中药菟丝子水提取物促毛囊无色素黑素细胞分化的实验研究

目的观察中药菟丝子水提取物诱导毛囊无色素黑素细胞(amelanotic melanocytes,AMMC)分化的作用。方法以8-甲氧补骨脂素(8-MOP)作为阳性对照,选择系列浓度的中药菟丝子水提取物和8-MOP作用于体外培养的AMMC,然后测定酪氨酸酶活性、黑素含量和细胞增殖率的变化。透射电镜观察药物作用前后AMMC超微结构的变化。结果菟丝子水提取物明显促进AMMC生成黑素(P<0.01),且呈浓度依赖性,但是促进作用弱于阳性对照8-MOP(P<0.01)。电镜结果显示,无药物作用的AMMC主要含有I,II和III期黑素小体,药物作用以后,AMMC内出现了大量的IV期黑素小体。菟丝子水提取物对细胞增殖无明显影响,但明显增强酪氨酸酶的活性。结论菟丝子水提取物促进了AM-MC的分化,这种作用与其增强酪氨酸酶活性有关。     

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。     

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。     

1,25二羟基维生素D3对毛囊外毛根鞘无色素黑素细胞激活的实验研究

目的研究1,25二羟基维生素D3(1,25-Dihydroxyvitamin D3,VID)对毛囊外毛根鞘无色素黑素细胞(amelanotic melanocytes,AMMC)的激活作用。方法以高、中、低3种不同浓度的1,25(OH)2D3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,然后收集细胞,测定细胞酪氨酸酶活性和黑素含量,透射电镜观察药物作用前后AMMC内黑素小体的变化,最后通过Western blot方法半定量分析药物作用前后AMMC内酪氨酸酶(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 relatedprotein-2,TRP-2)表达的变化。结果 VID能增加AMMC内酪氨酸酶活性,促进AMMC生成黑素,电镜结果显示药物作用后的AMMC内出现大量Ⅲ、Ⅳ期黑素小体。Western blot结果显示VID可以促进AMMC中TYR和TRP-1的表达,但对TRP-2的表达无明显影响。结论 VID通过促进黑素生成相关酶TYR和TRP-1的表达激活了AMMC。     

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。     

维A酸对A375细胞黑素合成及酪氨酸酶蛋白表达的影响

目的探讨维A酸对人A375黑素瘤株细胞黑素含量、酪氨酸酶活性及蛋白表达的影响。方法将培养的A375细胞分为维A酸组、8-甲氧补骨脂素(8-MOP)组、氢醌组、阴性对照组(A375细胞只加入相应溶媒,不加入药物)分别培养,于24 h、48 h和72 h测定黑素含量及酪氨酸酶活性;培养24 h Western检测酪氨酸酶蛋白的变化。结果 A375细胞加入维A酸24 h、48 h、72 h后黑素含量及酪氨酸酶活性均较阴性对照组下降(P0.05)。阴性对照组A375细胞酪氨酸酶蛋白含量测定值为(0.89±0.085),维A酸组A375细胞酪氨酸酶蛋白含量为(0.56±0.044)(t=3.811,P0.05)。结论维A酸能抑制人A375黑素瘤株细胞黑素含量、酪氨酸酶活性及蛋白表达。     

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。     

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。     

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。     

Integral hair lipid in human hair follicle

Integral hair lipid (IHL) is bound to the keratinized cell surface to make an environmentally resistant lipid envelope. It is mainly positioned on the hair cuticle and inner root sheath. IHL in the hair follicle may regard as hair barrier to be similar to the epidermal lipid layer functioning as skin barrier. Major constituents of IHL are fatty acid, phytosphingosine, ceramide in decreasing order. Minor constituents of IHL are cholesterol, cholesterol sulfate and cholesterol oleate. Cuticle or cortical cell surface in hair are abundant in fatty acids unlike the keratinized area of epidermis or sebaceous gland, and about 30-40% of such fatty acids are composed of 18-methyl-eicosanoic acid which is known to be bound to proteins by ester or thioester bond. Various factors including moisture, solvent, oxidative damage during bleaching or permanent waving affect IHL. Photochemical changes also can occur in IHL as well as in hair protein and hair pigment. Lipid metabolism is thought to play an essential role in lipid envelope of hair, but also involvement in hair development and function.     

Epidermis reconstructed from the outer root sheath of human hair follicle. Effect of retinoic acid

In the present paper, we show that a multilayered and well-differentiated epidermis can easily and rapidly be generated in vitro from the outer root sheath of human hair follicles deposited on de-epidermized demis. Histologically, this epidermis presented characteristic features of normal human epidermis in vivo. Moreover, markers specific for interfollicular keratinocyte terminal differentiation, such as the K10 keratin, involucrin, membrane-bound transglutaminase, filaggrin and loricrin, were expressed in the reconstructed tissue. By in situ hybridization, keratin K5 and K10 mRNAs were detected in the basal and suprabasal cells, respectively, as in normal human epidermis. The differentiation pattern achieved in this reconstructed epidermis confirms the already reported phenotypical shift from outer root sheath cells to interfollicular keratinocytes and shows that this transition takes place in the absence of living fibroblasts. The differentiation of the reconstructed epidermis thus obtained was modulated by retinoic acid in a dose-dependent manner. This culture system on dead dermis is easier to handle than similar cultures on collagen-fibroblast lattices because of the resistance of dermis to mechanical forces and to collagenolysis. It could represent a valuable wound-healing model and a promising tool for pharmacological studies on in vitro reconstructed skin.     

A primer for studying cell cycle dynamics of the human hair follicle

The cell cycle is of major importance to human hair follicle (HF) biology. Not only is continuously active cell cycling required to facilitate healthy hair growth in anagen VI HFs, but perturbations in the cell cycle are likely to be of significance in HF pathology (i.e. in scarring, non‐scarring, chemotherapy‐induced and androgenic alopecias). However, cell cycle dynamics of the human hair follicle (HF) are poorly understood in contrast to what is known in mouse. The current Methods Review aims at helping to close this gap by presenting a primer that introduces immunohistological/immunofluorescent techniques to study the cell cycle in the human HF. Moreover, this primer encourages the exploitation of the human HF as a powerful and clinically relevant tool to investigate mammalian cell cycle biology in situ. To achieve this, we describe methods to study markers of general ‘proliferation’ (nuclei count, Ki‐67 expression), apoptosis (terminal deoxynucleotidyl transferase dUTP nick‐end labelling, cleaved caspase 3), mitosis (phospho‐histone H3, ‘pS780’), DNA synthesis (5‐ethynyl‐2′‐deoxyuridine) and cell cycle regulation (cyclins) in the human HF. In addition, we provide specific examples of dual immunolabelling for instructive cell cycle analyses and for investigating the cell cycle behaviour of specific HF keratinocyte subpopulations, such as keratin 15+ stem/progenitor cells.     

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。     

Desmocollin 1 expression and desmosomal remodeling during terminal differentiation of human anagen hair follicle: an electron microscopic study

The terminal differentiation (TD) program of keratinocytes of the human hair follicle (HF) occurs with specific temporal and spatial features in the various layers of the inner root sheath (IRS) and in the innermost layer of the outer root sheath (companion layer). This process is characterized by complex nuclear and cytoplasmic morphological changes, accompanied by profound modifications in intercellular junctions. As no correlation exists between the structure and the molecular composition of desmosomes during TD of the IRS/companion unit, the aim of our study was to investigate by transmission electron microscopy the remodeling of desmosomes in keratinizing cells of these compartments. By immunogold post embedding technique, we studied in anagen HFs the modulation of the synthesis of desmocollin 1 (Dsc1), a transmembrane glycoprotein specifically synthesized in the IRS and in the companion layer. Dsc1 immunoreactivity was actually confined to these compartments and tended to increase just before the level of TD, particularly in the Henle's layer and in the IRS cuticle. In Huxley's layer, the immunolabeling was patchy and in the companion layer Dsc1 synthesis was detected above the level of keratinization of Huxley's layer. In the whole IRS, concomitantly with TD, there was an abrupt and almost complete disappearance of Dsc1 synthesis. An asymmetric distribution of Dsc1 was noticed (i) between cells at different stages of differentiation and (ii) between cells belonging to layers with different spatial/temporal features of TD. Our results show that the ultrastructural modifications of desmosomes during TD of HF are paralleled by the modulation of the synthesis of desmocollin 1.     

Skin-on-a-chip strategies for human hair follicle regeneration

The number of hair loss patients increases every year, and hair loss treatment has several limitations, so research on hair is attracting attention recently. However, most current hair follicle research models are limited by their inability to replicate several key functions of the hair follicle microenvironment. To complement this, an in vitro culture system similar to the in vivo environment must be constructed. It is necessary to develop a hair-on-a-chip that implements a fully functional hair follicle model by reproducing the main characteristics of hair follicle morphogenesis and cycle. In this review, we summarize the gradation of hair follicle morphogenesis and the roles and mechanisms of molecular signals involved in the hair follicle cycle. In addition, we discuss research results of various in vitro organoid products and organ-on-a-chip–based hair follicle tissue chips for the treatment of alopecia and present future research and development directions.     

毛囊间充质干细胞的研究进展

 毛囊间充质干细胞（HFMSCs）是一类具有自我再生能力、高度增殖潜能、可多向分化且来源丰富的慢周期标记滞留细胞,可促进表皮、毛囊和皮脂腺再生。得益于其来源丰富、易获得、可于体外扩增、无需基因操作、不会形成肿瘤和无伦理限制等特点,毛囊间充质干细胞在再生医学中具有重要优势。Wnt、Shh、Notch和BMP等信号通路之间的协同和拮抗作用在干细胞稳态调节、表皮发育和毛囊再生过程中发挥至关重要的作用,这些途径的失调可能导致毛发脱落或者肿瘤的发生。本文综述毛囊间充质干细胞的分类及其特异性生物标记物、毛囊间充质干细胞的活化以及影响毛发再生的生物分子途径,为毛发疾病的治疗提供新的线索和靶点。     

毛乳头细胞对毛囊前体黑素细胞作用的评价

目的：确定毛乳头细胞（DPC）对毛囊前体黑素细胞（MP）的激活和趋化作用。方法：采用三维分离式共培养体系,将MP与毛囊外毛根鞘角质形成细胞（KC）以1：10比例于6孔培养板中接触式共培养,DPC以1×10^6个／孔分离培养于Transwell inserts。检测DPC对MP的趋化作用。MP内黑素小体、酪氨酸酶（TYR）、酪氨酸酶相关蛋白1（TRP-1）和酪氨酸酶相关蛋白2（TRP-2）的表达。结果：MP与KC共培养后MP内出现Ⅲ期黑素小体,当三种细胞共培养后MP内主要为Ⅲ和Ⅳ期黑素小体。DPC可促进MP中TYR和TRP-1的表达,但对TRP-2的表达无明显影响,对MP具有明显趋化作用。结论：构建了MP、KC和DPC的三维培养体系,发现DPC对MP具有明显激活和趋化移行作用。