研究最新进展

白癜风的色素恢复过程中,白斑边缘正常表皮基底层黑素细胞和毛囊外毛根鞘无活性黑素细胞(AMMC)是白癜风恢复色素的两个途径,外毛根鞘无黑素细胞是黑素细胞重要储库。该细胞以及其中的黑素干细胞的活化、增殖、黏附、移行受多种因素影响,8-MOP促进黑素细胞活化,内皮素一1可促进黑素细胞黏附,在黑素细胞增殖方面,环孢A促进黑素细胞增殖,米诺地尔通过降低细胞内钙离子和扩张血管促进黑素细胞增殖,女贞子可促进毛囊干细胞生长因子mRNA的表达而促进黑素细胞增殖,紫外线,补骨脂,白芷促进黑素细胞移行,这些发现为白癜风以毛囊为靶点治疗提供了科学依据。     

外毛根鞘无色素性黑素细胞的免疫组化研究

目的初步探讨外毛根鞘处于静息状态的无色素性黑素细胞(AMMC)生物学特性。方法取正常人头皮,采用NK I/beteb,HMB-45和TYR(酪氨酸酶),TRP1(酪氨酸酶相关蛋白1),TRP2(酪氨酸酶相关蛋白2)抗体特异染色黑素细胞后,研究AMMC分布、形态和NK I/beteb,HMB-45,TYR,TRP1和TRP2等黑素细胞分化标记分子的表达情况。结果外毛根鞘HMB-45,TYR,TRP1,TRP2四种抗体呈阴性;NK I/beteb阳性染色的细胞位于生长期毛囊外毛根鞘的中段,通常平皮脂腺水平或在其之下,这些NK I/beteb阳性细胞就是AMMC,AMMC数目较少,常成群集中分布,外观胞体偏圆,树突不明显,形态类似于周边的毛皮质细胞。结论AMMC位于外毛根鞘的中下段,形态和功能上均不成熟。     

外毛根鞘黑素细胞研究进展

外毛根鞘存在未活化无黑素性黑素细胞。与表皮黑素细胞一样同源于神经嵴。但它们之间在树突形态、黑素合成、抗原表达、分化程度等方面不尽相同。现认为无黑素性黑素细胞不仅可能为生长期毛囊提供黑素干细胞 ,而且在白癜风色素再生时 ,它可被紫外线辐射、生长因子、擦皮术等因素激活后迁至白斑区起黑素细胞储库作用。     

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。     

黑素细胞干细胞与外毛根鞘黑素细胞

干细胞是一种具有无限或长期的自我更新能力，至少可向一种成熟功能细胞分化的细胞群体。目前认为，胚胎干细胞即全能干细胞，可以向包括生殖嵴在内的所有组织分化。定向干细胞具有单向分化的功能。多向干细胞则介于胚胎干细胞与定向干细胞之间，可以向多种组织分化。外毛根鞘存在黑素细胞干细胞，他在生长期具有不成熟、慢周期性、自我更新以及活化后完全再生潜力等特征。他与表皮黑素细胞一样同源于神经嵴，但很多方面不尽相同。而人外毛根鞘存在未活化的无黑素性黑素细胞，与表皮黑素细胞也有很大差别：外毛根鞘存在未活化的无黑素性黑素细胞到底是不是黑素细胞干细胞，二者的关系如何，目前仍在研究之中。     

外毛根鞘黑素细胞研究进展

外毛根鞘存在未活化无黑素性黑素细胞。与表皮黑素细胞一样同源于神经嵴。但它们之间在树突形态、黑素合成、抗原表达、分化程度等方面不尽相同。现认为无黑素性黑素细胞不仅可能为生长期毛囊提供黑素干细胞,而且在白癜风色素再生剂,它可被紫外线辐射、生长因子、擦皮术等因素激活后迁至白斑区起黑素细胞储库作用。     

毛囊与干细胞

摘要皮肤和毛囊的干细胞生物学已经成为生物学、医学热点之一。目前一些实验表明毛囊中很多数量的成体细胞群具有更为广泛的干细胞活性。外毛根鞘、隆突部位甚至结缔组织鞘中的某些细胞具有干细胞活性。毛囊是毛囊干细胞生长、发育的重要的组织结构：毛囊不仅为毛囊干细胞储库提供了保护环境，而且还为其生长、发育和分化提供了内环境及其所需的各种生长因子。     

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。     

无黑素性恶性黑素瘤1例

恶性黑素瘤(MM)是一种高度恶性的肿瘤,多发生于皮肤,占皮肤恶性肿瘤的6.8%～20%,好发于30岁以上的成年和老年人,儿童罕见.恶性黑素瘤的类型较多,临床表现亦各异.现将作者所见到的1例报告如下.     

分离酶法体外分离和培养人毛囊黑素细胞

越来越多的研究发现,人毛囊黑素细胞(hair follicle melanocyte,HFM)在白癜风的色素恢复中起重要作用.1995年Tobin等^[1]首次报道胶原酶法体外分离培养人HFM成功,我们加以改进后发现分离酶法更易成功.这为更好地研究人HFM的生物学特性提供了重要基础.     

A modified method for purifying amelanotic melanocytes from human hair follicles

We describe a modified method for establishing long-term pure cultures of amelanotic melanocytes (AMMC) derived from human hair follicles. Normal human corpse scalp (just after death, 1 h) was transected 1 mm below the epidermis, and hair follicles in the remaining dermis were isolated by a two-step enzyme treatment. Hair follicle cell suspensions were prepared by 0.50% trypsin treatment for 30 min and cultured in an optimized melanoblast proliferation nature mitogen medium. Cells attached to the substratum were mostly amelanotic melanocytic in character with small, bipolar shapes in the early stage; only a few keratinocytes and rare fibroblasts were observed. Keratinocytes were easily removed by differential trypsinization. After the third passage, the proliferating cells were all amelanotic melanocytes as confirmed by immunostaining with polyclonal antibodies to alphaPEP7h, which recognized the tyrosinase protein located on melanosomes and NKI/beteb, which is a pre-melanosomal antigen against synthetic peptides corresponding to the carboxyl termini of human melanosomal protein GP100. Cultured AMMC were highly positive to L-dopa reactivity after the addition of IBMX to the culture medium for 7 days. Many stage I and II melanosomes and occasional stage III melanosomes without stage IV melanosomes were found in the cytoplasm by transmission electron microscope. This modified technique is potentially more suitable for cultivating amelanotic melanocytes. The availability of pure cultures of hair-follicle amelanotic melanocytes will facilitate investigations of the roles of those cells in migration and differentiation during treatment of vitiligo.     

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。     

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。     

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.     

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。     

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.     

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。