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。     

人毛囊外根鞘及毛乳头细胞的迁移和生长观察

目的连续观察成人头皮毛囊外根鞘(ORS)和毛乳头(DP)细胞的生长及形态学变化。方法利用两步酶消化获得正常人头皮毛囊的ORS和DP,采用添加HKGS和HMGS的K-SFM进行体外培养,倒置显微镜下连续观察毛囊ORS和DP的迁移和生长。结果将单个毛囊置于预先涂布血清的培养板2h,然后添加少量培养基,毛囊黏附于培养板的几率增加,数小时后,即可见细胞从ORS及DP处迁移生长,以角质形成细胞(KCs)、成纤维细胞(FBs)为主,黑素细胞(MCs)较少。并且,ORS和DP的迁出和生长有明显不同。如果培养基过多,则多数毛囊悬浮,无细胞迁出,最终死亡。结论预先涂布血清和开始仅添加少量培养基,有利于毛囊黏附,随后细胞迁移生长。     

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。     

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。     

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。     

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。     

双氢睾酮对小鼠毛囊外根鞘体外培养再生的影响

目的 筛选小鼠触须毛囊外根鞘(ORS)在体外培养再生的最适双氢睾酮(DHT)浓度,通过检测Dermo-1、CD11b探索其对毛囊外根鞘再生的影响。方法 利用冰冻切片技术HE染色观察毛囊外根鞘在不同DHT浓度培养条件下的再生程度,利用免疫荧光技术鉴定Dermo-1、CD11b在最适DHT浓度条件下在毛囊外根鞘中的表达情况。结果 浓度为10^-5mol/L、10^-6mol/L的DHT对毛囊外根鞘体外再生起抑制作用,但10^-9mol/L的DHT促进其体外再生。并且Dermo-1、CD11b在毛囊外根鞘再生过程中均有表达。结论 毛囊外根鞘在体外培养第5天时再生的结构最完整,10^-9mol/L的DHT与体内浓度最为接近,毛囊干细胞对创伤修复和组织结构再生都发挥着重要作用。     

人头发毛囊外根鞘中有二个不同的K19阳性区:干细胞储库的统一假说?

至今，人生长期毛囊中干细胞的定位依靠三种互补的检查方法：即检测缓慢周期生长细胞、高集落形成细胞和分化细胞的免疫组化染色。由于干细胞既可以是位于隆突部位的长期标记的保留细胞，也可以是位于毛囊下三分之一处的高集落形成细胞，所以这些技术往往产生相互矛盾的结果。为了阐明毛发生长周期中人毛囊干细胞的分布情况，我们研究了通常被认为是上皮干细胞标志的细胞角蛋白19(K19)的表达。发现人生长期毛囊中有两个干细胞储库，分别位于毛囊的上、下三分之一处。这两个储库在毛囊的退行期一休止期的过渡阶段发生融合，在新形成的生长期毛囊中再次分开。     

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.     

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。     

The innermost cell layer of the outer root sheath in anagen hair follicle: Light and electron microscopic study

Summary To investigate cell differentiation in the outer root sheath (ORS) of the human anagen hair follicle, scalp skin specimens from individuals with normal hair were examined using light and electron microscopes. In the bulbar portion, the ORS was composed of two cell layers. The cells in the outer layer gradually increased in number upwards and finally underwent so-called trichilemmal keratinization, which proceeded toward the hair canal. On the other hand, the inner cells in the bulb formed a single cell layer along the outside of Henle's layer during cell differentiation; this unique layer was referred to as the innermost cell (IMC) layer of the ORS. With the use of hematoxylin and eosin stain, at the suprabulbar portion, where Henle's cells were keratinizing, an eosinophilic substance was deposited in the inner (Henle's) side of the IMC cytoplasm. The IMCs gradually became entirely eosinophilic and often produced keratohyaline granules. Ultrastructurally, the IMCs of the ORS showed an oblong shape forming a regularly arranged single-cell layer along the keratinizing Henle's layer and accumulated tonofilaments in the cytoplasm. They produced a few small electron-dense keratohyaline granules and were keratinized at the level at which Henle's layer still preserved its cell structure. From these findings, it is suggested that there are two types of keratinization of the ORS: trichilemmal keratinization and IMC keratinization.     

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。     

VEGF165 modulates proliferation,adhesion, migration and differentiation of cultured human outer root sheath cells from central hair follicle epithelium through VEGFR-2 activation in vitro

BackgroundThe functional state of vasculature is tightly controlled by vascular endothelial growth factor receptor-2 (VEGFR-2). Recent studies revealed that VEGFR-2 is expressed on hair follicle keratinocytes.ObjectiveWe proposed to investigate its effect on proliferation, adhesion and migration of cultured human outer root sheath cells from central hair follicle epithelium.MethodsThese studies were undertaken in vitro using human outer root sheath cells from central hair follicle epithelium, immunohistochemistry analysis, immunofluorescence microscopy, western blot analysis, MTT, trans well analysis, and RT-PCR.ResultsOur results show that VEGFR-2 is expressed in these cells in vivo and in vitro. Furthermore, proliferation and migration of cultured human outer root sheath cells from central hair follicle epithelium is increased by VEGF₁₆₅, while homotypic adhesion is decreased but heterotypic adhesion is increased. VEGF₁₆₅ upregulates integrin β1 but dowregulates lgr6 expression. In addition, phosphorylation of VEGFR-2, Erk1/2, c-Jun and p38, are increased following VEGF₁₆₅ treatment and these effects are reversed by a VEGFR-2 neutralizing antibody.ConclusionOur results suggest a role of VEGF/VEGFR-2 beyond angiogenesis in hair follicle regulation.