黑素小体代谢与相关皮肤病

黑素小体是皮肤、眼、内耳、脑膜等处色素细胞特有的、溶酶体样的膜性细胞器,是合成和储存黑色素的亚细胞结构,其代谢过程包括黑素小体的生成、转运和降解三个过程。其代谢过程的异常可导致一些皮肤病的发生。文中简要地综述了黑素小体的代谢过程以及与代谢异常有关的皮肤病。     

黑素小体转运机制的研究进展

色素的生物合成过程已基本明确,但黑素小体从黑素细胞转运至邻近的角质形成细胞的机制尚未完全明了.目前认为许多分子和信号转导途径参与此过程,如KGF、PAR-2、外源凝集素、拟糖蛋白、Rab-27a、肌球蛋白Va、Rho、Ca2+、cAMP/PK等,另外紫外线照射也影响黑素小体转运.了解黑素小体转运机制对于治疗色素异常疾病及开发美白产品有重要意义.     

黑素小体特异性蛋白Pmel17的研究进展

Pmel17是黑素小体特异性蛋白,在早期黑素小体中形成纤维性基质,以利于黑素沉积.在Pmel17合成代谢过程中,适配器蛋白与Rab7参与调控其从高尔基体到黑素小体的投递,去整合素金属蛋白酶与pH值参与调控其在黑素小体内形成纤维基质.Pmel17与黑素小体的结构形成,分级组装,转运功能等密切相关,其表达受小眼相关转录因子、眼白化病1型因子、α黑素细胞刺激素等因素的调节.Pmel17参与了白癜风发病,为白癜风的免疫机制研究提供新的方向,Pmel17还可作为黑素瘤的治疗靶点.     

黑素小体在不同肤色皮肤类型角质形成细胞内的分布:黑素小体簇不是具有降解功能的细胞器

黑素小体是人体皮肤黑素细胞、视网膜色素上皮细胞和脉络膜黑素细胞内合成黑素的细胞器:虽然与溶酶体一样都有溶酶体膜蛋白和水解酶,但黑素小体是起源于胞吞途径的一种独立的细胞器类型,且不能通过胞吞示踪剂检测到,另外其pH值较高,因此被归类为溶酶体相关细胞器:在表皮内,将黑素小体转运至邻近的角质形成细胞至少存在3种机制:细胞吞噬作用、胞吐/胞吞作用,或经丝状伪足转移。     

脂溢性角化病皮损组织蛋白酶L2表达及活性对黑素小体降解的影响北大核心CSCD

目的检测脂溢性角化病（SK）皮损中组织蛋白酶L2（CTSL2）表达及活性,观察sK皮损中黑素小体超微结构的变化,研究CTSL2对黑素小体降解的影响。方法2016年1—8月招募武汉大学人民医院皮肤科门诊SK患者20例,取患者皮损和周围正常皮肤组织,其中15例用HE染色、Fontana-Masson嗜银染色观察黑素颗粒分布,透射电镜（TEM）观察黑素小体超微结构变化,Ki67免疫组化染色检测细胞增殖状态。5例用RT-PCR和荧光底物裂解法分别检测CTSL2mRNA表达水平及酶活性;用蔗糖梯度离心法从废弃人眼球视网膜色素上皮组织分离纯化黑素小体,并与sK皮损表皮裂解物共孵育,TEM观察孵育后黑素小体膜结构的变化。两组间均数比较采用配对t检验,P〈0．05为差异有统计学意义。结果SK皮损中可见大量黑素颗粒沉积,而正常皮肤仅在基底层见线形黑素颗粒沉积。TEM观察显示,SK皮损中黑素小体破损率为24．33％±3．06％,正常皮肤为49．00％±4．00％,两组比较,t=8．49,P〈0．05。RT-PCR显示,5例SK皮损中CTSL2mRNA相对表达水平为0．35±0．09,正常皮肤组织为0．43±0．08,两组比较,t=3．17,P〈0．05。SK皮损中CTSL2酶活性（17．46±0．45）也低于正常皮肤组织（28．78±0．58）,t=34．29,P〈0．05。TEM还观察到,SK皮损裂解物处理的黑素小体破损率为32．33％±4．93％,低于正常皮肤43．00％±2．65％,两组比较,t=3．30,P〈0．05。结论SK皮损中CTSL2表达水平减低影响角质形成细胞对黑素小体的降解,是否直接参与SK皮损的病理发生有待进一步证实。     

~(14)C-2-硫脲嘧啶掺入显示:人毛囊色素单位可作为黑素生成调节剂的直接靶点

毛发色素由拉于毛乳头上部基底层中的一群黑素细胞合成,这些有活性的黑素细胞在黑素小体内生成黑素并将其转运至毛发纤维的前皮质角质形成细胞田(precortical kerationcyted).     

用灵敏的荧光测定及~(14)C标记酪氨酸掺入黑素的方法证实白癜风皮肤内的酪氨酸酶

白癜风的发炳占世界人口的0.14～3%.白癜风皮肤内是否存在黑素细胞仍有争论.过去检查黑素细胞是否存在用电镜或组织化学的方法,但比较理想的方法是测定白癜风表皮内的酪氨酸酶.酪氨酸酶可催化酪氨酸的羟基化,形成β3.4—二羟基苯     

The effect of niacinamide on reducing cutaneous pigmentation and suppression of melanosome transfer

BACKGROUND: Cutaneous hyperpigmentation occurs in multiple conditions. In addition, many Asian women desire a lighter skin colour. Thus, there is a need for the development of skin lightening agents. Niacinamide is a possible candidate. OBJECTIVES: To investigate the effects of niacinamide on melanogenesis in vitro and on facial hyperpigmentation and skin colour in vivo in Japanese women. METHODS: Melanin production was measured in a purified mushroom tyrosinase assay, cultured melanocytes, a keratinocyte/melanocyte coculture model, and a pigmented reconstructed epidermis (PREP) model. The clinical trials included 18 subjects with hyperpigmentation who used 5% niacinamide moisturizer and vehicle moisturizer in a paired design, and 120 subjects with facial tanning who were assigned to two of three treatments: vehicle, sunscreen and 2% niacinamide + sunscreen. Changes in facial hyperpigmentation and skin colour were objectively quantified by computer analysis and visual grading of high-resolution digital images of the face. RESULTS: Niacinamide had no effect on the catalytic activity of mushroom tyrosinase or on melanogenesis in cultured melanocytes. However, niacinamide gave 35-68% inhibition of melanosome transfer in the coculture model and reduced cutaneous pigmentation in the PREP model. In the clinical studies, niacinamide significantly decreased hyperpigmentation and increased skin lightness compared with vehicle alone after 4 weeks of use. CONCLUSIONS: The data suggest niacinamide is an effective skin lightening compound that works by inhibiting melanosome transfer from melanocytes to keratinocytes.     

p21小干扰RNA对低浓度过氧化氢诱导的黑素细胞提前衰老和黑素传递的影响

目的:探讨小干扰RNA(siRNA)抑制p21表达对低浓度过氧化氢(H₂O₂)诱导的黑素细胞提前衰老的影响,以及黑素细胞提前衰老与黑素传递功能间的关系。方法:p21 siRNA序列转染黑素细胞,采用荧光显微镜、实时荧光定量聚合酶链式反应(qRT-PCR)及western blot检测抑制效率。将正常人黑素细胞分为对照组(阴性siRNA转染处理)、p21 siRNA组(p21 siRNA转染处理)、阴性siRNA+H₂O₂组(阴性siRNA转染24 h后加入400μmol/L H₂O₂处理)及p21 siRNA+H₂O₂组(p21 siRNA转染24 h后加入400μmol/L H₂O₂处理)。采用β-半乳糖苷酶染色检测细胞衰老;EdU细胞增殖检测细胞增殖能力;western blot检测各组p21和树突调节蛋白[RhoA、Rac1及细胞分裂周期蛋白42(Cdc42)]表...     

谷氨酸受体非竞争性拮抗剂及激动剂调节表皮细胞伪足形成及黑素小体转运的观察

【摘要】 目的 探讨谷氨酸信号通路在黑素细胞及角质形成细胞伪足形成及黑素小体转运中的作用机制。 方法 原代培养并纯化黑素细胞、角质形成细胞,建立黑素细胞与角质形成细胞体外共培养体系。扫描电镜观察离子型谷氨酸受体N-甲基-D-天冬氨酸（NMDA）受体非竞争性拮抗剂MK801及激动剂NMDA作用下黑素细胞、角质形成细胞的伪足形态变化,以311 nm中波紫外线（UVB）照射为阳性对照。免疫荧光双染色激光共聚焦显微镜下观察MK801及NMDA对黑素小体转运的调节。 结果 扫描电镜观察发现,100 μmol/L MK801作用24 h后黑素细胞树突末端明显变细,树突变长,树突数量无明显变化而细胞表面的丝状伪足数量明显变少,且伪足长度变短;100 μmol/L NMDA作用24 h后黑素细胞树突的末端明显变宽、树突变短,树突数量无明显变化而细胞表面丝状伪足数量明显增多,丝状伪足长度增加。黑素细胞与角质形成细胞共培养体系中,未用药组黑素细胞与角质形成细胞间有丝状伪足相连接,黑素细胞的角质形成细胞侧丝状伪足数量多于对侧。100 μmol/L MK801作用于共培养体系24 h后,黑素细胞与角质形成细胞之间的丝状伪足数量及黑素细胞伸向角质形成细胞的丝状伪足数量减少;100 μmol/L NMDA作用于共培养体系24 h后,黑素细胞与角质形成细胞之间的丝状伪足数量及黑素细胞伸向角质形成细胞的丝状伪足数量增多。激光共聚焦显微镜观察显示,共培养体系下,未用药组角质形成细胞中存在黑素小体;100 μmol/L MK801作用24 h后角质形成细胞中的黑素小体数量减少;100 μmol/L NMDA作用24 h后角质形成细胞中的黑素小体数量增多,且与黑素细胞不相邻的角质形成细胞中也发现黑素小体存在。 结论 谷氨酸信号通路可能通过调节黑素细胞树突的形态及丝状伪足的形成参与调节黑素小体自黑素细胞至角质形成细胞的转运。 【关键词】 角蛋白细胞; 黑素细胞; 谷氨酸; 伪足; 黑色素小体; 树突     

Efficacy of quantifying melanosome transfer with flow cytometry in a human melanocyte–HaCaT keratinocyte co‐culture system in vitro

Please cite this paper as: Efficacy quantifying melanosome transfer with flow cytometry in a human melanocyte–HaCaT keratinocyte co‐culture system in vitro. Experimental Dermatology 2010; 19 : e282–e285. Abstract: In this study, we describe a simple, specific, reproducible and quantitative assay system to assess melanosome transfer. We first established a co‐culture model of normal human epidermal melanocytes and HaCaT keratinocytes. The cells were co‐cultured for 72 h in a serum‐free keratinocyte growth media and double labelled with Fluorescein isothiocyanate (FITC)‐conjugated antibody against the melanosome‐specific protein gp100, and with Phycoerythrin (PE)‐conjugated antibody against the keratinocyte‐specific marker cytokeratin. Then, the cells were examined using co‐focal microscope and flow cytometry. The increased melanosome transfer from melanocytes to HaCaT keratinocytes was observed in a time‐dependent manner. To verify the accessibility of this method, two known melanosome transfer inhibitors and two known melanosome transfer stimulators were applied. Consistent with previous investigation, soybean trypsin inhibitor (STI), niacinamide inhibited melanosome transfer, α‐melanocyte stimulating hormone (α‐MSH) and keratinocyte growth factor (KGF) increased melanosome transfer, respectively, in a dose‐dependent manner. The model used in this study could thus represent a rapid and reliable tool to identify modulators of human melanosome transfer.     

The silver locus product (Silv/gp100/Pmel17) as a new tool for the analysis of melanosome transfer in human melanocyte-keratinocyte co-culture

Abstract:  Melanosomes are melanocyte-specific lysosome-related organelles that are transferred to keratinocytes of the epidermis and anagen hair bulb. Transferred melanin forms supra-nuclear caps that protect epidermal keratinocytes against UV irradiation. The mechanism(s) responsible for melanosome transfer into keratinocytes and their subsequent intra-keratinocyte distribution has long remained one of the most enigmatic of heterotypic cell interactions. Although there have been many attempts to study this process, significant progress has been hindered by the absence of an adequate in vitro model. During our ongoing study of melanocyte–keratinocyte interactions in skin and hair follicle, we have developed a novel in vitro assay that exploits the specificity of Silv/Pmel17/gp100 expression for melanosome/melanin granules. Using matched cultures of keratinocytes and melanocytes isolated from normal healthy epidermis together with double immunofluorescence, we have determined that gp100 is a surprisingly useful tracker of transferred melanin. Moreover, transferred gp100 stained melanin granules emit a bright fluorescence signal, facilitating ready quantification of melanin transfer levels between melanocytes and keratinocytes. This quantitative approach was validated using known inducers and inhibitors of the melanocyte phenotype. This assay further confirmed that cytophagocytosis of melanocyte components (e.g. dendrite tips) by keratinocytes is one route for melanin incorporation into keratinocytes. Lastly, a role for the recently proposed filopodium as a direct conduit for melanin transfer was substantiated using this novel approach. In conclusion, this assay promises to significantly aid our investigations of the molecular basis of melanosome transfer and offers a new tool for the clinical evaluation of melanocyte modulators.     

A new spectrophotometric method for simple quantification of melanosomal transfer from melanocytes to keratinocytes

Three major difficulties must be overcome to establish a quantitative method for melanosomal transfer analysis: (i) establishing a three-dimensional co-culture reassuring direct melanocyte to keratinocyte transfer, (ii) separation of melanocytes and keratinocytes following co-culture and (iii) melanosome quantification in each cell population. Melanocytes and keratinocytes are cultured on the opposite sides of the porous membrane of hanging cell inserts (1μm pores, 2×10(6) pores/cm(2) ). Cell separation is performed after 3days of co-culture by simple trypsinisation. Melanosome quantification in separated cell populations was accomplished by an ELISA-like method using gp-100 as the antigen. Melanocytes and keratinocytes come into 'direct' contact through the pores, and melanosomal transfer is accomplished without cell passage through the membrane. Cell separation by simple trypsinisation results in pure melanocyte and keratinocyte populations. Melanosome quantification by the ELISA-like method proved to be sensitive and specific to distinguish the known inhibitors and inducers of melanosomal transfer.     

Demonstration of melanosome transfer by a shedding microvesicle mechanism

The process of melanosome transfer has fascinated pigment cell biologists for decades. Whole-organelle transfer is a unique property of melanocytes, suggesting that the biologic underpinnings of the process reflect melanocyte- and keratinocyte-specific proteins and pathways. Although several mechanisms of melanosome transfer are likely to occur in the skin, Ando et al. focused on a new mechanism of melanosome transfer that involves release of melanosome-containing globules, similar to shedding vesicles into the extracellular space, followed by uptake by keratinocytes. This model adds further complexity to the process of melanosome transfer in the skin.