In vitro growth characteristics of melanocytes obtained from adult normal and vitiligo subjects

The in vitro growth characteristics of melanocytes obtained from uninvolved and perilesional skin of vitiligo vulgaris subjects have been investigated in comparison to those from healthy adult donors. Normal human melanocytes have been found to grow exponentially in the presence of 10(-11) M cholera toxin and 10 ng/ml of 12-O-tetradecanoylphorbol-13-acetate in routine tissue culture media. They could be trypsinized up to 3-4 passages. Melanocytes of the uninvolved skin of vitiligo subjects manifested a lag of 8-11 days for the onset of growth and they could not be passaged. Melanocytes obtained from both hypo- and hyper-pigmented perilesional skin failed to grow under these conditions. Only in a few cases where the perilesional skin was normally pigmented did the melanocytes manifest some growth after a lag of 15 days. The initial seeding capacity of the melanocytes from uninvolved and perilesional skin of vitiligo patients were, respectively, 50% and 25% of the normal individuals. Vitiligo lesions themselves gave rise to unidentified dendritic cells that survived for 10-15 days without manifesting any growth. Our results suggest that melanocytes of individuals with vitiligo are defective. This fact has to be taken into account in any theory on the etiology of vitiligo.     

Culture of normal adult human melanocytes

Melanocytes isolated from normal adult human skin were cultured in vitro. Separation of the epidermis from the dermis by trypsin flotation proved better than collagenase treatment for providing viable cultures of melanocytes with a minimum of fibroblast contamination. Centrifugation on a discontinuous, rather than a continuous Percoll gradient, was more efficient in separating the epidermal cell types. Most of the melanocytes were usually found in one particular layer, and most of the viable keratinocytes were in the sediment. None of the layers produced a uniformly high percentage of melanocytes on routine culture, but enriched melanocyte cultures could be obtained by seeding the epidermal cells in magnesium- and calcium-free medium for 24 to 48 hours, and then transferring them to fibroblast-conditioned medium containing horse serum and polyamines. Melanocytes were identified by their dendritic morphology, ultrastructure, reaction to cholera toxin and pigment production after treatment with melanocyte stimulating hormone. Pure cultures of melanocytes have been cultivated by this method for more than 43 weeks (ten passages).     

Selective cultivation of human melanocytes from newborn and adult epidermis

Development of adequate culture systems for the human epidermal melanocyte is critical to further advances in pigment cell biology. We now report selective growth and long-term maintenance of melanocytes derived from both newborn and adult skin specimens. Disaggregated epidermal cell suspensions were plated in a hormone-supplemented medium containing cholera toxin and a hypothalamic extract treated to remove keratinocyte growth-promoting activity. After 3-4 weeks, pure melanocyte populations could be harvested and serially passaged up to 6 times over several months for a total of 10 or more cumulative population doublings in vitro. Electron microscopic studies revealed metabolically active cells with abundant melanosomes in various stages of melanization throughout the culture lifespan. Differences in size and number of melanosomes attributable to race of the tissue donor were readily apparent, and pigment content of melanocytes from both black and Caucasian donors appeared to increase with time in culture. Newborn melanocytes proliferated more rapidly and survived longer than did adult melanocytes, but there were no consistent morphologic differences as a function of donor age. Comparison of growth potential for the 3 major skin-derived cell types in this hormone-supplemented medium revealed striking specificity for melanocytes, with total elimination of keratinocytes over 1-2 weeks, and no fibroblast proliferation whatever in the absence of serum supplementation. This system promises to facilitate in vitro investigation of epidermal melanocytes in normal and diseased human skin.     

Effect of dicarboxylic acids on normal human melanocytes in dispersed tissue culture

Since dicarboxylic acids are competitive inhibitors of tyrosinase, and effective in treatment of hyperpigmentary disorders, such as chloasma and lentigo maligna, probably due to a cytotoxic effect on abnormal melanocytes, it is of interest to examine their effect on normal melanocytes in tissue culture. Azelaic or dodecandioic acids were added (150-200 micrograms/ml) to dispersed cultures of epidermal cells, and melanocytes were examined by electron microscopy after 7, 10, 15, 20 and 30 days. Apart from a stimulation of melanogenesis, the presence of dicarboxylic acids in the culture medium caused no detectable damage to melanocytes, nor did they prevent growth of a second generation of cells.     

自体黑素细胞培养移植治疗白癜风疗效评价

目的：评价黑素细胞培养移植治疗白癜风的疗效。方法：采用从发疱壁上获取黑素细胞、纯黑素细胞培养与增殖、移植区刮除种植法,进行自体黑素细胞培养移植治疗白癜风;应用计算机皮肤数字图像分析系统进行疗效评价。结果：23例白癜风患者中28块皮损进行了自体黑素细胞培养移植,总有效率为89.29%;移植后的平均灰度与正常皮肤平均灰度相接近。结论：此操作方法较简单,治疗面积大,色素分布均匀,值得临床参考应用;计算机图像分析系统的疗效评价更具有客观性。     

Transplantation of melanocytes in vitiligo

Summary We report the results of a study on 100 patients (aged 12–68) with vitiligo, who were treated by transplantation of cultured autologous melanocytes to the depigmented areas, after removal of the epidermis at the recipient site by dermabrasion. The melanocytes were cultured from a 2x3 cm² superficial shave biopsy taken from pigmented buttock skin. After 2–3 weeks in culture, 700–1000 cells per mm² were applied on 60–500 cm² dermabraded areas, and occluded for 1 week. The repigmented portion of the total treated area amounted to 95–100% in 40 patients, 65–94% in 32, 20–64% in 22, and 0–19% in six. It was more difficult to achieve complete pigmentation on the fingers, elbows and knees. In the first few months following the procedure, the treated areas were often hypo- or hyperpigmented, but after 6–8 months they had acquired the same colour as the surrounding skin. No scarring or other side-effects occurred. The donor site had repigmented after 3–6 months in all but two patients, who also showed poor pigmentation in the transplanted areas. At follow-up after 1 and 2 years in 50 and 10 patients, respectively, the repigmented areas remained unchanged. The method is time-consuming, but the results obtained indicate that the procedure can be valuable in motivated patients, when the extent of vitiligo does not exceed 30% of the total body area, and when the areas to be treated are not actively extending.     

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。     

Cultured human melanocytes from black and white donors have different sunlight and ultraviolet A radiation sensitivities.

Short-term and long-term survival of cultured neonatal foreskin melanocytes from black and white individuals were assessed following a single exposure to simulated sunlight or ultraviolet A (UVA) radiation. Melanocytes from black individuals contained significantly more melanin than melanocytes from white individuals (p less than 0.05). Black and white melanocytes had similar survival profiles following simulated sunlight exposure, whereas black melanocytes were significantly more resistant to UVA cytotoxicity than melanocytes from white subjects (p less than 0.05) at UVA doses above 15 J/cm2. There was no difference in unscheduled DNA synthesis in the black or white melanocytes following simulated sunlight exposure and no unscheduled DNA synthesis was measurable following melanocyte exposure to UVA radiation. Low-dose UVA (1 or 5 J/cm2) was mitogenic to both black and white melanocytes. By analysis of co-variance, the melanin content of melanocytes of black and white subjects was significantly (p less than 0.05) associated with susceptibility to UVA killing; melanocytes with high melanin content had high resistance to UVA cytotoxicity and those with low melanin content had low resistance to UVA cytotoxicity. From these data we suggest that the higher melanin content of melanocytes of black subjects confers increased resistance to UVA damage. This is likely to be of importance in epidermal photodamage.     

人黑素细胞体外培养条件的研究进展

从培养人黑素细胞的基本培养液、添加剂、紫外线以及共培养系统等方面综述安全、有效的黑素细胞体外培养条件的研究进展;同时总结了人毛囊无色素性黑素细胞、永生化黑素细胞的培养条件,对色素疾病的基础研究及临床治疗有重要意义。     

Effects of dicarboxylic acids on normal and malignant melanocytes in culture

We have shown that dicarboxylic acids (C9 and C12), known competitive inhibitors of tyrosinase, are selectively cytotoxic to malignant melanogenic melanocytes but not to normal pigmented cells or to amelanotic or non-melanogenic melanoma cells. The main target of this toxicity appears to be the mitochondria, which become markedly swollen and vacuolated. The mechanism of their action has been thought to be due to interference with oxidoreductases in the mitochondria. However, our results suggest that this cytotoxicity most probably does not result simply from inhibition of mitochondrial enzymes, but is closely related to the melanin biosynthesis pathway.     

Behavioral differences between donor site-matched adult and neonatal melanocytes in culture

Abstract Little is known about the biologic behaviors of cultured melanocytes in relation to donor age. To investigate age-dependent differences, neonatal and adult melanocytes were isolated from the same anatomical site, the foreskin, and cultured in the same growth medium supplemented with cAMP inducers (choleratoxin and 3-isobutyl-methylxanthine). The morphology, melanin content, pattern of melanosome distribution, and growth rate were then compared. Neonatal melanocytes were bipolar in appearance, whereas adult melanocytes were highly dendritic in appearance. Image analysis showed that adult melanocytes were larger and longer, and had a greater number of dendrites than neonatal melanocytes. When the growth medium was replaced by a medium without cAMP inducers, adult melanocytes showed a change in their morphology from dendritic to spindle-shaped, while the morphology of neonatal melanocytes remained unchanged. Melanosomes of adult melanocytes were distributed singly along the dendrites, and extracellular secretion of melanosomes was also found. In contrast, melanosomes of neonatal melanocytes were aggregated near the nuclei. No age-dependent differences in melanin content and growth rate were noted in the donor site-matched cultured melanocytes. These results suggest that donor age is one of the factors involved in determining melanocyte dendricity and melanosome distribution, and that increased dendricity of adult melanocytes is due to increased sensitivity to cAMP inducers. In addition, the adult melanocytes established in our culture system, which resembled dendritic melanocytes in vivo, could be considered a desirable model for studying the mechanisms of adult-onset hyperpigmentary disorders and melanogenesis. Received: 2 June 1998 / Revised: 29 November 1999 / Accepted: 10 December 1999     

Application of the tyrosinase assay to normal melanocytes in culture

Using a special selection technique, normal guinea-pig melanocytes were maintained in highly purified but sparse cultures (approximately 10(4) cells/25 cm2 culture vessel) which showed little proliferative activity. The applicability of the Pomerantz tyrosinase assay was tested in this in vitro model system using three different approaches, namely crude cell extracts and viable cell cultures either in situ or in suspension. The latter modifications both proved too insensitive, whereas crude cell extracts allowed accurate measurements of the basal tyrosinase activity and its stimulation by various agents. In unstimulated cultures basal tyrosinase activities ranged from 30% to 700% (mean 260%) above the blank values; intra-assay and inter-assay variability were 4.2% and 77.5%, respectively. Stimulation with alpha-MSH (10(-5) M, 10(-6) M), beta-MSH (10(-5) M), choleratoxin (10(-11) M) and cAMP (10(-4) M) plus theophylline (10(-4) M) resulted in an increase of tyrosinase activity 30-65% above basal values. Melanotropin potentiating factor (10(-8) M) enhanced the effects of alpha-MSH (10(-6) M) by 20%. This assay modification provides a sensitive tool for comparative studies of melanogenesis in normal melanocytes, malignant melanocytes and otherwise altered melanocytes.     

NB-UVB对白癜风患者黑素细胞生物学特性的影响

目的：明确窄谱中波紫外线(narrow-band UVB, NB-UVB)对白癜风患者的黑素细胞生物学特性的影响。方法：提取34例白癜风患者皮损处黑素细胞进行体外培养。每例样本分组后分别接受不同的照射剂量（0、20、40、60、80、100 mJ/cm2）的NB-UVB处理，照射后1 d采用免疫印迹法对黑素细胞中微管相关蛋白轻链3 II/I(LC3 II/I)、酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)、自噬信号磷酸化磷酸腺苷蛋白激酶(p-AMPK)、P26蛋白的表达情况进行测定。照射后2d、3d、4d采用MTT法对黑素细胞的增殖活性进行测定，采用NaOH染色法对黑素细胞中黑素含量进行测定。结果：随着NB-UVB照射剂量的增加,LC3 II/I和p-AMPK表达水平逐渐上调，p-mTOR和P26的表达水平逐渐下降。不同NB-UVB剂量下，黑素细胞增殖水平增加，但随剂量的增加，增殖的程度逐渐减弱。不同NB-UVB剂量黑素细胞中黑素水平升高，且呈剂量依赖式递增。结论：NB-UVB能够有效调节白癜风患者黑素细胞自噬相关蛋白表达水平，活化自噬信号通路，促进黑素细胞增殖及黑素合成能力。     

Study of CCN3 (NOV) and DDR1 in normal melanocytes and vitiligo skin

We have hypothesised that melanocytes disappear in vitiligo because they are weakly attached to the epidermal basal membrane (melanocytorrhagy). In the epidermis, attachment of melanocytes to collagen IV is mediated through DDR1, which is under the control of CCN3. DDR1 genetic variants have been associated with vitiligo in patients of different ethnic origin. In vitro studies have shown that inhibition of CCN3 induces the detachment of melanocytes. We have studied in parallel the expression of CCN3 and DDR1 in lesional and perilesional skin of patients with vitiligo and the impact of the silencing of CCN3 and DDR1 in normal human melanocytes on their behaviour in epidermal reconstructs. Our in vivo study provides evidence of a dysregulation of the DDR1-CCN3 interaction in vitiligo skin as melanocytes remaining in perilesional skin did not express CCN3. Expression of DDR1 was decreased in lesional versus perilesional vitiligo skin in the majority of patients, and the expression of collagen IV was found decreased in all patients. Silencing of CCN3 in melanocytes induced a significant inhibition of cell adhesion to collagen IV whereas melanocytes transduced with shDDR1 still adhered well on collagen IV and did not increase melanocyte loss in epidermal reconstructs as compared with normal melanocytes. Melanocyte detachment was observed but not in all reconstructs using CCN3 silenced melanocytes. Overall, our study confirms that a downregulation of CCN3 is implicated in melanocyte adhesion in part through DDR1. In vitiligo skin, the interaction of CCN3 with other molecules, such as TGFβ and CCN2, needs to be addressed.     

Expression and modulation of apoptosis regulatory molecules in human melanocytes: significance in vitiligo

Although the aetiology of the hypopigmentary disorder vitiligo is ill understood, it is clear that pigment producing cells are absent from vitiliginous lesional skin. The present study was designed to investigate the possible role of melanocyte-expressed apoptosis regulatory molecules in melanocyte disappearance. Flow cytometric evaluation of p53, p21, Bcl-2 and Bax revealed no differences in in vitro expression levels between normal control and non-lesional melanocytes. Moreover, no in situ immunohistological differences were observed in melanocytes present in control, non-lesional and perilesional skin. However, an enhanced number of p53+ nuclei, in the absence of detectable p21 expression, was detected in involved areas. The observed p53 expression pattern did not involve melanocytes and could be the result of ultraviolet (UV) A irradiation. Further, we showed that UVB is capable of modulating melanocyte-expressed apoptosis regulatory molecules. Consequently, a lethal dose of UVB was given to two groups of cultured normal control and non-lesional melanocytes. No significant differences were found when comparing the percentages and kinetics of UVB-induced apoptosis in these groups. In conclusion, our results indicate that the relative apoptosis susceptibility of melanocytes in vitiligo is comparable with that of normal control cells. It is therefore unlikely that vitiligo is causally related to dysregulation of apoptosis regulatory molecules.     

白癜风表皮黑素细胞超微结构及小眼畸形相关转录因子转录调控研究

目的 研究白癜风黑素细胞超微结构和小眼畸形相关转录因子 （MITF）及其转录调控的酪氨酸酶相关蛋白（TRP）与白癜风临床类型与病程的相关性。方法 选择不同病程的寻常型白癜风（VV）12例和节段型白癜风（SV）8例,分别取白斑区、白斑边缘正常肤色区和远离白斑正常肤色区的表皮片,经组织学确定其表皮的完整性。透射电镜观察10例患者（VV 6例,SV 4例）不同区表皮黑素细胞的超微结构特点。对所有20例远离白斑正常肤色区的表皮片黑素细胞进行培养,应用免疫印迹方法检测 MITF及其转录调控的酪氨酸酶（TYR）、酪氨酸酶相关蛋白1（TYRP1）和酪氨酸酶相关蛋白2（TYRP2）的表达水平。结果 白癜风表皮黑素细胞超微结构病理改变：10例中7例白斑区表皮内未见黑素细胞,1例短病程和2例长病程VV分别可见少量黑素体显著减少或缺失的黑素细胞;白斑边缘正常肤色区,6例VV中,3例病程小于15个月者可见黑素细胞超微结构异常,而4例SV中仅1例异常;远离白斑正常肤色区,10例黑素细胞超微结构均正常。白癜风表皮黑素细胞MITF及其转录调控TRP的表达：VV的MITF表达下调与TYR、TYRP1、TYRP2的表达下调一致;SV存在MITF显著表达下调,而TYR、TYRP1、TYRP2几均正常表达。结论 VV和SV可能存在不同的表皮黑素细胞超微结构病理改变和MITF转录调控机制。